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.

Lack of activity of 15-epi-lipoxin A4 on FPR2/ALX and CysLT1 receptors in interleukin-8-driven human neutrophil function.

Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15-epi-lipoxin (LX)A interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti-inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15-epi-LXA4 analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK-571 binds to FPR2/ALX, so cross-reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15-epi-LXA4 are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15-epi-LXA4-mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX-mediated signalling, enhancing guanosine triphosphate-gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15-epi-LXA4 was inactive. Furthermore, 15-epi-LXA4 showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15-epi-LXA4 showed a moderate reduction of interleukin (IL)-8-mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15-epi-LXA4 is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL-8-induced neutrophil survival and produces only moderate inhibition in IL-8-mediated neutrophil migration. Our data do not support an anti-inflammatory role of 15-epi-LXA4- FPR2/ALX interaction in IL-8-induced neutrophil inflammation.

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.

Synthesis and biological evaluation of 2,5-dihydropyrazol.

A novel series of 2,5-dihydropyrazolo[4,3-c]quinolin-3-ones has been prepared. These compounds showed good PDE 4 inhibitory activity and weak affinity for rolipram's binding site. They also exhibited a good anti-inflammatory profile without emetic side effects.

Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors.

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

Pharmacological characterization of almotriptan: an indolic 5-HT receptor agonist for the treatment of migraine.

Almotriptan (3-[2-(dimethylamino)ethyl]-5-(pyrrolidin-1-ylsulfonylmethyl )-1H-indo le) has been studied in several models predictive of activity and selectivity at 5-HT receptors. Almotriptan showed low nanomolar affinity for the 5-HT(1B) and 5-HT(1D) receptors in several species, including the human, while affinity for 5-HT receptors other than 5-HT(1B/1D) was clearly less. Affinity for 5-HT(7) and 5-HT(1A) receptors was approximately 40 and 60 times lower than that for 5-HT(1B/1D) receptors, respectively. Almotriptan did not exhibit significant affinity for several non-5-HT receptors studied up to 100 microM. Almotriptan inhibited forskolin-stimulated cyclic AMP accumulation in HeLa cells transfected with 5-HT(1B) or 5-HT(1D) human receptors. In this model, almotriptan had the same efficacy as serotonin and an affinity in the low nanomolar range. It induced vasoconstriction in several vessels in which it was compared with sumatriptan. In isolated dog saphenous veins, almotriptan elicited concentration-dependent contractions with an EC(50) of 394 nM. In both these systems, almotriptan behaved as a full agonist. Infusion of almotriptan into the porcine meningeal vasculature induced vasoconstriction. In contrast, in the pig renal and rabbit mesenteric arteries, it had a very low maximal efficacy even at 100 microM, with similar results obtained in the rabbit renal artery. The results suggest that almotriptan is a potent and selective 5-HT(1B/1D) receptor agonist, with selectivity for the cranial vasculature as compared with peripheral vessels.

Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes.

1. Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function. 2. Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 microM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50 approximately 100 microM). 3. Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2 approximately 4.5). Glaucine (10 microM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isoprenaline. The glaucine-induced relaxation was resistant to H-89, a protein kinase A inhibitor. Glaucine depressed the contractile responses to Ca2+ (pD'2 approximately 3.62) and reduced the sustained rise of [Ca2+]i produced by histamine in cultured human airway smooth muscle cells (-log IC50 approximately 4.3). 4. Glaucine augmented cyclic AMP levels in human polymorphonuclear leukocytes challenged with N-formyl-Met-Leu-Phe (FMLP) or isoprenaline, and inhibited FMLP-induced superoxide generation, elastase release, leukotriene B4 production, [Ca2+]i signal and platelet aggregation as well as opsonized zymosan-, phorbol myristate acetate-, and A23187-induced superoxide release. The inhibitory effect of glaucine on superoxide generation by FMLP was reduced by H-89. 5. In conclusion, Ca2+ channel antagonism by glaucine appears mainly responsible for the relaxant effect of glaucine in human isolated bronchus while PDE4 inhibition contributes to the inhibitory effects of glaucine in human granulocytes. The very low PDE4/binding site ratio found for glaucine makes this compound attractive for further structure-activity studies.

Design, synthesis, and biological activities of new thieno[3,2-d] pyrimidines as selective type 4 phosphodiesterase inhibitors.

A common pharmacophore for compounds structurally related to nitraquazone has been derived. Using this pharmacophore, new structures have been designed, synthesized, and evaluated for their inhibitory potencies against cyclic adenosine 5'-monophosphate (cAMP) specific phosphodiesterase (PDE 4). From these compounds, 4-benzylamino-2-butylthieno[3,2-d]pyrimidine (4) was selected for optimization. The effects of changes to the lipophilic groups and the amino linkage on the PDE 4 activity have been investigated. As a result, some potent PDE 4 inhibitors, selective with respect to PDE 3, have been identified. A selected group of compounds have been further evaluated for their ability to displace [3H]rolipram from its binding site and also to potentiate isoprenaline-induced cAMP accumulation in isolated guinea pig eosinophils. Of these, 2-butyl-4-cyclohexylaminothieno[3,2-d]pyrimidine (33) has an interesting profile, with an important improvement in PDE 4/[3H]rolipram ratio with respect to reference drugs, and good activity in cAMP potentiation, consistent with efficient cell penetration.

Functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type 4 inhibitor.

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.

Phosphodiesterase inhibitory properties of losartan. Design and synthesis of new lead compounds.

A 4-centre PDE4 pharmacophore search has been carried out in several 3D-databases containing compounds belonging to different therapeutic areas. Losartan, an angiotensin-II antagonist, has been identified as a new lead compound for developing PDE4 inhibitors. New families of compounds derived from losartan has been synthesized and their PDE inhibition has been measured.

Characterization of human serotonin 1D and 1B receptors using [3H]-GR-125743, a novel radiolabelled serotonin 5HT1D/1B receptor antagonist.

The study of serotonin (5-HT) receptors from the points of view of their anatomical localization and pharmacological characterization has been linked to the availability of highly selective radioligands exhibiting high affinity for their targets. This is particularly so in the case of serotonin receptors, since many different subtypes with overlapping pharmacological profiles have been described. Of these, the serotonin 5-HT1 receptor family appears to be the most complex in terms of molecular diversity and pharmacological properties. The lack of appropriate tools to characterize the different receptor subtypes included in this family has hampered progress in the understanding of biological function. In the case of serotonin 5-HT1D receptors all the radioligands used so far in their characterization behave as agonists from the functional point of view. This agonistic character is regarded as a disadvantage for radioligands since their interaction with the receptors under study depends on factors other than the abundance of the receptor, such as the coupling of the receptors with G-proteins. We describe here the binding properties of [3H]-GR-125743, a new radiolabelled derivative of a compound that exhibits selective antagonistic properties with respect to the serotonin human (h5-HT1D) and human (h5-HT1B) receptors. The compound has been characterized for its ability to label the cloned h5-HT1D and h5-HT1B receptors. The binding obtained in both cases was specific, saturable and reversible, whereas the percentage of specific binding depended on the level of expression of the receptors. Using saturation analysis we have found that, on the specific clones used in this study, the compound labels a receptor population 5 to 10-fold higher that the one revealed using [3H]-5-carboxamidotryptamine, a compound with agonist properties for these receptors in functional assays. Using [3H]-GR-125743 as a radioligand we have characterized the pharmacological profile of the same cloned h5-HT1D and h5-HT1B receptor preparations for a range of serotonin reference compounds by means of displacement assays. The affinities found have been compared, using regression analysis, with those obtained for the same radioligand and compounds in membranes obtained from human substantia nigra, a tissue known to be rich in hS-HT(1B/1D) receptors. We have found a better correlation, both in terms of correlation coefficient and of slope, between the substantia nigra data and the h5-HT1B data compared with the h5-HT1D data (0.94 and 1.05 vs. 0.86 and 0.64 respectively). Finally, the addition of 100 microM GTP reduced the binding of [3H]-GR-125743 to h5-HT1D and h5-HT1B receptor subtypes by approximately 20% without affecting the affinities obtained for different displacers. Therefore, [3H]-GR-125743 appears to be a suitable radioligand for the characterization of h5-HT1D and h5-HT1B receptor subtypes, being potentially more useful than previously existing compounds.

Novel heterocyclic-fused pyridazinones as potent and selective phosphodiesterase IV inhibitors.

A series of 6-aryl-4,5-heterocyclic-fused pyridazinones were designed and synthesized as selective phosphodiesterase (PDE) IV inhibitors. Biological evaluation of these compounds demonstrated a good selectivity profile toward the PDE IV family and greatly attenuated affinity for the Rolipram high-affinity binding site that seems to be responsible for undesiderable side effects. Structure-activity relationships (SARs) studies showed that the presence of an ethyl group at pyridazine N-2 is associated with the best potency and selectivity profile.

Characterization of 5-HT receptors on human pulmonary artery and vein: functional and binding studies.

1. This study aimed to investigate the 5-hydroxytryptamine (5-HT) receptors mediating contraction of ring preparations isolated from human pulmonary arteries and veins. In functional studies, the responses to 5-HT, sumatriptan, ergotamine, serotonin-O-carboxymethyl-glycyl-tyrosinamide (SCMGT), alpha-methyl 5-HT (alpha-Me) and 2-methyl 5-HT (2-Me) were studied with WAY100635, GR127935, ritanserin, zacopride and SB204070 as antagonists. 2. All agonists produced concentration-dependent contractions of human pulmonary artery and vein preparations. The order of potency (-log ECS0 values) was ergotamine (6.88) > 5-HT (6.41) > or = SCMGT (6.20) = sumatriptan (6.19) > or = alpha-Me (6.04) in the artery, and ergotamine (7.84) > 5-HT (6.96) > sumatriptan (6.60) = alpha-Me (6.56) > SCMGT (6.09) in the vein. The potency of each agonist, except for SCMGT, was greater in vein than in artery preparations. Contractile responses to 5-HT were similar in intact and endothelium-denuded preparations but responses to sumatriptan were enhanced in artery rings without endothelium. 3. GR127935 (1 nM to 0.5 microM) produced an unsurmountable antagonism of the response to 5-HT, sumatriptan, ergotamine and SCMGT. Ritanserin (1 nM to 1 microM) also reduced the maximum contractile responses to 5-HT, ergotamine and alpha-Me in artery and vein preparations without affecting those to sumatriptan and SCMGT. In endothelium-denuded preparations, surmountable antagonism of sumatriptan by GR127935 (in the presence of ritanserin) and of alpha-Me by ritanserin (in the presence of GR127935) allowed for the calculation of the apparent pK(B) values of GR127935 (9.17+/-0.11 in artery and 9.11+/-0.05 in vein) and ritanserin (8.82+/-0.09 in artery and 8.98+/-0.12 in vein). 4. WAY100635 (1 nM to 1 microM), zacopride (1 nM to 1 microM), or SB204070 (1 nM) did not significantly alter the concentration-response curves for 5-HT, sumatriptan, ergotamine, SCMGT or 2-Me in human pulmonary artery or vein thus indicating that 5-HT1A, 5-HT3 and 5-HT4 receptors are presumably not involved in the contractile response to these agonists. 5. Binding studies using selective radioligands for different 5-HT receptors could not detect the presence of 5-HT1A receptor binding in human pulmonary blood vessels whereas the 5-HT(1B/1D) radioligand [3H]-5CT significantly labelled a population of specific binding sites in both vessel types. The presence of 5-HT2A receptors could also be inferred from the level of binding of [3H]-ketanserin to membranes obtained from human pulmonary vessels, although significance could not be reached for arteries. 5-HT4 specific receptor binding was scarce in veins and absent in the case of arteries. 6. These findings indicate that the human pulmonary artery and vein have a mixed functional population of 5-HT(1B/1D) and 5-HT2A receptors mediating the contractile response to 5-HT which is consistent with results of the binding studies.

Identification and characterization of serotonin 5-HT4 receptor binding sites in human brain: comparison with other mammalian species.

Specific binding for the 5-HT4-selective radioligand [3H]GR 113808 has been identified in human and calf brain membranes. Using human tissue the distribution of the binding was heterogeneous throughout different brain regions, being highest in the caudate nucleus. For this region a Kd value of 0.59 +/- 0.08 nM and a Bmax of 225 +/- 2.6 fmol/mg were obtained. Other regions with substantial densities were the lenticular nucleus, the substantia nigra, the hippocampus and the frontal cortex, whereas no binding could be detected in the cerebellum. The ability of several standard compounds in displacing the radioligand was compatible with the labelling of 5-HT4 receptors. Correlation analysis showed no significant differences amongst data obtained for these compounds using human, calf and guinea-pig membranes.

Pancopride, a potent and long-acting 5-HT3 receptor antagonist, is orally effective against anticancer drug-evoked emesis.

Pancopride ((+-)N-(1-azabicyclo-[2,2,2]-oct-3-yl)-2-cyclopropylmethoxy-4-ami no-5-chlorobenzamide) is a new potent and selective 5-HT3 receptor antagonist, orally and parenterally effective against cytotoxic drug-induced emesis. In vitro, pancopride displayed high affinity (Ki = 0.40 nM) for [3H]GR65630-labelled 5-HT3 recognition sites in membranes from the cortex of rat brains. In vivo, pancopride antagonized 5-HT-induced bradycardia in anaesthetized rats when administered i.v. 5 min (ID50 = 0.56 microgram/kg) or p.o. 60 min (ID50 = 8.7 micrograms/kg) before 5-HT challenge. A single oral dose (10 micrograms/kg) of pancopride produced a significant inhibition of the bradycardic reflex over an 8-h period. Pancopride dose dependently inhibited the number of vomiting episodes and delayed the onset of vomiting induced by cisplatin in dogs (ID50 = 3.6 micrograms/kg i.v. and 7.1 micrograms/kg p.o.). Pancopride was also effective in blocking mechlorethamine- and dacarbazine-induced emesis. Unlike metoclopramide, pancopride was shown to lack any measurable antidopaminergic activity both in vitro and in vivo. These results support clinical data, indicating that pancopride will be a useful drug for treating cytostatic-induced emesis in humans.

A sensitive direct calorimeter for small mammals.

A sensitive direct calorimeter for small animals is presented. Its principle is based on the measurement of the heat transfer from the animal chamber to a heat sink. The system gives repetitive measurements with a high efficiency and allows a detailed time-related measurement of the heat production by the whole animal. Its low response time can be advantageously used for the study of post-prandial heat generation and diet-induced thermogenesis. Data on the heat production by Wistar and lean and obese Zucker rats is also included.