High-affinity Rolipram Binding Research Articles

Suppressive effects of rutin, quercitrin, and isoquercitrin on atypical allergic asthma in an animal model

Quercetin and isoquercitrin were reported to have anti-inflammatory effects on typical asthma. Therefore, we investigated quercetin-3-O-glycosides to clarify the potential for treating atypical asthma. The inhibitions of quercetin-3-O-glycosides on PDE1–5 activities and on high-affinity rolipram binding sites were measured. The sensitized guinea-pig trachealis was challenged by ovalbumin (OVA) to induce contractions. Ten female BABL/c mice in each group were sensitized by OVA on days 0 and 14. On day 21, these mice were injected a mixture of 1% OVA and Freund’s complete adjuvant (1:1). Mice were challenged using 1% OVA in saline for 30 min on days 28, 29, and 30 by ultrasonic nebulization. Twenty-four hours after the last nebulization, the blood and bronchoalveolar lavage fluid (BALF) of these mice were collected. Total immunoglobulin (Ig)E or IgG2a level in the serum and cytokines in the both were determined. The number of inflammatory cells was counted using a hemocytometer. Rutin (30 and100 μmol/kg, p.o.) and quercitrin (100 μmol/kg, p.o.) significantly suppressed the increases in the inflammatory cells and cytokines in the BALF of mice. In contrast to rutin, quercitrin significantly increased the IFN-γ level. Both significantly increased the IgG2a level in the serum and suppressed total and OVA-specific IgE levels in the serum and BALF of mice. Rutin, quercitrin, and isoquercitrin did not affect xylazine/ketamine-induced anesthesia. In conclusion, the above results suggest that quercetin-3-O-glycosides have few adverse effects and that rutin and quercitrin, but not isoquercitrin, may have the potential for treating atypical asthma.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model

Background and PurposecAMP and pharmacological inhibition of PDE4, which degrades it, are promising therapeutic targets for the treatment of spinal cord injury (SCI). Using our previously described in vitro SCI model, we studied the mechanisms by which cAMP modulators promote neurite outgrowth and myelination using enantiomers of the PDE4-specific inhibitor rolipram and other modulators of downstream signalling effectors.Experimental ApproachRat mixed neural cell myelinating cultures were cut with a scalpel and treated with enantiomers of the PDE4-specific inhibitor rolipram, Epac agonists and PKA antagonists. Neurite outgrowth, density and myelination were assessed by immunocytochemistry and cytokine levels analysed by qPCR.Key ResultsInhibition of the high-affinity rolipram-binding state (HARBS), rather than the low-affinity rolipram binding state (LARBS) PDE4 conformer promoted neurite outgrowth and myelination. These effects were mediated through the activation of Epac and not through PKA. Expression of the chemokine CXCL10, known to inhibit myelination, was markedly elevated in astrocytes after Rho inhibition and this was blocked by inhibition of Rho kinase or PDE4.Conclusions and ImplicationsPDE4 inhibitors targeted at the HARBS conformer or Epac agonists may provide promising novel targets for the treatment of SCI. Our study demonstrates the differential mechanisms of action of these compounds, as well as the benefit of a combined pharmacological approach and highlighting potential promising targets for the treatment of SCI. These findings need to be confirmed in vivo.

Disease Progression in MRL/lpr Lupus-Prone Mice Is Reduced by NCS 613, a Specific Cyclic Nucleotide Phosphodiesterase Type 4 (PDE4) Inhibitor

Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFα secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC50 = 1.4 nM) than the other subtypes (PDE4A, IC50 = 44 nM; PDE4B, IC50 = 48 nM; and PDE4D, IC50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (Ki = 148 nM) in comparison to rolipram (Ki = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFα secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.

GSK256066, an Exceptionally High-Affinity and Selective Inhibitor of Phosphodiesterase 4 Suitable for Administration by Inhalation: In Vitro, Kinetic, and In Vivo Characterization

Oral phosphodiesterase (PDE) 4 inhibitors such as roflumilast have established the potential of PDE4 inhibition for the treatment of respiratory diseases. However, PDE4 inhibitor efficacy is limited by mechanism-related side effects such as emesis and nausea. Delivering the inhibitor by the inhaled route may improve therapeutic index, and we describe 6-({3-[(dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy) phenyl]amino}-3-quinolinecarboxamide (GSK256066), an exceptionally high-affinity inhibitor of PDE4 designed for inhaled administration. GSK256066 is a slow and tight binding inhibitor of PDE4B (apparent IC(50) 3.2 pM; steady-state IC(50) <0.5 pM), which is more potent than any previously documented compound, for example, roflumilast (IC(50) 390 pM), tofimilast (IC(50) 1.6 nM), and cilomilast (IC(50) 74 nM). Consistent with this, GSK256066 inhibited tumor necrosis factor α production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes with 0.01 nM IC(50) (compared with IC(50) values of 5, 22, and 389 nM for roflumilast, tofimilast, and cilomilast, respectively) and by LPS-stimulated whole blood with 126 pM IC(50). GSK256066 was highly selective for PDE4 (>380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and >2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (>17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED(50) values of 1.1 μg/kg (aqueous suspension) and 2.9 μg/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED(50) 9.3 μg/kg). Thus, GSK256066 has been demonstrated to have exceptional potency in vitro and in vivo and is being clinically investigated as a treatment for chronic obstructive pulmonary disease.

Genistein, a competitive PDE1–4 inhibitor, may bind on high-affinity rolipram binding sites of brain cell membranes and then induce gastrointestinal adverse effects

The affinities of genistein on phosphodiesterase (PDE)1–4 and cause of gastrointestinal adverse effects of genistein remain unclear. Female BALB/c mice were actively sensitized by intraperitoneal injections of ovalbumin and challenged by aerosolized ovalbumin (1%). After secondary challenge, aerosolized methacholine (6.25–50 mg/ml) induced increases of enhanced pause (P enh) values in conscious mice in a concentration-dependent manner. Genistein (30–100 μmol/kg, i.p.) markedly inhibited methacholine (12.5–50 mg/ml)-induced increase of P enh value in the sensitized and challenged mice. In addition, genistein significantly reduced total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid, with the exception that lymphocytes and neutrophils were not significantly inhibited by genistein at the lowest dose (10 μmol/kg). Genistein also markedly attenuated the release of cytokines, including interleukin (IL)-2, IL-4, IL-5, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Genistein competitively inhibited PDE1–4, with a K i value ranging from 4.3 to 13.7 μM. Genistein (3–300 μM) concentration-dependently displaced 2 nM [ 3H]-rolipram bound on high-affinity rolipram binding sites of brain cell membranes. The therapeutic ratio of genistein was calculated to be 7.9. Genistein (100 μmol/kg, s.c.) significantly shortened xylazine/ketamine-induced anesthesia, suggesting that genistein administered at a higher dose may have gastrointestinal adverse effects. In conclusion, owing to the low therapeutic ratio of genistein, the gastrointestinal adverse effects may be induced via the binding of genistein on high-affinity rolipram binding sites of brain cell membranes, when it is used for a long term or at higher doses for treating allergies, asthma or chronic obstructive pulmonary disease.

Luteolin, a non-selective competitive inhibitor of phosphodiesterases 1–5, displaced [ 3H]-rolipram from high-affinity rolipram binding sites and reversed xylazine/ketamine-induced anesthesia

The aim of the present study was to investigate the mode of action of luteolin on phisphodiesterase (PDE) 1–5, and the possible adverse effects, such as nausea, vomiting, and gastric hypersecretion, determined by replacing [ 3H]-rolipram binding and reversing xylazine/ketamine-induced anesthesia. The reversing effect was reported to occur through a presynaptic α 2-adrenoceptor inhibition and trigger vomiting in ferrets. In contrast, clonidine, an α 2-adrenoceptor agonist, prevented emesis induced by PDE4 inhibitors in ferrets. According to the Lineweaver–Burk analysis, luteolin (3–30 μM) competitively inhibited PDE1–5 activities, with K i values of 15.0, 6.4, 13.9, 11.1, and 9.5 μM, respectively, which did not significantly differ from each other. The equilibrium dissociation constant ( K d) and maximal density ( B max) for [ 3H]-rolipram binding at high-affinity rolipram binding sites of guinea pig brain cell membranes were 10.1 nM and 3.7 pmol/g of tissue, respectively. The EC 50 (PDE4 H) values of luteolin and Ro 20-1724, a selective PDE4 inhibitor, for displacing 2 nM [ 3H]-rolipram binding were 11.2 μM and 45.6 nM, respectively. The therapeutic (PDE4 H/PDE4 L) ratios of luteolin and Ro 20-1724 were calculated to be 0.6, and 0.004, respectively. Both luteolin (10–30 μmol/kg, s.c.) and Ro 20-1724 (0.1–1 μmol/kg, s.c.) significantly reversed the xylazine/ketamine-induced anesthesia in mice. Although luteolin non-selectively and competitively inhibited PDE1–5, only PDE4 inhibition contributed to a reversing effect. In conclusion, because of the low therapeutic (PDE4 H/PDE4 L) ratio of luteolin, the gastrointestinal adverse effects such as nausea, vomiting and gastric hypersecretion should be carefully monitored, whenever luteolin is used for treating allergies, asthma or chronic obstructive pulmonary disease.

Inhibitory effects of quercetin derivatives on phosphodiesterase isozymes and high-affinity [3 H]-rolipram binding in guinea pig tissues

Rolipram has high (PDE4(H)) and low (PDE4(L)) affinities for phosphodiesterase (PDE)-4, respectively. In general, it is believed that inhibitions by PDE4(H) and PDE4(L) are respectively associated with an adverse response and with anti-inflammatory and bronchodilating effects. This has provided a rational basis for designing new compounds with high PDE4(H)/PDE4(L) ratios. In the present study, we attempted to determine the PDE4(H)/PDE4(L) ratios of quercetin (1), qercetin-3-O-methylether (3-MQ, 2), quercetin-3,7,4'-O-trimethylether (ayanin, 3), quercetin-3,7,3',4'-O- tetramethylether (QTME, 4), quercetin-3,5,7,3',4'-O-petamethylether (QPME, 5), quercetin-3,5,7,3',4'-O-pentaacetate (QPA, 6), and quercetin-3-O-methyl-5,7,3',4'-O-tetraacetate (QMTA, 7). The activities of PDE1 approximately 5, which were partially separated from homogenates of guinea pig lungs and hearts, were measured by a two-step procedure using adenosine 3',5'-cyclic monophosphate (cAMP) with [(3) H]-cAMP or guanosine 3',5'-cyclic monophosphate (cGMP) with [(3) H]-cGMP as substrates. The IC(50) values of all of these compounds except quercetin (1), 3-MQ (2), and QMTA (7) on PDE1 approximately 5 inhibition were determined. The anti-inflammatory effects of PDE4 inhibitors were reported to be associated with inhibition of PDE4 catalytic activity. Therefore, these IC(50) values for PDE4 inhibition were taken as the PDE4(L) values. The effective concentration (EC(50)), at which one half of the [(3) H]-rolipram bound to high-affinity rolipram binding sites (HARBSs) of brain cell membranes was replaced, was defined as the PDE4(H) value. In the present results, the PDE4(H)/PDE4(L) ratios of quercetin (1), ayanin (3), and QPME (5) were >30, >19, and 11, respectively (Table 1), which are higher than or equal to that of AWD12-281, the selective PDE4 inhibitor with the greatest potential currently undergoing clinical trials for treating asthma and chronic obstructive pulmonary disease.

Correlation between emetic effect of phosphodiesterase 4 inhibitors and their occupation of the high-affinity rolipram binding site in Suncus murinus brain

We employed an ex vivo [(3)H]rolipram binding experiment to elucidate the mechanism of emetic activity of phosphodiesterase 4 inhibitors. In Suncus murinus (an insectivore used for evaluation of emesis), emetic potential as well as ability to occupy the high-affinity rolipram binding site in brain membrane fraction in vivo were determined for phosphodiesterase 4 inhibitors. In vitro, [(3)H]rolipram bound to the membrane fraction of S. murinus brain with high affinity and its value was comparable to that for rat brain (K(d)=3.6 nM and 3.5 nM, respectively). The test compounds included denbufylline, rolipram, piclamilast, CDP840 and KF19514, each of which possessed similar affinities for the rolipram binding sites in both S. murinus and rat brain. In S. murinus, these compounds induced emesis via intraperitoneal administration. Their ED(50) values were as follows: denbufylline (1.4 mg/kg), rolipram (0.16 mg/kg), piclamilast (1.8 mg/kg), CDP840 (20 mg/kg), and KF19514 (0.030 mg/kg). In addition, these compounds occupied the high-affinity rolipram binding site in vivo as detected by dose-dependent reduction in capacity of ex vivo [(3)H]rolipram binding in brain membrane fractions. A clear correlation was observed between dose required to induce emesis and that to occupy the high-affinity rolipram binding site for individual phosphodiesterase 4 inhibitors. We conclude that the emetic effect of phosphodiesterase 4 inhibitors is caused at least in part via binding to the high-affinity rolipram binding site in brain in vivo.

Tetomilast (OPC-6535)

Purpose: Tetomilast, an anti-inflammatory compound, is being developed for the treatment of inflammatory bowel disease (IBD). Although its mechanism of action has not been fully elucidated, it is known to inhibit phosphodiesterase 4 (PDE4) activity. To investigate the mechanisms involved in tetomilast's anti-inflammatory action, we compared its effects on CD4 T cells, which have been associated with the pathogenesis of IBD, with that of several known PDE4 inhibitors. Methods: The effect of tetomilast on recombinant human PDE4 subtypes was determined by PDE assay. IC50 was calculated from dose-response curves for tetomilast and for the PDE4 inhibitors cilomilast, rolipram, and roflumilast. Human CD4 T cells were purified from the blood of healthy donors using a commercial CD4 T cell isolation kit. CD4 T cells were stimulated using anti-CD3 and anti-CD28 antibodies and incubated in the absence or presence of test compounds for 72 hr at 37°C. For determination of T cell proliferation, [3H]-thymidine (0.2 μCi/well) was added for the last 18 hr of the incubation period. Interferon-γ (INF-γ) released into the culture medium was determined by ELISA. Results: Tetomilast was found to be a potent inhibitor of PDE4 subtypes. Its selectivity for inhibiting the high-affinity rolipram-binding (HARB) conformer of PDE4 was reduced compared with that of rolipram. Tetomilast's PDE4 inhibitory potency was similar to that of rolipram and cilomilast and was at least 100× less than that of roflumilast. At 10 μM, tetomilast caused a significantly greater inhibition of CD3/CD28-stimulated T cell proliferation than did the equivalent PDE4-inhibitory concentrations of 10 μM cilomilast, 10 μM rolipram, and 0.1 μM roflumilast. Tetomilast also demonstrated greater inhibition of INF-γ release from CD3/CD28-stimulated T cells as compared with the other PDE4 inhibitors. Conclusions: Compared with rolipram, tetomilast showed a reduced selectivity for inhibition of the HARB conformer of PDE4, predicting a reduction in side effects, which have been associated with binding of PDE4 inhibitors to the HARB site in the CNS and gastrointestinal tract. Tetomilast's inhibition of CD3/CD28-stimulated CD4 T cell proliferation and INF-γ release was significantly greater than that of the other PDE4 inhibitors tested, suggesting that other mechanisms beyond PDE4 inhibition may account for the pharmacologic activity of tetomilast.

Antidepressant-like effects of PDE4 inhibitors mediated by the high-affinity rolipram binding state (HARBS) of the phosphodiesterase-4 enzyme (PDE4) in rats

Phosphodiesterase-4 (PDE4) has two conformation states based on rolipram binding, the high-affinity rolipram binding state (HARBS) and the low-affinity rolipram binding state (LARBS); their functions remain to be fully explained. Experiments were carried out to determine the roles of the HARBS and LARBS in the mediation of antidepressant-like effects on behavior. Two animal models sensitive to antidepressant drugs, the forced-swim test (FST), and the differential-reinforcement-of-low-rate (DRL) 72-s operant schedule, were used to examine the antidepressant-like effects of rolipram, CDP840, and piclamilast, PDE4 inhibitors that interact differentially with the HARBS and LARBS, and MEM1018 and MEM1091, two novel PDE4 inhibitors. Drug discrimination vs rolipram and rolipram competition binding assays also were carried out. In the FST, rolipram and piclamilast, both at 0.1 mg/kg, produced an antidepressant-like effect, i.e., reduced immobility and increased swimming, whereas, 1 mg/kg of CDP840 or 0.5 mg/kg of MEM1018 or MEM1091 was required to produce a similar effect. Consistent with this, only rolipram and piclamilast produced antidepressant-like effects in rats under the DRL schedule of reinforcement, as evidenced by decreased response rates and increased reinforcement rates. In addition, in rats trained to discriminate rolipram from its vehicle, only rolipram and piclamilast substituted. Finally, [(3)H]rolipram and [(3)H]piclamilast binding analysis revealed that CDP840 and the two novel PDE4 inhibitors MEM1018 and MEM1091 exhibited a lower affinity for the HARBS than did rolipram. These results suggest that the HARBS of PDE4 is the primary conformation important for antidepressant-like effects on behavior.

The Oligomerization State Determines Regulatory Properties and Inhibitor Sensitivity of Type 4 cAMP-specific Phosphodiesterases

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Antidepressant-induced increase in high-affinity rolipram binding sites in rat brain: dependence on noradrenergic and serotonergic function.

The effects of antidepressant treatment on the high- and low-affinity rolipram binding sites on type 4 phosphodiesterase (PDE4) were determined; previous work had shown that repeated antidepressant treatment increases the overall expression of PDE4. Rats were administered different doses of the antidepressant drugs desipramine or fluoxetine, or saline, for 1, 7, or 14 days. [3H]Rolipram and [3H]piclamilast were used to assess the high-affinity rolipram binding sites (HARBS) and low-affinity rolipram binding sites (LARBS) on PDE4 in the hippocampus and cerebral cortex. Repeated, but not acute, treatment with the antidepressants increased [3H]rolipram binding to membrane fractions in a dose-dependent manner; the HARBS component of [3H]piclamilast binding also was increased by these treatments. By contrast, the LARBS component of [3H]piclamilast binding was not altered. [3H]Rolipram and [3H]piclamilast binding to the cytosolic fractions of rat cerebral cortex and hippocampus was not altered by the antidepressant treatments. 6-Hydroxydopamine (6-OHDA; 300 microg i.c.v.) and 5,7-dihydroxytryptamine (5,7-DHT; 200 microg i.c.v.) were used to lesion noradrenergic and serotonergic neurons, respectively. The effects of desipramine, but not fluoxetine, on [3H]rolipram and [3H]piclamilast binding to rat hippocampal membranes were blocked by the 6-OHDA-induced lesion. By contrast, the effects of fluoxetine, but not desipramine, were reduced by the 5,7-DHT-induced lesion. This indicates that the up-regulation of the HARBS by desipramine and fluoxetine requires the integrity of noradrenergic and serotonergic neurons, respectively. Collectively, these results suggest that antidepressants, although acting through different pathways, may eventually lead to the regulation of components of the cAMP signal transduction system.

Cyclic AMP-specific PDE4 Phosphodiesterases as Critical Components of Cyclic AMP Signaling

Cyclic AMP-specific PDE4 Phosphodiesterases as Critical Components of Cyclic AMP Signaling

Airway relaxant and anti-inflammatory properties of a PDE4 inhibitor with low affinity for the high-affinity rolipram binding site.

Inhibitors of phosphodiesterase 4 (PDE4) possess bronchospasmolytic and anti-inflammatory properties, which make them very attractive drugs for the treatment of asthma and COPD. Unfortunately, many PDE4 inhibitors also produce central nervous and gastrointestinal side effects, which have limited their clinical application. PDE4 has two binding sites for the archetypal PDE4 inhibitor rolipram, and it has been suggested that binding to the high-affinity rolipram binding site (HARBS) is responsible for the side effects of PDE4 inhibitors. Recently, we have synthesised the PDE4 inhibitor CC3 which shows low affinity to the HARBS. In the present study we investigated the bronchospasmolytic and anti-inflammatory properties of this novel compound in comparison to rolipram and the PDE3 inhibitor motapizone. The airway-relaxant properties of the PDE inhibitors were analysed in rat precision-cut lung slices (PCLS) in which airways were contracted by methacholine or in passively sensitised PCLS exposed to ovalbumin. The anti-inflammatory properties were investigated by measuring the release of TNF from endotoxin-treated human monocytes.Up to concentrations of 10 microM none of the PDE inhibitors significantly affected bronchoconstriction elicited by 10 microM methacholine. However, if rolipram or CC3 were given in combination with motapizone, methacholine-induced bronchoconstriction was concentration-dependently attenuated. Allergen-induced bronchoconstriction in passively sensitised PCLS was attenuated by CC3 (IC(50) 2.7 microM), rolipram (0.23 microM) and motapizone (8 microM). Combination of equimolar concentrations of motapizone and CC3 (0.34 microM) or rolipram (0.005 microM) showed an additive effect. Endotoxin-induced TNF release from human monocytes was attenuated by all three PDE inhibitors, i.e. CC3 (IC(50) 4.6 microM), rolipram (0.18 microM) and motapizone (5.8 microM). Our findings suggest that PDE4 inhibitors with only low affinity for the HABRS have bronchospasmolytic and anti-inflammatory properties.

Suppression of anti-CD3-induced interleukin-4 and interleukin-5 release from splenocytes of mesocestoides corti-infected BALB/c mice by phosphodiesterase 4 inhibitors

We investigated the suppressive effects of rolipram, RP 73401 (piclamilast), and other structurally diverse inhibitors of adenosine 3′5′-cyclic monophosphate (cAMP)-specific phosphodiesterase (PDE4) on anti-CD3-stimulated interleukin (IL)-4 and IL-5 generation by splenocytes from BALB/c mice infected with Mesocestoides ( M) corti. RP 73401 ( ic 40: 0.011 ± 0.004 μM) was a very potent inhibitor of anti-CD3-induced IL-4 release, being ∼40-fold more potent than (±)-rolipram ( ic 40: 0.43 ± 0.09 μM). A maximal inhibition of 60–70% of the response was achieved at the top concentrations of RP 73401 (1 μM) and rolipram (100 μM). These PDE inhibitors also suppressed IL-5 generation over the same concentration ranges, but the maximal suppression achieved was only 30–40%. R-(−)-rolipram ( ic 40: 0.39 ± 0.09 μM) was ∼6-fold more potent than S-(+)- rolipram ( ic 40: 2.6 ± 0.95 μM) in inhibiting IL-4 release. A close correlation ( r 2 = 0.82) was observed between suppression of IL-4 release by PDE inhibitors and inhibition of CTLL cell PDE4, a form against which R-(−)-rolipram displayed relatively weak inhibitory potency. A poorer correlation ( r 2 = 0.26) was observed between suppression of IL-4 release and affinities of cAMP PDE inhibitors for the high-affinity rolipram binding site in mouse brain membranes. The cGMP-inhibited PDE (PDE3) inhibitor, siguazodan, had little or no effect ( ic 40 > 100 μM) on anti-CD3-stimulated release of either IL-4 or IL-5 and did not significantly enhance the inhibitory action of RP 73401 on the release of either of these cytokines. Finally, RP 73401 ( ic 50: 0.41 ± 0.19 nM) inhibited anti-CD3-stimulated DNA synthesis in splenocyte preparations from M. corti-infected mice and siguazodan (10 μM) had no effect on this response, either alone or in combination with the PDE4 inhibitor. The results show that PDE4 inhibitors suppress the release of Th2 cytokines from anti-CD3-stimulated murine spenocytes and that this effect is correlated with inhibition of a low-affinity PDE4 form.

Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no β-adrenergic receptors (β-AR), was transiently transfected with β-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3′,5′-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A 330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Novel, potent, and selective phosphodiesterase-4 inhibitors as antiasthmatic agents: synthesis and biological activities of a series of 1-pyridylnaphthalene derivatives.

The structural requirements for potent and selective PDE4 inhibition were revealed in a 1-pyridylnaphthalene series, and the best compound (3kg, T-2585.HCl) was chosen for further biological evaluation (PDE4 inhibition IC50 = 0.13 nM, selectivity PDE3/4 ratio = 14 000). Compound 3kg showed potent antispasmogenic activities (ED50 = 0.063 mg/kg for reduction of antigen-induced bronchoconstriction, intravenously; ED50 = 0.033 mg/kg for reduction of histamine-induced bronchoconstriction, intraduodenally) in guinea pigs with little cardiovascular effects. Furthermore, 3kg induced significantly weaker emetic effects than RP73401 after oral administration in ferrets and intravenous administration in dogs (3kg, none of 4 ferrets vomited at a dose of 10 mg/kg, po and none of 8 dogs vomited at a dose of 0.3 mg/kg, iv; RP73401, 4 of 8 ferrets vomited at a dose of 3 mg/kg, po and 6 of 8 dogs vomited at a dose of 0.3 mg/kg, iv); that is compatible with the lower affinity for the high-affinity rolipram binding site (3kg, 2.6 nM; RP73401, 0. 85 nM). This may imply that 3kg has an improved therapeutic ratio because of a broad margin between the Ki value of binding affinity and the IC50 value of PDE4 inhibition (ratio = 0.050).

Aryl sulfonamides as selective PDE4 inhibitors

A series of novel selective phosphodiesterase 4 (PDE4) inhibitors has been developed which displays activity both in vitro and in vivo. These compounds possess good selectivity for the catalytic site of PDE4 over the high affinity Rolipram binding site. In vivo studies demonstrate a reduced propensity to display the emetic side effects which are commonly observed with PDE4 inhibitors.

Evidence that cyclic AMP phosphodiesterase inhibitors suppress interleukin-2 release from murine splenocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer.

1. We have investigated the suppressive effects of rolipram, RP 73401 (piclamilast) and other structurally diverse inhibitors of cyclic AMP-specific phosphodiesterase 4 (PDE4) on interleukin (IL)-2 generation from Balb/c mouse splenocytes exposed to the superantigen, Staphylococcocal enterotoxin-A (Staph. A). The purpose was to determine whether their potencies are more closely correlated with inhibition of PDE4 from CTLL cells, against which rolipram displays weak potency (low-affinity PDE4), or displacement of [3H]-(+/-)-rolipram from its high-affinity binding site (HARBS) in mouse brain cytosol. 2. RP 73401 (IC50 0.46 +/- 0.07 nM, n = 4) was a very potent inhibitor of Staph. A-induced IL-2 release from Balb/c mouse splenocytes, being > 1100 fold more potent than (+/-)-rolipram (IC50 540 +/- 67 nM, n = 3). 3. A close correlation (r = 0.95) was observed between suppression of IL-2 release by PDE inhibitors and inhibition of PDE4. In contrast, little correlation (r = 0.39) was observed between suppression of IL-2 release and their affinities for the high-affinity rolipram binding site (HARBS). 4. RP 73401 only inhibited partially (30-40%) Staph. A-induced incorporation of [3H]-thymidine into splenocyte DNA. The PDE3 inhibitor, siguazodan (10 microM), had little or no effect on IL-2 release or DNA synthesis. This concentration of siguazodan did not enhance the inhibitory action of RP 73401 on IL-2 release but potentiated its effect on DNA synthesis, increasing potency and efficacy. 5. Staph. A-induced DNA synthesis was only partially inhibited by anti-IL-2 neutralizing antibody, whereas dexamethazone (100 nM) and cyclosporine A (100 nM) completely blocked the response. 6. RP 73401 (IC50 6.3 +/- 1.9 nM, n = 4) was 140 fold more potent than rolipram (IC50 900 +/- 300 nM, n = 3) in inhibiting Staph. A-induced [3H]-thymidine incorporation into splenocyte DNA. 7. The results implicate a low-affinity form of PDE4 in the suppression of Staph. A-induced IL-2 release from murine splenocytes by PDE inhibitors. The data also indicate that mitogenic factors other than IL-2, whose elaboration or responses to which are regulated by PDE3 as well as PDE4, contribute to the superantigen-induced DNA synthesis.