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 μM to 100 μM, was devoid of effect on the contractile force in this preparation.
3. Conversely, diazepam (5 μM–100 μM) produced a consistent positive inotropic response in the presence of a concentration (1 μM), 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 γ-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 μM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and β-carboline-3-carboxylic acid methyl ester (βCCMe), or by the inverse agonists, β-carboline-3-carboxylic acid N-methylamide (βCCMa) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, 0.1 μM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 μM), 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.

     

The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue

The inhibitory effect of the flavonoid dioclein was assessed on purified vascular cyclic nucleotide phosphodiesterase isoforms (EC 3.1.4.17, PDE1-5) in comparison with 8-methoxymethyl-isobutylmethylxanthine (8-MM-IBMX) and vinpocetine which are currently used as PDE1 inhibitors. The mechanism underlying the vasorelaxant effect of dioclein was investigated in human saphenous vein. Dioclein inhibited PDE1 more selectively than vinpocetine and 8-MM-IBMX, with IC₅₀ values of 2.47 ± 0.26 and 1.44 ± 0.35 µM, respectively in basal- and calmodulin-activated states. Dioclein behaved as a competitive inhibitor for cGMP hydrolysis by PDE1 in basal- and calmodulin-activated states (K_i = 0.62 ± 0.14 and 0.55 ± 0.07 µM, respectively), indicating this inhibitory effect to be independent of calmodulin interactions. In addition, dioclein induced a concentration-dependent relaxation of human saphenous vein which was independent on the presence of functional endothelium (EC₅₀ values of 7.3 ± 3.1 and 11 ± 2.7 µM, respectively with and without endothelium). 8-MM-IBMX relaxed human saphenous vein with an EC₅₀ = 31 ± 16 µM, whereas vinpocetine did not cause any vasorelaxation at concentrations up to 100 µM. Rp-8-pCPT-cGMPS, which inhibits cGMP-dependent protein kinase (PKG), blocked the vasodilator effect of dioclein, whereas H-89, which is a cAMP-dependent protein kinase (PKA) inhibitor, had a minor inhibitory effect. Our data show that dioclein is a potent calmodulin-independent selective inhibitor of PDE1 and that inhibition of PDE1 is involved in the PKG-mediated vasorelaxant effect of dioclein in human saphenous vein. Furthermore, dioclein may represent a new archetype to develop more specific PDE1 inhibitors.     

磷酸二酯酶3抑制剂的功能和临床应用

磷酸二酯酶3（PDE3）是cAMP的水解酶,参与人体许多生理活动的调节。磷酸二酯酶3抑制剂升高细胞内cAMP水平,具有强心、血管舒张、抗血小板凝聚、抗增殖等作用,在心力衰竭和间歇性跛行等症的治疗中具有临床价值。     

Effects of SK&F 94120, an inhibitor of cyclic nucleotide phosphodiesterase type III, on human platelets

Elevation of cyclic AMP concentrations in platelets inhibits agonist-induced responses. Pharmacological interventions which could increase the levels of platelet cyclic AMP include activation of the synthesis of cyclic AMP or inhibition of its breakdown. In this study we have investigated the effects of SK&F 94120 on human platelet phosphodiesterase (PDE) activities separated by ion-exchange chromatography, and studied the effects of this agent on platelet responses caused by the agonists collagen, U44069 and ADP. Four PDE activities were identified from human platelet preparations. The PDE activities found comprised a cyclic GMP selective PDE, a Ca2+/calmodulin stimulated PDE, a cyclic GMP stimulated PDE and a "low Km" PDE activity called PDE III by analogy with activities described in other tissues. SK&F 94120 was found selectively to inhibit the "low Km" PDE III activity with an IC50 of 10.8 microM, which is consistent with the effects of this compound on cardiac ventricle PDE activities. Exposure of human platelets to SK&F 94120 produced concentration dependent increases in cyclic AMP, showing that inhibition of PDE III activity alone can cause an increase in the level of platelet cyclic AMP. SK&F 94120 also caused an inhibition of platelet responses to collagen, U44069 and ADP. However, SK&F 94120 was much less effective as an inhibitor of aggregation induced by ADP (IC50 greater than 100 microM) than by collagen (IC50 = 24.1 microM) or by U44069 (IC50 = 1.7 microM). Isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, was less effective than SK&F 94120 as an inhibitor of platelet responses for the same measured increase in cyclic AMP levels. M&B 22948 and rolipram, inhibitors of PDE I and PDE IV respectively, had no significant effect on platelet responses. These data suggest that selective inhibition of PDE III is the primary mechanism of action of SK&F 94120 as an inhibitor of agonist-induced platelet responses, and that increased cyclic AMP in the pool controlled by PDE III has important consequences on platelet responses. Moreover, these data suggest that some form of compartmentalization of cyclic AMP and/or PDE activity exists in human platelets.     

KF31327, a new potent and selective inhibitor of cyclic nucleotide phosphodiesterase 5.

The effects of KF31327 (3-ethyl-8-[2-(4-hydroxymethylpiperidino)benzylamino]-2,3-dihydro-1H-imidazo[4,5-g]quinazoline-2-thione dihydrochloride) on phosphodiesterase 5 (cyclic GMP-specific phosphodiesterase) activity and platelet aggregation were investigated and compared with those of sildenafil, a well-known phosphodiesterase 5 inhibitor. KF31327 inhibited phosphodiesterase 5 from canine trachea (K(i)=0.16 nM) more potently than sildenafil (K(i)=7.2 nM). The kinetic analysis revealed that KF31327 was a non-competitive inhibitor. In the presence of nitroglycerin (nitric oxide generator), both compounds inhibited the collagen-induced aggregation of rabbit platelets at less than 0.1 microM, augmenting intracellular cyclic GMP level without affecting cyclic AMP. In contrast, in the absence of nitroglycerin, a higher concentration (10 microM) of KF31327 was required to inhibit platelet aggregation and increased both cyclic nucleotide levels. However, 10 microM sildenafil did not affect aggregation despite elevation of cyclic GMP comparable to that in the presence of nitroglycerin. These results indicate that in the presence of nitroglycerin, the inhibition of platelet aggregation by KF31327 is due to the elevation of cyclic GMP, whereas the mechanism underlying the inhibition without nitroglycerin might be related to a rise in intracellular cyclic AMP.     

Aflatoxin B1 is an inhibitor of cyclic nucleotide phosphodiesterase activity.

Aflatoxin B1 (AFB1) action on cyclic nucleotide phosphodiesterase (PDE) activity has been tested on tissue extracts of various organs. In the presence of 100 microM AFB1 a significant inhibition of cAMP and cGMP hydrolytic activity is observed in all tested tissue extracts. However, cGMP hydrolytic activity appears more sensitive to AFB1 inhibition than cAMP hydrolytic activity and a considerably higher inhibition is observed in lung and spleen, than in liver, brain, kidney, and heart. When cGMP is used as substrate, the inhibitory response reaches 72% in lung and spleen extracts. We have also tested AFB1 effects on lung and liver PDE activity peaks separated by DEAE-cellulose chromatography. These data confirm the poor sensitivity to the toxin of all PDE activities present in liver, while the lung peak (where PDE V in present) shows a higher sensitivity to AFB1. In order to establish whether PDE V is in fact more sensitive to AFB1, we have used mouse neuroblastoma cells, in which cGMP hydrolytic activity has been shown to be due to PDE V only. In this case, the calculated IC50 is 24 microM and Dixon plot analysis shows a competitive inhibitory effect with a Ki of 16.7 microM. We have also used aflatoxin B2 and M2, and they proved to be much less effective than AFB1: AFB2 inhibits PDE V with an IC50 of 117 microM, while AFM2 does not show any effect. These results provide the first evidence of a competitive inhibition of AFB1 on an enzymatic activity and suggest that an alteration of cellular cyclic nucleotide levels may play a role in the mechanism of aflatoxin action.     

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 μM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 μM.
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.

     

Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain injuries

Cilostazol, a selective inhibitor of phosphodiesterase 3, exerts neuroprotective effects on acute brain injury after cerebral ischemia in rats. However, it is unknown whether cilostazol affects the subacute or chronic ischemic injury. In the present study, we evaluated the dose- and time-dependent effects of cilostazol on acute ischemic brain injury and the long-lasting effect on the late (subacute/chronic) injury in mice with focal cerebral ischemia induced by transient middle cerebral artery occlusion. We found that pre-treatment of cilostazol (injected i.p. at 30 min before ischemia) significantly ameliorated the acute injury 24 h after ischemia, and the effective doses were 3-10 mg/kg. The post-treatment of cilostazol (10 mg/kg) was effective on the acute injury when it was injected 1 and 2 h after ischemia. In addition, for the late injury, post-treatment of cilostazol (10 mg/kg, i.p., for 7 consecutive days after ischemia) attenuated neurological dysfunctions, brain atrophy and infarct volume. It also inhibited astrocyte proliferation/glial scar formation and accelerated the angiogenesis in the ischemic boundary zone 7 and 28 days after ischemia. Thus, we conclude that cilostazol protects against not only the acute injury, but also the late injury in mice with focal cerebral ischemia; especially it can modify brain remodeling, astrogliosis and angiogenesis.     

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) in the ferret

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) has been investigated in the ferret. The PDE IV inhibitors studied were: RS14203, R-rolipram and CT-2450 (i.e. (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl ]N'-ethylurea), in addition to the less active enantiomers S-rolipram and CT-3405. Following oral administrations, different emetic profiles were observed with time. Emesis induced by RS14203 exhibited a dose-response relationship but no such relationship was seen for R-rolipram or CT-2450. The incidence of emesis was positively influenced by the dose of PDE IV inhibitors administered, allowing a rank order of potency: RS14203 > R-rolipram > S-rolipram > CT-2450 > CT-3405. PDE IV inhibitor-induced emesis was abolished by the tachykinin NK1 receptor antagonist, CP-99,994. No peripheral release of substance P by PDE IV inhibitors seems to be involved in triggering the emetic reflex since L-743,310, which only has peripheral NK1 receptor antagonist activity, was without effect. The implication of 5-HT3 receptors in PDE IV inhibitor-induced emesis was variable. Our results suggest that the PDE IV inhibitors studied are mixed peripheral-central emetogens. PDE IV inhibition itself could be plausible mechanism of action of these agents. However, whether emesis is mediated via a specific isoform of PDE IV remains to be established.     

LY195115: a potent, selective inhibitor of cyclic nucleotide phosphodiesterase located in the sarcoplasmic reticulum

LY195115 selectively inhibited the peak III isozyme of cardiac cyclic nucleotide phosphodiesterase (PDE) eluted from DEAE-cellulose columns. Inhibition curves were biphasic, suggesting heterogeneity within this preparation. Since peak III PDE is reported to be derived from membranes, effects of LY195115 upon PDE associated with cardiac membranes were examined. LY195115-sensitive PDE measured in the various membrane fractions correlated well with the sarcoplasmic reticulum marker Ca2+-ATPase (r = 0.94; p less than 0.001), but not with Na+,K+-ATPase or azide-sensitive ATPase. Membrane disruption failed to reveal latent LY195115-sensitive PDE in sarcolemmal vesicles known to be primarily right side out. The results suggest that LY195115-sensitive PDE is located within sarcoplasmic reticulum membranes with a distribution similar or identical to that of Ca2+-ATPase. Accordingly, LY195115-sensitive PDE was referred to as SR-PDE. A subfraction of sarcoplasmic reticulum vesicles (free SR vesicles) was sufficiently homogeneous with respect to SR-PDE activity to carry out steady state kinetic studies. Double reciprocal plots of cAMP hydrolysis were linear, yielding Km and Vmax values of 0.46 +/- 0.03 microM and 700 +/- 90 pmol/min/mg of vesicle protein, respectively. LY195115 was a linear competitive inhibitor of SR-PDE with a Ki of 80 +/- 10 nM. -LogIC50 values for inhibition of SR-PDE by a series of structural analogues of LY195115 correlated highly with published -logED50 values for stimulation of cardiac contractility in vivo (r = 0.91, p less than 0.001). Consequently, in vivo effects of LY195115 upon the heart appear to result primarily from competitive inhibition of SR-PDE, or from binding to a site with a topography similar or identical to that of the catalytic site of SR-PDE.     

Control of platelet activation by cyclic AMP turnover and cyclic nucleotide phosphodiesterase type-3

Prostaglandin-induced cAMP elevation restrains key signaling pathways in platelet activation including Ca(2+) mobilization and integrin alphaIIbbeta3 affinity regulation. We investigated how cAMP turnover by cyclic nucleotide phosphodiesterases (PDEs) regulates platelet activation. In washed human platelets, inhibition of all PDEs and also specific inhibition of PDE3 but not of PDE5 suppressed thrombin-induced Ca(2+) responses. The effect of general PDE or PDE3 inhibition was accompanied by an increase in cAMP, and potentiated by Gs stimulation with prostaglandin E(1). In platelet-rich plasma, general or PDE3 inhibition blocked platelet aggregation, integrin activation, secretion and thrombin generation. In contrast, inhibition of PDE5 increased the cGMP level, but without significant influence on aggregation, alphaIIbbeta3 activation, secretion or procoagulant activity. Nitroprusside (nitric oxide) potentiated the effect of PDE5 inhibition in elevating cGMP. Nitroprusside inhibited platelet responses, but this was accompanied by elevation of cAMP. Together, these results indicate that cAMP is persistently formed in platelets, independently of agonist-induced Gs stimulation. PDE3 thus functions to keep cAMP at a low equilibrium level and reduce the cAMP-regulated threshold for platelet activation. This crucial role of PDE3, but not of PDE5, extends to all major processes in thrombus formation: assembly of platelets into aggregates, secretion of autocrine products, and procoagulant activity.     

An update on cyclic nucleotide phosphodiesterase (PDE) inhibitors: phosphodiesterases and drug selectivity

In recent years, there has been a prodigious expansion of knowledge about cyclic nucleotide phosphodiesterases (PDEs). This isozyme superfamily has now become a major focus of drug discovery efforts owing to its diversity, molecular nature, differential regulation and expression in different cell types, and the range of biological functions. Inhibition of these isozymes can lead to an increase in cAMP and cGMP levels, which can affect a variety of physiological responses. Selective inhibitors for each of the multiple forms of PDE can offer an opportunity for desired therapeutic intervention and would be an extremely useful tool in drug discovery efforts for a medicinal chemist. This review details many key aspects of multiple forms of PDEs and their inhibitors with diversified chemical structures, which can act as leads for synthesis of novel drugs.     

Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model

The effects of cilostamide, a cyclic nucleotide phosphodiesterase 3 (PDE3) selective inhibitor, on vascular intimal hyperplasia were evaluated using a single-balloon injury model and a double-injury model in which the rat common carotid artery was subjected to a second injury at a site injured 14 days previously. In the double-injury model, the second balloon injury caused more severe intimal hyperplasia (intima/media (IM) ratio, 1.88+/-0.10) than in the single-injury model (1.09+/-0.08). Histopathological study revealed that vascular smooth muscle cells (VSMC) were the predominant cell-type in the affected neointimal area. Oral administration of cilostamide for 2 weeks after the second injury suppressed intimal hyperplasia in the double-injury model (30 mg kg(-1) bid, 83% inhibition in terms of the IM ratio, P<0.05; 100 mg kg(-1) bid, 69% inhibition, P<0.05). Similar effects were also observed in the single-injury model with oral administration of cilostamide for 2 weeks (100 mg kg(-1) bid, 36% inhibition, P<0.01). Cilostamide inhibited DNA synthesis of cultured VSMC stimulated by foetal calf serum or different kinds of growth factors, but did not affect their migration stimulated by platelet-derived growth factor (PDGF)-BB. Cilostamide significantly increased the cyclic AMP concentration of VSMC dose-dependently. These results indicate that cilostamide suppresses intimal hyperplasia both in the single- and double-injury models of rat, presumably by inhibiting proliferation rather than migration of VSMC. It is suggested that PDE3 inhibitors might find application in preventing intimal hyperplasia following angioplasty such as percutaneous transluminal coronary angioplasty (PTCA) or stent.     

Structure of a novel Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase inhibitor K-259-2

The structure of K-259-2, a potent inhibitor of Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase, was determined to be 3-(2Z-2-ethyl-2-butenyl)-1,6,8-trihydroxy-anthraquinone-2-carboxyl ic acid by chemical conversion and spectral studies.     

Structure of a novel Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase inhibitor KS-619-1

The structure of KS-619-1, a potent inhibitor of Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase, was determined to be 8,13-dioxo-3-(2-oxopropyl)-5,6,8,13-tetrahydro-1,7,9,11- tetrahydroxybenz[a]naphtacene-2-carboxylic acid by spectral studies of KS-619-1 and its methyl derivative.