The human area postrema and other nuclei related to the emetic reflex express cAMP phosphodiesterases 4B and 4D.

Phosphodiesterase 4 (PDE4) inhibitors, i.e. rolipram, are being extensively investigated as therapeutic agents in several diseases. Emesis is one of the most common side effects of PDE4 inhibitors. Given the fact that the area postrema is considered the chemoreceptor trigger zone for vomiting, the present study investigates the regional distribution and cellular localization of the four gene transcripts of the PDE4 subfamily (PDE4A, PDE4B, PDE4C and PDE4D) in human brainstem. In situ hybridization histochemistry was used to locate the mRNA distribution of the four PDE4 subfamilies in the area postrema and related nuclei of human postmortem brainstem. We have found that in the brainstem PDE4B and PDE4D mRNA expression is abundant and distributed not only in neuronal cells, but also in glial cells, and on blood vessels. The hybridization signals for PDE4B and PDE4D mRNAs in the area postrema were stronger than those in any other nuclei in the brainstem. They were also found in vomiting-related nuclei such as the nucleus of the solitary tract and the dorsal vagal motor nucleus. These findings suggest that cAMP signaling modification in the area postrema could mediate the emetic effects of PDE4 inhibitors in human brainstem.

Pharmacological blockade of CCR1 ameliorates murine arthritis and alters cytokine networks in vivo.

BACKGROUND AND PURPOSE: The chemokine receptor CCR1 is a potential target for the treatment of rheumatoid arthritis. To explore the impact of CCR1 blockade in experimental arthritis and the underlying mechanisms, we used J-113863, a non-peptide antagonist of the mouse receptor. EXPERIMENTAL APPROACH: Compound J-113863 was tested in collagen-induced arthritis (CIA) and three models of acute inflammation; Staphylococcus enterotoxin B (SEB)-induced interleukin-2 (IL-2), delayed-type hypersensitivity (DTH) response, and lipopolysaccharide (LPS)-induced tumour necrosis factoralpha (TNFalpha) production. In the LPS model, CCR1 knockout, adrenalectomised, or IL-10-depleted mice were also used. Production of TNFalpha by mouse macrophages and human synovial membrane samples in vitro were also studied. KEY RESULTS: Treatment of arthritic mice with J-113863 improved paw inflammation and joint damage, and dramatically decreased cell infiltration into joints. The compound did not inhibit IL-2 or DTH, but reduced plasma TNFalpha levels in LPS-treated mice. Surprisingly, CCR1 knockout mice produced more TNFalpha than controls in response to LPS, and J-113863 decreased TNFalpha also in CCR1 null mice, indicating that its effect was unrelated to CCR1. Adrenalectomy or neutralisation of IL-10 did not prevent inhibition of TNFalpha production by J-113863. The compound did not inhibit mouse TNFalpha in vitro, but did induce a trend towards increased TNFalpha release in cells from synovial membranes of rheumatoid arthritis patients. CONCLUSIONS AND IMPLICATIONS: CCR1 blockade improves the development of CIA, probably via inhibition of inflammatory cell recruitment. However, results from both CCR1-deficient mice and human synovial membranes suggest that, in some experimental settings, blocking CCR1 could enhance TNF production.

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.

Synthesis and biological evaluation of 3,4-diaryloxazolones: A new class of orally active cyclooxygenase-2 inhibitors.

A series of 3,4-diaryloxazolones were prepared and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2). Extensive structure-activity relationship work was carried out within this series, and a number of potent and selective COX-2 inhibitors were identified. The replacement of the methyl sulfone group on the 4-phenyl ring by a sulfonamide moiety resulted in compounds with superior in vivo antiinflammatory properties. In the sulfonamide series, the introduction of a methyl group at the 5-position of the oxazolone ring gave rise to very COX-2-selective compounds but with decreased in vivo activity. Selected 3,4-diaryloxazolones exhibited excellent activities in experimental models of arthritis and hyperalgesia. The in vivo activity of these compounds was confirmed with the evaluation of their antipyretic effectiveness and their ability to inhibit migration of proinflammatory cells. As expected from their COX-2 selectivity, most of the active compounds lacked gastrointestinal toxicity in vivo in rats after a 4-day treatment of 100 mg/kg/day. Within this novel series, sulfonamides 9-11 have been selected for further preclinical evaluation.

Pharmacological profile of LAS 31180, a new inotropic/vasodilator quinolone derivative.

LAS 31180 (3-methylsulphonylamino-1-methyl-4(1H)-quinolone, CAS 137338-43-3) was found to have positive inotropic and vasodilator properties through its inhibitory action on type 3 phosphodiesterase. The inotropic effects of LAS 31180 were demonstrated in vitro both in isolated guinea-pig ventricular strips (EC50 of 1.2 mumol/l) and in isolated guinea-pig working hearts. In conscious chronically instrumented Beagle dogs, LAS 31180 administered either by intravenous or oral route, showed a dose-dependent, long-lasting positive inotropic effect with minimal effects on heart rate. In hypertensive Beagle dogs LAS 31180 elicited a potent and long-lasting fall in blood pressure.

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.

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.

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.

Anticholinergic effects of desloratadine, the major metabolite of loratadine, in rabbit and guinea-pig iris smooth muscle.

Allergic conjunctivitis is the most common ocular allergic disease. Although very symptomatic it does not endanger vision, and topical antihistamines or chromones are the first choice treatment in clinical practice. Recently, equivalent nanomolar affinities for histamine H and muscarinic M 1 and M3 cloned human receptors have been reported for desloratadine, the active metabolite of loratadine, a widely prescribed antihistamine. This property might enhance its utility in the treatment of asthma, but could induce adverse anticholinergic effects after topical administration. In the present study, we compare the anticholinergic activity of desloratadine with other known muscarinic antagonists and antihistamines on rabbit and guinea-pig iris smooth muscle. Desloratadine was found to be a competitive antagonist (pA2 = 6.67+/-0.09) of carbachol-induced contractions in isolated rabbit iris smooth muscle. Atropine (pA2 = 9.44+/-0.02) and NPC-14695 (pA2 = 9.18+/-0.03) also behaved as competitive antagonists, whereas tiotropium bromide (pD'2 = 9.06+/-0.02) exhibited a non-competitive behaviour in this tissue. Carebastine (pA2 = 5.64+/-0.04) and fexofenadine (pA2 < 4.0) were also studied. After topical administration on the guinea-pig eye conjunctiva, desloratadine produced a potent (ED50 = 2.3 mg/ml) and long lasting mydriasis (> 120 min at the ED50) in conscious animals. Fexofenadine and carebastine were inactive even at the highest concentration tested (10 mg/ml). Atropine (ED50 = 30 microg/ml) and tiotropium bromide (ED50 = 10 microg/ml) were much more potent than desloratadine or pirenzepine (ED50 = 3 mg/ml) in this model. The competitive muscarinic antagonism of desloratadine in vitro, and its potency and duration of action in vivo, suggest that topical treatment of allergic conjunctivitis and rhinitis with desloratadine could produce undesirable peripheral anticholinergic side effects such as mydriasis and xerostomia.

Rolipram inhibits staphylococcal enterotoxin B-mediated induction of the human skin-homing receptor on T lymphocytes.

The cutaneous lymphocyte-associated antigen defines T lymphocytes with cutaneous tropism under inflammatory conditions. Bacterial infections participate in cutaneous inflammations, such as atopic dermatitis or psoriasis. Bacterial superantigens, such as staphylococcal enterotoxin B, can activate peripheral blood mononuclear cells to induce effector T cells bearing the T cell skin homing receptor cutaneous lymphocyte-associated antigen via enhancement of interleukin-12 production. We have identified and characterized the anti-inflammatory effects of different phosphodiesterase inhibitors on this system. Our data indicate that the selective type 4 phosphodiesterase inhibitor rolipram inhibits the Staphylococcal enterotoxin B-mediated generation of cutaneous lymphocyte-associated antigen positive CD3+ cells from peripheral blood mononuclear cells by reducing interleukin-12 production in a concentration-dependent manner. Conversely, type 3 phosphodiesterase or type 5 phosphodiesterase selective inhibitors were not effective. The rolipram inhibitory effect was on interleukin-12 production, as exogenously added interleukin-12 could revert rolipram suppression. These results suggest that selective type 4 phosphodiesterase inhibition may have beneficial effects on T cell mediated skin inflammatory processes characterized by the presence of bacterial infections, that are thought to exacerbate ongoing skin inflammation.

Synthesis and biological evaluation of a conformationally free seco-analogue of the immunosuppressant FR901483.

The synthesis of an azaspirocyclic analogue of FR901483, phosphate 2, is described based on the implementation of a key 5-endo aminocyclization promoted by iodine for direct functionalization of the 1-azaspiro[4.5]decane ring at the C-3 atom. Compound 2 has no inhibitory activity in the cell proliferation assays reported.

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.

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram.

1. The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated. 2. Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity > 99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L-selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay. 3. Our results indicate that rolipram inhibited eotaxin-induced CD11b up-regulation up to 60.6 +/- 7.6% at the highest tested dose (10 microM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 microM. 4. We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen-induced eosinophil infiltration in different animal models of allergic inflammation.

Selective induction of cyclo-oxygenase-2 activity in the permanent human endothelial cell line HUV-EC-C: biochemical and pharmacological characterization.

1. Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX-1 and COX-2 which are encoded by different genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other nonsteroidal anti-inflammatory drugs. COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the development of inflammation has been claimed. 2. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX-2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. 3. The HUV-EC-C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1 beta (IL-1 beta). 4. Both PMA and IL-1 beta produced dose- and time-dependent increases of the synthesis of the COX-derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. 5. The increase of COX activity by PMA or IL-1 beta correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. 6. Treatment of the cells with PMA induced a time-dependent increase in the expression of both COX-1 and COX-2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX-2 isoenzyme at the protein level. 7. The enzymatic activity of the PMA-induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX-2 inhibitors (SC-58125, NS-398 and nimesulide) showed higher potency towards the COX of PMA-treated HUV-EC-C. 8. Overall, it appears that the stimulation of the HUV-EC-C line with PMA selectively induces the COX-2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.

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.