Silymarin: potent inhibitor of cyclic AMP phosphodiesterase

Silymarin, the active principle of the Milk Thistle (Silybum marianum Gaertner), is a very potent inhibitor of cyclic AMP breakdown in vitro by a commercial beef heart phosphodiesterase preparation. Its main constituents, silybin, silydianin and silychristin, are 12.66 to 52.06 times more active than theophylline and 0.77 to 3.17 times more active than papaverine in this respect. Using a novel HPLC technique, the enzyme kinetical analysis can be performed much faster than by the classical methods.     

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.     

Inhibition of cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA receptor antagonism.

Rolipram, a selective inhibitor of type 4 cyclic AMP phosphodiesterase (PDE4), completely reversed the amnesic effects of MK-801 on working and reference memory (F[4,64] = 11.10; p <.0001 and F[4,64] = 2.53; p <.05, respectively) at doses of 0.01-0.1 mg/kg in the radial-arm maze task. Similar antagonism by rolipram of the effects of MK-801 was observed on inhibitory avoidance behavior (F[3,35] = 190.8; p <.0001). In vitro evidence suggests that an increase in cAMP concentrations may mediate the observed behavioral effects of rolipram. In the absence of PDE4 inhibition, NMDA did not increase cAMP concentrations in primary cultures of rat cerebral cortical neurons. However, when PDE4 was inhibited with rolipram, NMDA markedly elevated cAMP. These observations suggest that PDE4 is an integral component of the NMDA receptor-mediated signal transduction pathway involved in memory processes. Inhibitors of PDE4 may act on this pathway to produce their effects on memory and may represent a new class of cognitive enhancers.     

Vascular smooth muscle cell phosphodiesterase (PDE) 3 and PDE4 activities and levels are regulated by cyclic AMP in vivo

Prolonged incubation of several cell types, including cultured vascular smooth muscle cells (VSMC), with cyclic AMP-elevating agents increases cAMP phosphodiesterase (PDE) activity and levels. In this work, we describe for the first time an increase in arterial VSMC cAMP PDE activity and levels caused by cAMP-elevating agents when these agents are administered to rats in vivo. Injections of rats with dibutyryl cAMP (dbcAMP) or forskolin increased both PDE3 and PDE4 activities in aortic and femoral artery VSMC. Consistent with the idea that cAMP-elevating agents increased PDE3 and PDE4 activities by acting directly on VSMC, local delivery of dbcAMP or forskolin to femoral arteries using a pluronic gel-based approach increased femoral artery VSMC PDE3 and PDE4 activities to levels similar to those observed after injection of these agents. Consistent with a role for de novo mRNA and protein synthesis in the cAMP-elevating agent induced increase in PDE3 and PDE4, 1) systemic administration of forskolin increased PDE3A, PDE3B, and PDE4D mRNA levels in aortic VSMC and femoral artery VSMC, 2) local delivery of dbcAMP increased PDE3A, PDE3B, and PDE4D3 protein levels in femoral artery VSMC, and 3) local administration of either actinomycin D or cycloheximide attenuated the effect of dbcAMP. In addition, our results indicate that the PDE3 and PDE4 variants increased by cAMP-elevating agents in arterial VSMC in situ were distinct from those elevated by these agents in cultured arterial VSMC. Consistent with the effect of increased VSMC cAMP PDE on blood vessel function, inhibition of PDE3 and PDE4 activities potentiated the relaxant effect of forskolin in dbcAMP-treated femoral artery rings to a greater extent than in untreated control blood vessels. We propose that our findings are consistent with the concept that cAMP regulates VSMC cAMP PDE activity and levels in vivo and that VSMC phenotype influences the choice of cAMP PDE variant that is elevated. Our findings are discussed in the context that agents aimed at specific PDE3 or PDE4 variants could perhaps allow greater control of cAMP-mediated regulation of VSMC behaviors that are phenotype-dependent.     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

A potent and selective inhibitor of cyclic AMP phosphodiesterase with potential cardiotonic and antithrombotic properties

Some biochemical and pharmacological properties of a novel, potent inhibitor of cyclic AMP phosphodiesterase, N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo[2,1-b] quinazolin-2-one) butyramide (RS-82856), were investigated. RS-82856 selectively inhibits the high affinity form of cyclic AMP phosphodiesterase (type IV) isolated from human platelets (Ki = 0.5 nM) with only weak effects on both the nonspecific and cyclic GMP-sensitive phosphodiesterases. The inhibitor reduces both maximum velocity and substrate affinity of the type IV enzyme. This mixed pattern of partial competitive and noncompetitive inhibition was also obtained with one of the two high affinity forms of phosphodiesterase found in dog heart (Ki = 0.75 nM). Of several human and dog tissues examined, RS-82856 exhibits marked selectively for the platelet high affinity enzyme. It also has significant inhibitory effects on cardiac membrane-bound phosphodiesterase. RS-82856 inhibits the aggregation of human platelets in response to adenosine 5'-diphosphate (IC50 = 0.11 microM) in vitro and is active ex vivo for at least 2 hr following oral administration (10 mg/kg) to rhesus monkeys. Administration of RS-82856 to instrumented, anesthetized dogs by either intravenous or intraduodenal routes increases cardiac contractile force and reduces afterload. These data suggest that RS-82856 may be useful as an agent to increase cardiac output in the treatment of congestive heart failure.     

Molecular structure of the dihydropyridazinone cardiotonic 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-pyridazinyl)- 2H-indol-2-one, a potent inhibitor of cyclic AMP phosphodiesterase

The cardiotonic 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3- pyridazinyl)-2H-indol-2-one (1, LY195115) is a potent, competitive inhibitor (Ki = 80 nM) of sarcoplasmic reticulum derived phosphodiesterase (SR-PDE). Moreover, the compound is a potent positive inotrope both in vitro and in vivo. To assist further cardiotonic drug-design studies, we have mapped the three-dimensional structure of 1 using X-ray crystallography. From a global viewpoint, this drug was essentially planar, but two small regions of nonplanarity were apparent. These involved the geminal methyl substituents in the indol-2-one moiety and the C5' methylene unit of the dihydropyridazinone ring. Because of our previous studies involving the bipyridine cardiotonics amrinone and milrinone, the conformational relationship between the plane of the phenyl ring and the horizontal symmetry plane defined by N2', C3', and C4' of 1 was of particular interest. The C6-C5-C3'-C4' dihedral angle was -2.7 degrees, whereas the C6-C5-C3'-N2' dihedral angle was 174.6 degrees. Therefore the two rings maintain a high degree of coplanarity. Compound 4, the congener of 1 possessing a completely unsaturated pyridazinone ring was also studied. In terms of inotropic activity, this compound, devoid of any puckering in the pyridazinone moiety, was equipotent with 1. Methyl substitution at the 4-position of the dihydropyridazinone and pyridazinone rings provided disparate results. Compound 2, the 4-methyl analogue of 1, was 2-fold more potent than 1, and the methyl substituent probably caused only minor perturbations in overall molecular topology. However 5, the 4-methyl analogue of the pyridazinone 4, was 4.4-fold less active than 4, perhaps as a result of methyl-induced molecular nonplanarity.     

Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodiesterase (Type 5) by sildenafil and related compounds.

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) degrades cGMP and regulates the intracellular level of cGMP in many tissues, including the smooth muscle of the corpus cavernosum of the penis. Sildenafil (Viagra), a specific PDE5 inhibitor, promotes penile erection by blocking the activity of PDE5, which causes cGMP to accumulate in the corpus cavernosum. In the present study, sildenafil, like other PDE5 inhibitors, stimulates cGMP binding to the allosteric sites of PDE5 by interacting at the catalytic site of this enzyme, but the drug does not compete with cGMP for binding at the allosteric sites. Both sildenafil and zaprinast are competitive inhibitors of PDE5, and double-inhibition analysis shows that these two inhibitors added together interact with the catalytic site of PDE5 in a mutually exclusive manner. After site-directed mutagenesis of each of 23 conserved amino acid residues in the catalytic domain of PDE5, the pattern of changes in the IC50 values for sildenafil or UK-122764 is similar to that found for zaprinast. However, among the three inhibitors, sildenafil exhibits the most similar pattern of changes in the IC50 to that found for the affinity of cGMP, implying similar interactions with the catalytic domain. This may explain in part the stronger inhibitory potency of sildenafil for wild-type PDE5 compared with the other inhibitors [sildenafil (Ki = 1 nM) > UK-122764 (Ki = 5 nM) > zaprinast (Ki = 130 nM)]. The affinity of each of these inhibitors for PDE5 is much higher than that of cGMP itself (Km = 2000 nM). It is concluded that residues such as Tyr602, His607, His643, and Asp754 may form important interactions for sildenafil in PDE5, but because these amino acids are conserved in all mammalian PDEs, the selectivity and potency of sildenafil is likely to be provided by a nonconserved residue or residues in the PDE5 catalytic domain.     

8-Substituted analogues of 3-(3-cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine: highly potent and selective PDE4 inhibitors

3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine V11294 (1) has been identified as a lead structure, which selectively inhibits human lung PDE4 (436 nM) and is also active in a number of in vitro and in vivo models of inflammation. Here we describe the synthesis and pharmacology of a series of highly potent 8-[(benzyloxy)methyl]-substituted analogues, with potencies in the range 10-300 nM. In addition, several compounds showed interesting PDE4 subtype specificities, for example, the 3-thienyl derivative 5v, which showed 6-10 nM potency at PDE4B, D3, and D5 and a 20- to 200-fold selectivity over A and D2, respectively.     

Anxiogenic-like behavioral phenotype of mice deficient in phosphodiesterase 4B (PDE4B).

Phosphodiesterase-4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP and plays a critical role in controlling its intracellular concentration, has been implicated in depression- and anxiety-like behaviors. However, the functions of the four PDE4 subfamilies (PDE4A, PDE4B, PDE4C, and PDE4D) remain largely unknown. In animal tests sensitive to anxiolytics, antidepressants, memory enhancers, or analgesics, we examined the behavioral phenotype of mice deficient in PDE4B (PDE4B-/-). Immunoblot analysis revealed loss of PDE4B expression in the cerebral cortex and amygdala of PDE4B-/- mice. The reduction of PDE4B expression was accompanied by decreases in PDE4 activity in the brain regions of PDE4B-/- mice. Compared to PDE4B+/+ littermates, PDE4B-/- mice displayed anxiogenic-like behavior, as evidenced by decreased head-dips and time spent in head-dipping in the holeboard test, reduced transitions and time on the light side in the light-dark transition test, and decreased initial exploration and rears in the open-field test. Consistent with anxiogenic-like behavior, PDE4B-/- mice displayed increased levels of plasma corticosterone. In addition, these mice also showed a modest increase in the proliferation of neuronal cells in the hippocampal dentate gyrus. In the forced-swim test, PDE4B-/- mice exhibited decreased immobility; however, this was not supported by the results from the tail-suspension test. PDE4B-/- mice did not display changes in memory, locomotor activity, or nociceptive responses. Taken together, these results suggest that the PDE4B subfamily is involved in signaling pathways that contribute to anxiogenic-like effects on behavior.     

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.     

Effects of 1-benzylxanthines on cyclic AMP phosphodiesterase 4 isoenzyme

Based on our results regarding the structure-activity relationships of alkylxanthines and imidazo[2,1-i]purines as phosphodiesterase 4 (PDE4) inhibitors, we designed new 1-benzylxanthines and investigated their PDE4 inhibitory activities. 3,7-Dihydro-7-acetonyl-1-(2,4-dichlorobenzyl)-3-propyl-1H-purine-2,4-dione (2h) exhibited both more selective and more potent PDE4 inhibitory activity than rolipram and XT-611.     

1-[4-(4-Chlorophenyl)-2-(2,6-dichlorophenylthio)-n-butyl]-1H-imidazole nitrate, a new potent antifungal agent.

The preparation and antifungal properties of 1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenylthio)-n-butyl]-1H-imidazole nitrate 1 are described. It is particularly effective against in vivo Candida albicans infections (mice), maintaining good activity down to 0.25% formulation strength and showing unusually low reinfection rates after treatment is ended.     

Bemoradan--a novel inhibitor of the rolipram-insensitive cyclic AMP phosphodiesterase from canine heart tissue.

Canine cardiac muscle contains a type IV cyclic AMP (cAMP) phosphodiesterase (PDE) that is composed of two subtypes. One subtype is sensitive to rolipram inhibition (RSPDE), whereas the other is not inhibited significantly by rolipram (RIPDE). The RIPDE is inhibited by several cardiotonic agents operating by a PDE-inhibitory mechanism. Bemoradan [RWJ-22867; 7-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-2H-1,4-benzoxazin -3(4H)-one], a novel, potent positive inotropic agent, demonstrated biphasic inhibition of the fraction III enzyme from canine cardiac muscle. Inhibition by rolipram of the RSPDE converted the IC50 curves of bemoradan, indolidan, pimobendan, and imazodan to sigmoidal, monophasic curves. Lineweaver-Burk analysis yielded competitive inhibition KI values of 0.023, 0.09, 0.065 and 0.60 microM, respectively, for these compounds. The cardiotonic compounds, however, were not potent inhibitors of the Type I and Type II cAMP PDEs found in canine ventricular muscle. The order of potency for inhibiting the RIPDE cAMP PDE subtype was bemoradan greater than pimobendan greater than indolidan greater than imazodan. Bemoradan is, therefore, a potent inhibitor of the cardiac muscle cAMP PDE which could, in part, be responsible for its cardiotonic activity.     

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。