Selective Inhibitor Of Phosphodiesterase Type Research Articles

Histopathologic results of long-term sildenafil administration on rat inner ear

ObjectivesSildenafil, a selective inhibitor of phosphodiesterase type 5, is widely used for the treatment of erectile dysfunction. Although cochlear effects of phosphodiesterase type 5 inhibitors remain still unclear because of inadequate data, some evidence that recently emerged indicates that these medications may be responsible for hearing impairment. In the present study, we aimed to examine the histopathologic effects of long-term sildenafil use on the cochlea in a rat model. MethodsThe study was performed with adult male Wistar albino rats. The control group was fed on standard laboratory diet. The study group was applied orally with sildenafil therapy, 1.5 mg/kg once a day for 45 days. Rats were anesthetized and decapitated. Each temporal bone was dissected, and the cochleas were removed en bloc. The inner-ear biopsy specimens were examined histologically with hematoxylin and eosin and caspase 3 immunoreaction under light microscopy. ResultsHematoxylin and eosin staining showed no distinctive difference between the control group and the sildenafil group. With immunohistochemical examination, caspase 3 immunoreactivity was observed in the sildenafil group. In the control group, caspase 3 immunoreactivity was not observed. ConclusionsThe caspase 3 immunoreactivity in the sildenafil group was strongly associated with an increase in apoptotic events in the cochlea. Long-term use of sildenafil can cause hearing impairment through increased apoptosis.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Phosphodiesterase type 5 (PDE5) inhibition improves object recognition memory: Indications for central and peripheral mechanisms

A promising target for memory improvement is phosphodiesterase type 5 (PDE5), which selectively hydrolyzes cyclic guanosine monophosphate (cGMP). In rodents, PDE5 inhibitors (PDE5-Is) have been shown to improve memory performance in many behavioral paradigms. However, it is questioned whether the positive effects in animal studies result from PDE5 inhibition in the central nervous system or the periphery. Therefore, we studied the effects of PDE5 inhibition on memory and determined whether compound penetration of the blood-brain barrier (BBB) is required for this activity. Two selective PDE5-Is, vardenafil and UK-343,664, were tested in the object recognition task (ORT) in both a MK-801- and scopolamine-induced memory deficit model, and a time-delay model without pharmacological intervention. Compounds were dosed 30min before the learning trial of the task. To determine if the PDE5-Is crossed the BBB, their concentrations were determined in plasma and brain tissue collected 30min after oral administration. Vardenafil improved object recognition memory in all three variants of the ORT. UK-343,664 was ineffective at either preventing MK-801-induced memory disruption or time-dependent memory decay. However, UK-343,664 attenuated the memory impairment of scopolamine. Vardenafil crossed the BBB whereas UK-343,664 did not. Further, co-administration of UK-343,664 and scopolamine did not alter the brain partitioning of either molecule. This suggests that the positive effect of UK-343,664 on scopolamine-induced memory decay might arise from peripheral PDE5 inhibition. The results herein suggest that there may be multiple mechanisms that mediate the efficacy of PDE5 inhibition to improve memory performance in tasks such as the ORT and that these involve PDE5 located both within and outside of the brain. To further elucidate the underlying mechanisms, the cellular and subcellular localization of PDE5 needs to be determined.

Simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetrahydro-1h-pyrido[3,4-b]indole-3-carboxylic acid methyl ester a key intermediate of tadalafil by chiral high-perfo

(1R, 3R)-1-(1, 3-Benzodioxol-5-yl)-2-(chloroacetyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester ((1R, 3R)-Cpe) is a key intermediate used in the synthesis of tadalafil, a highly selective phosphodiesterase type-5 inhibitor. In the present study, a chiral high-performance liquid chromatography method was developed for the simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe. Separation was performed on an Ultron ES-OVM chiral column (150 mm × 4.6 mm, 5 μm,) with a guard column at a column temperature of 30°C. The gradient elution used was acetonitrile (solvent A) and water (solvent B), and the following elution program was used at a flow rate of 1 ml/min: 0-5 min (80% B), 5-10 min (80-60% B), 10-12 min (60% B). The detection wavelength was 220 nm. The four isomers of Cpe were baseline separated in 12 min. The results of method validation indicated that the method was specific and sensitive and was suitable for the quality control of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe.

Is Inhibition of Phosphodiesterase Type 5 by Sildenafil a Promising Therapy for Volume-Overload Heart Failure?

Phosphodiesterase type 5 (PDE5) is an enzyme that belongs to a large family of cyclic nucleotide PDEs that catalyze cAMP and cGMP. cAMP and cGMP are 2 essential intracellular second messengers regulating many different cellular functions of living cells. PDE5 specifically breaks down the substrate cGMP. Inhibition of PDE5 increases intracellular cGMP levels by inhibiting its degradation. Sildenafil (Viagra) is a potent and selective inhibitor of cGMP-specific PDE5 and is the first oral treatment for men with erectile dysfunction; it was approved by Food and Drug Administration on March 27, 1998. Sildenafil binds to the catalytic site of PDE5 and inhibits the degradation of intracellular cGMP in smooth muscle within the corpus cavernosum, resulting in increased levels of cGMP, which cause smooth muscle cell relaxation and vasodilation and improve erectile function.1,2 Article see p 1390 In recent years, there has been growing interest in therapeutic applications of PDE5 inhibitors for cardiovascular diseases other than erectile dysfunction. Some experimental studies demonstrated direct cardioprotective effects of inhibition of PDE5 in experimental acute myocardial infarction models. In 2002, Ockaili et al3 treated rabbits with sildenafil (0.7 mg/kg given as an intravenous bolus) either 30 minutes or 24 hours before coronary occlusion. The rabbits were then subjected to 30 minutes of left anterior descending coronary artery occlusion followed by 3 hours of reperfusion, and the infarct size was measured by tetrazolium staining. The results demonstrated for the first time that sildenafil had a powerful preconditioning-like effect against myocardial ischemia/reperfusion injury. Our research group4 injected 1.45 mg/kg intravenous sildenafil or saline 30 minutes before ischemia in anesthetized open-chest rabbits subjected to 30 minutes of left anterior descending coronary occlusion followed by 3 hours of reperfusion. Compared with saline, sildenafil significantly decreased coronary resistance in the ischemic risk area, slightly increased …

Cilostazol Improves Outcome after Subarachnoid Hemorrhage: A Preliminary Report

Background: Cerebral vasospasm (VS) is the most common cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). Reversal of VS by intra-arterial infusion of cyclic adenosine monophosphate (cAMP)-elevating agents has been reported; however, the preventive role in the development of VS is not fully understood. This study is designed to evaluate the possible efficacy of using cilostazol, a selective inhibitor of phosphodiesterase type 3 and a cAMP-elevating agent, in patients with SAH. Methods: In this prospective randomized study, we enrolled 100 SAH patients who met the following criteria: neck clipping within 72 h after onset, Hunt and Hess (HH) score ≤4, modified Rankin scale (mRS) score ≤2 prior to ictus, and no serious cardiovascular complications. Patients were divided into control and cilostazol groups; we focused on the effects of cilostazol on the decrease in the incidence of symptomatic VS, cerebral infarction, and the mRS score at discharge. Result: Patients’ age, male/female ratio, mRS score prior to ictus, HH grade, Fisher group, site of the aneurysm, drugs prescribed during the observation period, and length of hospital stay were not different between the groups. Cilostazol did not significantly decrease the incidence of symptomatic VS (37.3% in the control vs. 22.4% in the cilostazol group, p = 0.183) and cerebral infarction (27.5% in control vs. 10.2% in the cilostazol, p = 0.091). However, mRS score was significantly improved at discharge (2.6 in controls vs. 1.5 in the cilostazol group, p = 0.041). Patients’ age being ≤65 years (OR = 8.47, 95% CI = 2.45–29.32, p = 0.0007), Fisher group ≤3 (OR = 4.64, 95% CI = 1.00–21.45, p = 0.049), HH grade ≤2 (OR = 4.31, 95% CI = 1.27–14.59, p = 0.019), no hydrocephalus (OR = 8.55, 95% CI = 1.72–19.23, p = 0.0046), and cilostazol use (OR = 5.52, 95% CI = 1.61–18.90, p = 0.0065) were independent predictors of good outcomes (mRS score ≤2). Conclusion: Cilostazol may improve outcomes after SAH, but further double-blind, placebo-controlled studies are required for a definitive conclusion.

Tadalafil for the treatment of pulmonary arterial hypertension

Tadalafil is a selective inhibitor of phosphodiesterase type-5 (PDE-5) that was originally developed for the treatment of male erectile dysfunction and recently approved for the treatment of pulmonary arterial hypertension (PAH). The antipulmonary hypertensive effects of nitric oxide and the natriuretic peptides are mediated via increasing intracellular cGMP and enzymatic degradation by PDE-5 is the major route of cGMP inactivation in the lung. Evidence is accruing that PDE-5 activity is increased in pulmonary vascular diseases and may contribute to the pathogenesis of PAH. The longer half-life of tadalafil allows for once-daily dosing as compared with three-times daily dosing for sildenafil, the only other PDE-5 inhibitor currently approved for treatment of PAH. This article reviews the role of cGMP and PDE-5 in PAH, presents the results of recent clinical trials and discusses the role of tadalafil in the treatment of this rare but difficult-to-treat disease.

Chronic Antigen Challenge Converts Bronchoconstriction by Inhaled Thromboxane‐mimetic, U46619, to a Bronchodilation in Conscious Sensitized Guinea‐pigs*

The study was undertaken to examine whether chronic repeated challenge of sensitized guinea-pigs with antigen would induce hyper-reactivity to the spasmogen U46619 (9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α). Airway function was measured in conscious guinea-pigs by whole-body plethysmography; leucocyte infiltration into the lungs was monitored using bronchoalveolar lavage. Male guinea-pigs were sensitized to ovalbumin by injection (i.p.) of a suspension (1 mL) containing ovalbumin (10 μg) and aluminium hydroxide (100 mg); they were used 14 days later. In sensitized guinea-pigs repeated inhaled antigen challenges (0.5% ovalbumin in saline for 10 min twice weekly for 4 weeks) induced a state of pulmonary inflammation characterized by an enhanced and prolonged late-phase response and a persistent eosinophilia. Inhalation of a threshold dose of the thromboxane-mimetic, U46619 (30 ng mL−1 for 60 s), 72 h after the final saline challenge of control animals caused a threshold bronchoconstriction. Seventy two hours after the final antigen challenge, however, there was a marked bronchodilation to U46619. A persistent hyper-reactivity was expected. Ro 20–1724 (3 mg kg−1 i.p. before and 6 h after each antigen challenge), a selective inhibitor of phosphodiesterase type IV, attenuated both early and late asthmatic responses and the airway eosinophilia associated with this chronic antigen challenge model. There was no evidence of a bronchodilatory response to the same dose of inhaled U46619 in the group treated with Ro 20–1724. These results suggest that inhaled low dose U46619 induces a bronchodilation when delivered to conscious guinea-pigs after a series of repeated antigen challenges and when the airways are presumably in a state of chronic inflammation. This effect is lost when the airway inflammation is attenuated by the concurrent administration of Ro 20–1724. The reason for this remains unexplained.

Cerebral venous sinus thrombosis following sildenafil administration; a case report

Background: Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5) which currently used for the treatment of erection dysfunction (ED). Medicine approved by the FDA for treatment of pulmonary hypertension. Although there is no evidence of hypercoagulation states being associated with the use of PDE5 inhibitors, several animal studies suggest that PDE5 inhibitors may cause arterial and venous thrombotic or embolic events. Case report: The patient was a 37 year old man who had suffered sever headache and tonic- clonic generalized seizure. In neurological examination, he had decreased level of consciousness and he had no other positive finding in examination. The patient has been constantly taken 100 mg of Sildenafil once a day without medical supervision over the last month. Brain computerized tomography (CT) showed a small hemorrhage in left frontal lobe with suspicious to cerebral venous sinus thrombosis (CVST) lumbar puncture was performed. Increased cerebrospinal fluid pressure suggested the probability of CVST. Brain MRI result confirmed superior sagittal sinus thrombosis. Conclusion: Cerebral venous sinus thromboses need to be considered a potential side effect of Sildenafil administration.

Avanafil: A New Phosphodiesterase Type 5 Inhibitor for the Treatment of Erectile Dysfunction

Phosphodiesterase type 5 inhibitors (PDE5i) revolutionized erectile dysfunction (ED). Avanafil is a new PDE5i expected to enter the market in forthcoming years. It is a highly selective PDE5i associated with rapid absorption (Tmax ∼35 min) and a short half-life (T1/2 &lt;1.5 h) without any significant accumulation of the drug with either once- or twice-daily dosing. Phase III studies evaluated 50, 100 and 200 mg of avanafil in the general ED population as well as in diabetic men. The International Index for Erectile Function-erectile function domain score, as well as the successful erection for vaginal penetration and successful intercourse, improved significantly compared with placebo in both groups. Moreover, it may be associated with a better safety profile. Patients must be aware of all treatment options for ED since no one treatment will be suitable for everyone.

Inhaled phosphodiesterase type 5 inhibitors restore chloride transport in cystic fibrosis mice

Sildenafil and vardenafil, two selective inhibitors of phosphodiesterase type 5 (PDE5) are able, when applied by intraperitoneal injection, to activate chloride transport in cystic fibrosis (CF) mice homozygous for the F508del mutation. Oral treatment with the drugs may be associated with adverse haemodynamic effects. We hypothesised that inhaled PDE5 inhibitors are able to restore ion transport in F508del CF airway epithelium. We developed a restraint-free mouse chamber for inhalation studies. PDE5 inhibitors were nebulised for 15 min at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. We measured in vivo nasal transepithelial potential difference 1 h after a single inhalation of sildenafil, vardenafil or tadalafil in F508del CF and normal homozygous mice. After nebulisation with the drugs in F508del mice, chloride transport, evaluated by perfusing the nasal mucosa with chloride-free buffer containing amiloride followed by forskolin, was normalised; the forskolin response was increased, with the largest values being observed with tadalafil and intermediate values with vardenafil. No detectable effect was observed on sodium conductance. Our results confirm the role of PDE5 inhibitors in restoring chloride transport function of F508del CF transmembrane conductance regulator protein and highlight the potential of inhaled sildenafil, vardenafil and tadalafil as a therapy for CF.

ChemInform Abstract: N-3-Substituted Imidazoquinazolinones: Potent and Selective PDE5 Inhibitors as Potential Agents for Treatment of Erectile Dysfunction.

Phosphodiesterase type 5 (PDE5) inhibitors with improved PDE isozyme selectivity relative to sildenafil may result in agents for the treatment of male erectile dysfunction (MED) with a lower incidence of PDE-associated adverse effects. This paper describes the discovery of 14, a PDE5 inhibitor with improved potency and selectivity in vitro compared to sildenafil. This compound shows activity in a functional assay of erectile function comparable to that of sildenafil.

Evidence for the use of combination targeted therapeutic approaches for the management of pulmonary arterial hypertension

The last decade has witnessed a remarkable increase in the number of effective treatment options available for the management of patients with pulmonary arterial hypertension. In this regard, agents belonging to the therapeutic classes that specifically target the prostacyclin, endothelin and nitric oxide pathways have shown the greatest efficacy in clinical studies to date. These various drug treatments have individually been shown to confer improvements in symptoms, exercise capacity, pulmonary haemodynamics and possibly survival in different patient subgroups. However, pulmonary arterial hypertension is characterised by dysregulation of a variety of pathways. In addition, disease worsening is inevitable for the majority of patients receiving monotherapy. As a consequence, there is increasing interest in the use of treatment combinations in order to target multiple targets with the aim of restoring normal pulmonary vascular function in order to improve clinical status. Indeed, use of multiple specific-treatment regimens is now part of routine clinical practice and is emphasized in recently published therapeutic guidelines. This review details the rationale for the different combination strategies and examines the clinical evidence in favour of some of the approaches that have been evaluated.

Simple and sensitive LC-ESI-MS method for the quantitation of sildenafil in plasma samples

Sildenafil is a selective inhibitor of phosphodiesterase type 5 enzyme. A simple and sensitive LC-MS assay was developed to determine the concentration of sildenafil in human plasma. Sildenafil and omeprazole (internal standard) were extracted from the plasma with diethyl ether. The extract was evaporated under nitrogen and the residue was constituted with ACN and injected onto Novapak C(18) column (75x3.9 mm, 4 microm). The mobile phase consisted of 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid and was delivered isocratically at a flow rate of 0.2 mL/min. Sildenafil and omeprazole were monitored using a positive electrospray mode with single-ion recording set at m/z 475 and 346, respectively, which are consistent with [M+H](+) molecular ions, and the run time was less than 5 min. The detection limit of sildenafil was 0.5 ng/mL, and the calibration curve was linear between 0.5 and 2000 ng/mL (R(2)>0.99). Within- and between-day coefficients of variation were less than 7%. This method has been successfully used to measure sildenafil plasma concentrations in a beagle dog model following an oral administration of the drug.

Continuing Medical Education: 2009 Update on Phosphodiesterase Type 5 Inhibitor Therapy Part 2: Updates on Optimal Utilization for Sexual Concerns and Rare Toxicities in This Class (CME)

IntroductionHighly selective inhibitors of phosphodiesterase type 5 (PDE5I) have been commercially available for over a decade. Our knowledge of these drugs continues to expand. AimsTo review recent (since 2007) developments on the utilization of PDE5I in clinical practice. The focus of this manuscript is on the use of PDE5I for sexual concerns. Also reviewed are recent reports of rare but potentially serious toxicity. MethodsPubmed search utilizing the search terms “phosphodiesterase type 5 inhibitor,”“PDE5 inhibitor,”“sildenafil,”“vardenafil,” and “tadalafil.” Articles were screened for their relevance to the clinical practice of sexual medicine and/or PDE5I toxicity. Publications on routine dose PDE5I for penile rehabilitation, lower urinary tract symptoms, and stuttering priapism are summarized in a separate manuscript in this series. Main Outcome MeasuresPeer-reviewed publications since the last major update on PDE5I published in the medical literature. ResultsRecent investigations have suggested a number of interventions to potentially improve patient compliance with PDE5I therapy. Additionally, the approval in the past year of tadalafil as a daily medication signifies a potential paradigm shift in our concept of this disorder. Daily dosing may be useful in some men; however, the other available PDE5I continue to show excellent efficacy in the management of erectile dysfunction (ED). In addition to direct effects on ED, several recent reports highlight the use of PDE5I for improvement of premature ejaculation, sexual relationship status, and sexual function in women. There have also been several recent reports of rare but serious toxicity, particularly ototoxicity, associated with PDE5I use. ConclusionsRecent studies have suggested new ways to optimize utilization of PDE5I not only for the management of ED but also for other sexual concerns in both men and women. Rare but serious toxicities have been reported with PDE5I and, therefore, judicious counseling is indicated before prescribing these medications. Shindel AW. 2009 update on phosphodiesterase type 5 inhibitor therapy part 2: Updates on optimal utilization for sexual concerns and rare toxicities in this class. J Sex Med 2009;6:2352–2364.

Differential effects of selective PDE5 inhibitors in rat cerebral arteries in vitro and in vivo

Compounds which increase cGMP levels are implicated in migraine pathophysiology as well as stroke recovery. Inhibitors of the cGMP degrading enzyme phosphodiesterase type 5 (PDE5) induce headache and migraine in humans, however surprisingly and unlike other migraine inducing drugs without measurable dilatation of cerebral arteries [1] or changes in hemodynamic response or excitability [2]. We aimed to investigate whether sildenafil and tadalafil induced dilatation of rat middle cerebral and meningeal arteries in vitro and in vivo.

Continuing Medical Education: 2009 Update on Phosphodiesterase Type 5 Inhibitor Therapy Part 1: Recent Studies on Routine Dosing for Penile Rehabilitation, Lower Urinary Tract Symptoms, and Other Indications (CME)

Highly selective inhibitors of phosphodiesterase type 5 (PDE5I) have been commercially available for over a decade. Our knowledge of these drugs continues to expand. To review developments within the past 18 months on the utilization of PDE5I in preclinical studies and clinical practice. The focus of this article is on updates on regular dosing regimens of PDE5I other than the newly approved daily dose tadalafil. PubMed search utilizing the terms "phosphodiesterase type 5 inhibitor," PDE5 inhibitor,""sildenafil,""vardenafil," and "tadalafil." Articles were screened based on whether or not they addressed issues of routine dosing of PDE5I. Manuscripts on the newly approved daily dose tadalafil for erectile dysfunction (ED) were deferred for analysis in a separate manuscript in this series. Peer reviewed publications on routine dosing of PDE5I published in the medical literature since 2007. There have been numerous publications in the past 2 years regarding routine dosing of PDE5I for three major urological indications; penile rehabilitation, stuttering priapism, and management of lower urinary tract symptoms (LUTS). Evidence from basic science investigations has indicated that daily dose PDE5I may improve erectile function and exert a number of beneficial tissue effects on the penis. Unfortunately, data from human series of routine dose PDE5I for penile rehabilitation after radical prostatectomy are conflicting, with the two largest studies showing no benefit to daily dose therapy in the post-radical prostatectomy and the general ED populations. PDE5I are generally helpful at reducing symptoms of LUTS, particularly when given in conjunction with alpha blockers. Routine dosing of PDE5I has also been utilized successfully for management of stuttering ischemic priapism and several other medical indications. PDE5I given as routine doses have clinical promise. Further research is required to clarify their safety and efficacy for various indications.

Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca 2+ current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca 2+ current ( I Ca). However, the NOS3 signaling pathway modulating I Ca is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I Ca. Myocytes were isolated from NOS3 knockout ( NOS3 −/− ) and wildtype (WT) mice. We measured I Ca (whole-cell voltage-clamp), and simultaneously measured Ca 2+ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)- I Ca, and Ca 2+ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I Ca, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3 −/− myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I Ca, or Ca 2+ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I Ca, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I Ca; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca 2+ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I Ca and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated I Ca, while limiting effects on cardiac contraction.

ERK phosphorylation mediates sildenafil-induced myocardial protection against ischemia-reperfusion injury in mice

Sildenafil, a selective inhibitor of phosphodiesterase type 5, induces powerful protection against myocardial ischemia-reperfusion injury through activation of cGMP-dependent protein kinase (PKG). We further hypothesized that PKG-dependent activation of survival kinase ERK may play a causative role in sildenafil-induced cardioprotection via induction of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and Bcl-2. Our results show that acute intracoronary infusion of sildenafil in Langendorff isolated mouse hearts before global ischemia-reperfusion significantly reduced myocardial infarct size (from 29.4 +/- 2.4% to 15.9 +/- 3.0%; P < 0.05). Cotreatment with ERK inhibitor PD98059 abrogated sildenafil-induced protection (31.8 +/- 4.4%). To further evaluate the role of ERK in delayed cardioprotection, mice were treated with sildenafil (ip) 24 h before global ischemia-reperfusion. PD98059 was administered (ip) 30 min before sildenafil treatment. Infarct size was reduced from 27.6 +/- 3.3% in controls to 7.1 +/- 1.5% in sildenafil-treated mice (P < 0.05). The delayed protective effect of sildenafil was also abolished by PD98059 (22.5 +/- 2.3%). Western blots revealed that sildenafil significantly increased phosphorylation of ERK1/2 and GSK-3beta and induced iNOS, eNOS, Bcl-2, and PKG activity in the heart 24 h after treatment. PD98059 inhibited the enhanced expression of iNOS, eNOS, and Bcl-2 and the phosphorylation of GSK-3beta. PD98059 had no effect on the sildenafil-induced activation of PKG. We conclude that these studies provide first direct evidence that PKG-dependent ERK phosphorylation is indispensable for the induction of eNOS/iNOS and Bcl-2 and the resulting cardioprotection by sildenafil.

Clinical deterioration after sildenafil cessation in patients with pulmonary hypertension

Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE-5). Its chronic administration has been shown to improve exercise capacity, World Health Organization functional class, and haemodynamics in patients with symptomatic pulmonary arterial hypertension (PAH). There is however, no data describing the clinical consequences of sudden cessation of sildenafil treatment. In this series, 9 patients with NYHA Class II-IV PAH who were stable on 2 months of sildenafil monotherapy, had their sildenafil ceased to accommodate a 2-week washout period, required for enrollment in research involving an endothelin receptor antagonist. Six minute walk distance (SMWD) and clinical assessments were performed before cessation of sildenafil, and again 2 weeks later. Over the course of this 2-week washout period, 6 of the 9 patients reported increased breathlessness and fatigue, 1 of these was hospitalized with worsening right heart failure. The SMWD fell in 6 patients, with falls of greater than 100 m recorded in 4 patients. This was accompanied by a worsening of NYHA Class from 2.5 +/- 0.2 to 3.1 +/- 0.1 (mean +/- SEM, p = 0.01). These data indicate that sudden cessation of sildenafil monotherapy, in patients with PAH, carries with it a significant and unpredictable risk of rapid clinical deterioration. We recommend that if sildenafil needs to be ceased, it would be more prudent to consider concurrent vasodilator therapy before the gradual cessation of sildenafil.