Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum

This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.     

Enzymological and pharmacological profile of T-0156, a potent and selective phosphodiesterase type 5 inhibitor

The enzymological and pharmacological properties of 2-(2-Methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), a new phosphodiesterase type 5 inhibitor, were studied in vitro and in vivo. The inhibitory effects of T-0156 on six phosphodiesterase isozymes isolated from canine tissues were investigated. T-0156 specifically inhibited the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5, at low concentration (IC(50)=0.23 nM), in a competitive manner. T-0156 also inhibited phosphodiesterase type 6 with IC(50) value of 56 nM, which was 240-fold higher than that for inhibition of phosphodiesterase type 5. T-0156 had low potencies against phosphodiesterase types 1, 2, 3, and 4 (IC(50)>10 microM). In the isolated rabbit corpus cavernosum, T-0156 at 10 and 100 nM increased cGMP levels (100 nM T-0156-treated: 6.0+/-1.5 pmol/mg protein, vehicle-treated: 1.1+/-0.4 pmol/mg protein, P<0.05), causing relaxation of the tissue. T-0156 at 1 to 100 nM potentiated the electrical field stimulation-induced relaxation in the isolated rabbit corpus cavernosum in a concentration-dependent manner (100 nM T-0156-treated: 76.9+/-19.8%, vehicle-treated: 12.3+/-10.1%, P<0.05). Intraduodenal administration of T-0156 at 100 to 1000 microg/kg potentiated the pelvic nerve stimulation-induced tumescence in anesthetized dogs (1000 microg/kg T-0156-treated: 279.0+/-38.4%, vehicle-treated: 9.8+/-4.5%, P<0.05). These results suggested that T-0156 enhanced the nitric oxide (NO)/cGMP pathway, probably through blockade of phosphodiesterase type 5 in vitro and in vivo experimental conditions. The present study clearly showed that T-0156 is a potent and highly selective phosphodiesterase type 5 inhibitor, which is a useful tool for pharmacological studies in vitro and in vivo.     

T-1032, a novel specific phosphodiesterase type 5 inhibitor, increases venous compliance in anesthetized rats.

Nitric oxide (NO) donors including organic nitrates dilate capacitance vessels. As inhibition of phosphodiesterase type 5 results in the accumulation of guanosine 3'5'-cyclic monophosphate (cGMP), specific phosphodiesterase type 5 inhibitors are expected to have a vasodilator property similar to that of NO donors. To test this hypothesis, we examined the effect of methyl2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel specific phosphodiesterase type 5 inhibitor, on mean arterial pressure and mean circulatory filling pressure (an index of venodilation) compared with that of nitroglycerin and diltiazem in mecamylamine- and noradrenaline-treated anesthetized rats. Intravenous infusion of T-1032 (0.1, 1, 10 microg/kg/min) dose-dependently decreased mean arterial pressure (-3.8+/-0.3%, -9.1+/-0.8%, -16.8+/-1.5% at doses of 0.1, 1 and 10 microg/kg/min, respectively) and mean circulatory filling pressure (-6.1+/-0.9%, -12.5+/-0.7%, -18.6+/-3.0% at doses of 0.1, 1 and 10 microg/kg/min, respectively). The mean circulatory filling pressure-mean arterial pressure relationship revealed that T-1032 had a selective action on the mean circulatory filling pressure compared with diltiazem (10, 100 microg/kg/min) and a similar or more selective effect than nitroglycerin (0.3, 3 and 30 microg/kg/min). In the next study, we calculated venous compliance and unstressed volume from the mean circulatory filling pressure-volume relationship. Intravenous infusion of T-1032 (3 microg/kg/min) increased venous compliance (3.35+/-0.40 in T-1032 vs. 2.31+/-0.15 ml/kg/mm Hg in vehicle, P<0.05) without changing the unstressed volume (37.2+/-2.80 in T-1032 vs. 42.6+/-2.37 ml/kg in vehicle, P>0.05). It was concluded that T-1032 increased venous capacitance by increasing venous compliance, and that this selective phosphodiesterase type 5 inhibitor appeared to have a different vasodilator action from that of an NO donor and a Ca(2+) channel antagonist in that it had a selective action on the mean circulatory filling pressure.     

T-0156, a novel phosphodiesterase type 5 inhibitor, and sildenafil have different pharmacological effects on penile tumescence and electroretinogram in dogs

T-0156 (2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride) is a newly synthesized phosphodiesterase type 5 inhibitor, and its potency and selectivity are higher than those of sildenafil in an enzyme assay. In the present study with anesthetized dogs, we examined the effects of intravenous T-0156 or sildenafil on the pelvic nerve stimulation-induced penile tumescence and light-adapted flicker stimulation-induced electroretinogram, parameters of which are reported to be indicators for inhibition of phosphodiesterase type 5 and type 6, respectively. Both compounds potentiated the penile tumescence in a dose-dependent manner. T-0156 at 10 microg/kg and sildenafil at 100 microg/kg showed almost the same potentiating percentage (181.5+/-31.1% and 190.0+/-37.9%) in spite of the plasma concentration of T-0156 being about five times lower than that of sildenafil (16.7+/-1.6 and 78.8+/-5.3 ng/ml), indicating that the effect of T-0156 on tumescence is more potent than that of sildenafil. While the high dose of T-0156 (1000 microg/kg) reduced the amplitude and increased the latency of the electroretinogram positive wave, the effects of T-0156 were conversely weaker than those of sildenafil (reduction of amplitude; T-0156: 41.1+/-8.0%, sildenafil: 71.7+/-3.9%, increase of latency; T-0156: 3.9+/-0.6%, sildenafil: 14.5+/-1.4%, at 1000 microg/kg). These results clearly showed the difference in the properties of T-0156 and sildenafil in pharmacological studies with anesthetized dogs, and the difference appeared to correspond with their inhibitory potencies for phosphodiesterase type 5 and type 6. It was concluded that T-0156 would be a useful pharmacological tool as a potent and highly selective phosphodiesterase type 5 inhibitor.     

Characterization and effects of methyl-2- (4-aminophenyl)-1, 2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.     

Acute and chronic effects of T-1032, a novel selective phosphodiesterase type 5 inhibitor,on monocrotaline-induced pulmonary hypertension in rats

We examined the hemodynamic property of T-1032 (methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridylmethoxy)-4-(3,4,5-trimethoxy-phenyl)-3-isoquinoline carboxylate sulfate), a novel selective phosphodiesterase type 5 (PDE5) inhibitor, and evaluated the chronic effect of T-1032 on cardiac remodeling and its related death in monocrotaline (MCT)-induced pulmonary hypertensive rats. T-1032 (1, 10, 100 micro g/kg, i.v.) significantly reduced mean arterial pressure (MAP) and right ventricular systolic pressure (RVSP) without a change in heart rate. The change in RVSP was more potent than that in MAP with 1 micro g/kg T-1032 treatment (RVSP: -8.2+/-1.2%, mean arterial pressure: -5.7+/-1.2%), and reductions in RVSP and MAP reached a peak at doses of 1 and 10 micro g/kg, respectively. In contrast, nitroglycerin (0.1, 1, 10 micro g/kg, i.v.) and beraprost (0.1, 1 micro g/kg, i.v.) did not cause a selective reduction in RVSP at any dose. When T-1032 (300 ppm in diet) was chronically administered, it delayed the death, and significantly suppressed right ventricular remodeling (T-1032-treated: 0.318+/-0.021 g, control: 0.401+/-0.013 g, p<0.05). Our present results suggest that T-1032 selectively reduces RVSP, and resulting in the suppression of right ventricular remodeling with a delay of the death in MCT-induced pulmonary hypertensive rats.     

The effect of sildenafil on corpus cavernosal smooth muscle relaxation and cyclic GMP formation in the diabetic rabbit

Sildenafil, a type V phosphodiesterase inhibitor, enhances smooth muscle relaxation in normal human and rabbit corpus cavernosum. We investigated the in vitro effects of sildenafil on non-adrenergic, non-cholinergic and nitric oxide (NO)-mediated cavernosal smooth muscle relaxation in diabetic rabbits, since alterations in this pathway are recognised in diabetic erectile dysfunction. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan. Cavernosal strips from age-matched control, 3- and 6-month diabetic animals were mounted in organ baths. Relaxation responses to electrical field stimulation (1-20 Hz) or sodium nitroprusside (10(-8)-10(-4) M) were assessed in the absence and presence of sildenafil (10(-8) and 10(-7) M). The effect of sildenafil on cGMP formation by the corpus cavernosum was also assessed following stimulation with sodium nitroprusside, A23187 and acetylcholine. Sodium nitroprusside-stimulated relaxations were significantly (P<0.03) impaired in the corpus cavernosum from both diabetic groups, (IC(50)=4.6 x 10(-6) M following 3 months of diabetes mellitus and 4.0 x 10(-6) M following 6 months of diabetes mellitus; compared to 7.5 x 10(-7) M for pooled age-matched controls). Sildenafil (10(-7) M) significantly enhanced sodium nitroprusside-stimulated relaxation in control (P<0.05) and diabetic groups (P<0.03). Electrical field stimulation-mediated relaxations of the corpus cavernosum were significantly impaired after 6-month diabetes mellitus and enhanced by sildenafil (10(-8) M). cGMP formation by the diabetic corpus cavernosum was impaired significantly, but restored towards normal by sildenafil. We suggest that the impairment of NO-mediated relaxation of the corpus cavernosum reflect, at least in part, a defect in guanylyl cyclase activity. These findings support the use of sildenafil as an effective, orally administered, treatment for diabetic erectile dysfunction.     

Inhibitory effect of sildenafil on rat duodenal contractility in vitro: putative cGMP involvement

1. Sildenafil citrate (Viagratrade mark; Pfizer, Sandwich, Kent, UK), a phosphodiesterase 5 inhibitor, rises cGMP levels in smooth muscle cells. It relaxes both vascular and visceral smooth muscle. In order to assess the intestinal effects of sildenafil, we decided to investigate its actions on rat duodenal motor activity in vitro. 2. In isolated duodenal segments maintained in Tyrode's solution, sildenafil exhibited a concentration-dependent antispasmodic effect on acetylcholine (ACh)-induced phasic contractions, with an IC50 value of 26.7 micromol/L (95% confidence interval (CI) 2.0-55.3 micromol/L). 3. Sildenafil also relaxed the carbamylcholine (CCh)-induced sustained contraction with an IC(50) of 16.2 micromol/L (95% CI 9.5-27.6 micromol/L). Sildenafil produced significant additional relaxation of 25.2 +/- 8.1% of the CCh-induced contraction, beyond basal tone. 4. Sildenafil reduced the amplitude of spontaneous duodenal contractions with an EC50 of 9.6 micromol/L (95% CI 5.7-16.2 micromol/L). This effect was significantly more potent than the effects of zaprinast and papaverine, which also reduced duodenal contractions with EC50 values of 91.6 micromol/L (95% CI 46.0-182.2 micromol/L) and 78.5 micromol/L (95% CI 37.1-166.3 micromol/L), respectively. 5. In preparations treated previously with methylene blue (10 micromol/L) or 1H-[1,2,4]oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10 micromol/L), the EC50 values for the sildenafil effect were significantly increased to 39.0 micromol/L (95% CI 23.9-63.4 micromol/L) and 43.8 micromol/L (95% CI 24.5-78.3 micromol/L), respectively. These values were significantly greater than those obtained with sildenafil alone. 6. In conclusion, sildenafil has myorelaxant and antispasmodic effects on rat duodenal segments in vitro. The contractile inhibitory effect of sildenafil on rat isolated duodenum is probably due to intracellular cGMP accumulation as a result of its decreased degradation.     

T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo

1. Cyclic GMP phosphodiesterase-5 inhibitors have been shown to alter blood flow in specific tissues by potentiating local NO-dependent vasodilatory mechanisms. Since the haemodynamic effects of physiologic insulin, particularly capillary recruitment, may be critical for muscle glucose uptake in vivo and are blocked by inhibitors of nitric oxide synthase, we have explored the acute effects of the specific cGMP phosphodiesterase-5 inhibitor T-1032 on physiologic insulin action in anaesthetized healthy rats in vivo. 2. Whole-body glucose infusion (GIR), femoral blood flow (FBF), hind leg vascular resistance (VR), hind leg glucose uptake (HGU), 2-deoxyglucose uptake into muscles of the lower leg (R'g), hind leg metabolism of infused 1-methylxanthine (1-MX), a measure of capillary recruitment, and muscle cGMP were determined. The experimental groups were T-1032 (10 microg min-1 kg-1) infused for 1 h before and during a euglycaemic insulin clamp (3 mU min-1 kg-1 x 2 h), T-1032 infused for 3 h with saline, T-1032 during a 2 h clamp, T-1032 with saline for 2 h, and a 2 h saline control. 3. Insulin increased GIR from zero to 13 mg min-1 kg-1, HGU from 0.1+/-0.01 to 0.43+/-0.05 micromol min-1, R'g and 1-MX, marginally increased FBF, and had no effect on blood pressure or heart rate. T-1032 alone had no effect on blood pressure, heart rate, FBF, VR, HGU, R'g or 1-MX, but increased muscle cGMP. T-1032 1 h before and during insulin completely blocked GIR (1 h), HGU (2 h), R'g (2 h), and 1-MX (2 h). T-1032 commenced with insulin had only partial blocking activity against insulin. 4. We conclude that T-1032 is a potent acutely acting inhibitor of the muscle effects of physiologic insulin on capillary recruitment and glucose uptake in vivo. These, together with inhibition of whole-body glucose infusion during insulin, may caution against the use of isoenzyme-5-specific cyclic GMP phosphodiesterase inhibitors as therapeutic agents.     

Endothelium-independent vasorelaxant effect of dioclein, a new flavonoid isolated from Dioclea grandiflora, in the rat aorta

We have investigated the endothelium-independent vasorelaxant effect of the new flavonoid dioclein (5,2',5'-trihydroxy-6-7-dimethoxyflavanone) in the rat aorta. In endothelium-denuded vessels, dioclein induced a concentration-dependent relaxation of aortic rings precontracted with noradrenaline (IC50 = 3.5+/-0.89 x 10(-4) M and KCl (IC50 = 5.2+/-1.2 x 10(-4) M). In the absence of extracellular calcium, dioclein reduced the contraction induced by noradrenaline (maximal reduction approximately 33%) but not that induced by caffeine. Dioclein also produced a concentration-dependent inhibition of the sustained contractions induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in normal (IC50 = 4.0+/-0.2 x 10(-4) M) and Ca2+-free solution (IC50 = 4.0+/-0.3 x 10(-4) M). The results indicate that the endothelium-independent vasorelaxant effect of dioclein may be explained by inhibition of contractions dependent on activation of protein kinase C, voltage-dependent Ca2+ influx and on the release of intracellular Ca2+ stores sensitive to noradrenaline.     

Effect of sildenafil on cyclic nucleotide phosphodiesterase activity,vascular tone and calcium signaling in rat pulmonary artery

(1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.     

Sildenafil does not improve nitric oxide-mediated endothelium-dependent vascular responses in smokers

AIMS: To examine the hypothesis that sildenafil, a phosphodiesterase type 5 inhibitor that inhibits cGMP breakdown, could enhance nitric oxide-mediated vasodilation and reverse endothelial dysfunction in chronic smokers. METHODS: Flow-mediated dilation of the brachial artery and forearm postischemic reactive hyperemia (both nitric oxide-mediated responses) were measured before and after sildenafil 50 mg and placebo in a double-blind, randomized, crossover study in 9 men who were chronic smokers (21 +/- 3 pack years). RESULTS: There was no significant change in flow-mediated dilation after either sildenafil (0.18%, 95%CI -1.7-2%) or placebo (0.24%, 95%CI -2.8-3.3%) (P = 0.88 and 0.8, respectively). Sildenafil had no significant effect on resting forearm blood flow or postischemic reactive hyperemia (P = 0.39 and 0.7, respectively). Resting heart rate and blood pressure were unaffected by sildenafil. CONCLUSIONS: Acute sildenafil administration did not improve endothelial function in chronic smoking men.     

Mechanisms of direct relaxant effect of sildenafil, tadalafil and vardenafil on corpus cavernosum

Sildenafil, tadalafil, vardenafil and verapamil induced concentration-dependent relaxation of the rabbit corpus cavernosum muscle precontracted with noradrenaline. The maximal relaxation (%) at 20 microM was 61.4 +/- 6.9, 32.4 +/- 5.4, 100.0 +/- 5.5 and 86.6 +/- 5.1 (n = 5 each) respectively. Pre-incubation of cavernosal muscle strips with N(omega)-nitro-L-arginine or guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but not adenylate cyclase inhibitor, cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] (MDL12330A) culminated in only a 20-30% reduction in muscle relaxant action of the 3 phosphodiesterase inhibitors. This suggests that another mechanism of relaxation independent of nitric oxide-cGMP or cAMP pathway was involved. Higher concentrations of sildenafil (100 microM) and vardenafil (10 and 100 microM) produced non-competitive antagonism of noradrenaline-induced contraction characterized by reduced maximal effect. In contrast, tadalafil was devoid of significant effect on noradrenaline. On K(+)-depolarized tissues, sildenafil was as potent as vardenafil whereas tadalafil was the least effective in relaxing K(+)-induced tone. The maximal relaxation (% of K(+)-induced tone) at 20 microM sildenafil, tadalafil and vardenafil was respectively 84.1 +/- 6.5, 9.0 +/- 19.9, and 88.9 +/- 6.2 (n = 5 each). In addition, verapamil, sildenafil and vardenafil were more efficacious than tadalafil in reversing tonic contractions by Ca(2+) channel activator, 1,4,dihydro-2,6-dimethyl-5-nitro-4-[2(triflouromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (BAY K-8644). These results indicate that vardenafil and sildenafil possess direct muscle relaxant potential possibly via inhibiting Ca(2+) influx through both receptor-operated and voltage-dependent Ca(2+) channels whereas tadalafil appears capable of inhibiting receptor-operated transmembrane Ca(2+) entry only.     

Inhibitors of phosphodiesterase 5 (PDE 5) inhibit the nerve-induced release of nitric oxide from the rabbit corpus cavernosum

BACKGROUND AND PURPOSE: Nitrergic neurons are important for erectile responses in the corpus cavernosum and impaired signalling results in erectile dysfunction, today treated successfully by oral administration of the selective phosphodiesterase 5 (PDE 5) inhibitors sildenafil, tadalafil and vardenafil. Although the importance of nitrergic neurons in urogenital function has become evident, it has not been investigated if the PDE 5 inhibitors affect the nerve-induced release of nitric oxide (NO). In a previous study we found that the soluble guanylate cyclase (sGC)/cyclic guanosine 3',5'-monophosphate (cGMP) pathway might modulate nerve-induced release of NO in isolated cavernous tissue. EXPERIMENTAL APPROACH: Electrical field stimulation (EFS 5 Hz, 40 V, 0.3 ms pulse duration, 25 pulses at intervals of 2 min) of rabbit isolated cavernous tissue elicited reproducible, nerve-mediated relaxations in the presence of scopolamine (10(-5) M), guanethidine (10(-5) M) and phenylephrine (3 x 10(-6) M). In superfusion experiments, nerve stimulation (20 Hz, 40 V, 1 ms) of the cavernous tissue evoked release of NO/NO2-, measured by chemiluminescence. KEY RESULTS: Sildenafil, tadalafil and vardenafil decreased the muscular tone and prolonged the relaxations to nerve stimulation. The evoked release of NO decreased to 72+/-11%, 55+/-16% and 61+/-14% of control, respectively after addition of sildenafil, tadalafil or vardenafil (all 10(-4) M, n=6-8, p<0.05). CONCLUSIONS AND IMPLICATIONS: Selective PDE 5 inhibitors influence the nerve-induced release of NO, probably via cGMP-mediated negative feedback. This negative feedback might explain why priapism is not seen during monotherapy with the PDE inhibitors.     

枸橼酸西地非尔对兔海绵体组织作用及其机制的离体研究

目的　研究枸橼酸西地非尔对兔海绵体组织舒张功能的影响及其作用机制。方法　 37℃温孵兔海绵体组织(RCC) ,用放射免疫方法监测在硝普钠存在下 ,西地非尔10、10 0、10 0 0nmol·L-1对cGMP水平的影响 ;采用离体器官水浴技术 ,将RCC用苯丙肾上腺素 (PE)收缩 ,观察西地非尔对NO供体硝普钠 (SNP)及促内皮依赖性NO释放剂氯化乙酰甲基胆碱介导RCC舒张作用的影响。结果　SNP可提高RCC中cGMP水平 ,西地非尔 10、10 0、10 0 0nmol·L-1可剂量依赖性使之升至更高水平。西地非尔使SNP、氯化乙酰甲基胆碱 (MCH)作用IC50 显著减小。结论　以上结果表明枸橼酸西地非尔通过NO/cGMP呈剂量依赖性增强MCH和SNP对RCC的舒张作用 ,对动物阴茎海绵体组织舒张功能有显著性改善作用 ,从而大大提高动物性功能。     

3H]sildenafil binding to phosphodiesterase-5 is specific,kinetically heterogeneous,and stimulated by cGMP

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP. PDE5 was the only [3H]sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [3H]sildenafil exchange dissociation yielded two components with t1/2 values of 1 and 14 min and corresponding calculated KD values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [3H]Sildenafil binding isotherm of PDE5 indicated KD was 8.3 to 13.3 nM, and low cGMP decreased the KD to 4.8 nM but only slightly increased Bmax to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.     

Pharmacological profiles of sildenafil (VIAGRA) in the treatment of erectile dysfunction: efficacy and drug interaction with nitrate

Penile erection follows relaxation of the corpus cavernosum in which nitric oxide (NO) released during sexual stimulation from non-adrenergic non-cholinergic nerve endings and from endothelial cells of the corpus cavernosum plays a crucial role. Sildenafil (VIAGRA) selectively inhibited phosphodiesterase type 5 (PDE5) activity in the human corpus cavernosum and increased cGMP concentrations in the rabbit cavernosum in the presence of NO. Sildenafil enhanced the NO-dependent relaxation of the isolated human corpus cavernosum and the intracavernosal pressure in the anesthetized dog without affecting systemic blood pressure and heart rate. In the patients with erectile dysfunction, an orally administered sildenafil enhanced the penile rigidity during visual sexual stimulation. Sildenafil did not affect the phenylephrine-induced contraction of the isolated rabbit aorta, but enhanced the relaxant effect of glyceryl trinitrate. The pharmacodynamic interaction with glyceryl trinitrate was also observed in human studies where sildenafil potentiated the hypotensive effect of the nitrate. These results indicate that sildenafil, which enhances the physiological process of penile erection during sexual arousal, is a novel orally effective treatment for erectile dysfunction. It should be noted, however, that sildenafil enhanced the hypotensive effect of glyceryl trinitrate, as a result of inhibition of PDE5 in vascular smooth muscle. Therefore, administration of sildenafil to patients who are using nitrates and NO donors is contraindicated.     

The pharmacology of sildenafil, a novel and selective inhibitor of phosphodiesterase (PDE) type 5

Sildenafil (1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d]pyrimidin-5-yl) phenylsulphonyl]-4-methylpiperazine) has been shown to be an effective oral treatment for male erectile dysfunction. Sildenafil is a potent competitive inhibitor of PDE5 (IC50 3.5 nM) and is selective over PDE1 to 4 (80 to 19,000-fold) and retinal PDE6 (10-fold). Sildenafil enhanced cGMP accumulation driven with sodium nitroprusside in the corpus cavernosum of rabbits without affecting cAMP formulation. In the absence of nitric oxide drive, sildenafil had no functional effect on the human and rabbit isolated corpus cavernosum, but potently potentiated the relaxant effects of nitric oxide on these tissues. In the anaesthetised dog, sildenafil (ED50: 12 to 16 micrograms/kg i.v.) enhanced the increase in intracavernosal pressure induced by electrical stimulation of the pelvic nerve or intracavernosal injection of sodium nitroprusside in the absence of meaningful effects on blood pressure. Consistent with its mode of action, sildenafil potentiated the vasorelaxant effects of glyceryl trinitrate on rabbit isolated aortic rings. However, unlike milrinone, sildenafil had no inotropic effects on the dog isolated trabeculae carneae. Thus it is unlikely to have the deleterious effects on cardiac function associated with PDE3 inhibitors. As a consequence of inhibition of PDE6 in the retina, sildenafil (1 to 100 microM) altered the kinetics of the light response of the dog isolated retina. In the anaesthetised dog, sildenafil modified the a- and b-wave of the electroretinogram induced by a flash of blue light. These effects were proportional to plasma concentrations, were fully reversible and only occurred following plasma concentrations higher (approximately 30-fold) than those active on intracavernosal pressure. These studies have shown that sildenafil is a potent and selective inhibitor of PDE5. It enhances the effect of nitric oxide on the corpus cavernosum and has been shown to be an effective oral treatment of erectile dysfunction.     

Phosphodiesterase 5 and effects of sildenafil on cerebral arteries of man and guinea pig

Sildenafil (Viagra), a selective inhibitor of phosphodiesterase 5 (PDE5), induces headache and migraine. Although previously supposed to be a "vascular" headache, no significant cerebral artery dilatation was found in vivo. Thus, we hypothesised that PDE5 may not be present or that sildenafil is less effective on the cGMP hydrolysis in cerebral arteries, and that sildenafil may not be an effective dilator of cerebral arteries under baseline conditions. We evaluated the presence of PDE5 mRNA and protein in human arteries. Furthermore, the effects of two selective PDE5 inhibitors, sildenafil and UK-114,542, and a PDE1 inhibitor UK-90,234 on cGMP hydrolysis were investigated in human and guinea pig cerebral arteries. The vasoactive responses of the compounds were evaluated in guinea pig basilar arteries in vitro, with concomitant measurements of cAMP and cGMP. PDE5 was found in human middle cerebral arteries. Sildenafil and UK-114,542 inhibited cGMP hydrolysis concentration-dependently in both species. In guinea pig arteries, sildenafil induced an endothelium-dependent vasodilatation only at concentrations above 10 nM, which was augmented by sodium nitroprusside and attenuated by reduction of cGMP, but was cGMP independent at high concentrations. UK-114,542 was more and UK-90,234 was less potent than sildenafil. In conclusion, PDE5 is present in human and guinea pig cerebral arteries, and is inhibited by sildenafil at micromolar levels. Sildenafil in vitro is a poor dilator of guinea pig cerebral arteries unless a nitric oxide donor is co-administered, corresponding to the previous findings in vivo.     

Phosphodiesterase type 5 inhibition enhances vasorelaxation caused by nitroprusside in guinea pig intact heart and isolated aorta

Increased vascular smooth muscle cyclic guanine monophosphate (cGMP) results in vascular relaxation. The vascular effects of stimulating cGMP production with 10(-8)-10(-4) M nitroprusside (NP) and inhibiting cGMP hydrolysis with 10(-8)-10(-4) M zaprinast (ZAP), a selective type V inhibitor of cGMP phosphodiesterase (PDE), were assessed in isolated guinea pig hearts and aortic rings. Coronary flow (CF) IC50 values for NP and ZAP, respectively, were 0.8+/-0.1 x 10(-6) M and 3.6+/-0.1 x 10(-6) M; for coronary sinus pO2 IC50 values were 0.7+/-0.1 x 10(-6) M and 3.7+/-0.1 x 10(-6) M. CF increased by 13+/-2% with 10(-6) NP, and by 12+/-2% with 10(-5) M ZAP; percentage O2 extraction (%O2E) decreased by 17+/-3% with NP and 28+/-4% with ZAP. Together, 10(-6) M NP + 10(-5) M ZAP augmented the increased in CF to 23+/-3% of control, and the decrease in percentage O2 extraction (%O2E) to 40+/-4% of control. Other cardiac effects of NP and ZAP were minimal. In norepinephrine preconstricted aortic rings, the IC50 for relaxation was elicited at 0.4+/-0.1 x 10(-6) M NP and 6.1+/-0.1 x 10(-6) M ZAP. NP given with ZAP gave a logarithmic relation so that IC50 [NP] = -(57 log10 [ZAP]) + 416; R2 = 0.95. NP, 3 x 10(-7) M; ZAP, 3 x 10(-6) M; and NP + ZAP combined increased aortic tissue cGMP by eight-, nine-, and 15-fold, respectively. Inhibiting cGMP hydrolysis may be an effective approach to augment vasorelaxation elicited by cGMP synthesis in the heart.