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2, 3 In the Massachusetts Male Aging Study, a random sample survey of 1290 men aged 40 to 70 years, the age-adjusted prevalence of complete ED (no erections) was 15% in those with treated hypertension and 14% in those taking antihypertensive agents compared with a prevalence of 9.6% in the entire sample.

Sildenafil, an orally active and selective inhibitor of cGMP-specific phosphodiesterase type 5, is an effective and well-tolerated treatment for ED of various causes. 4 By inhibiting the degradation of cGMP in the corpus cavernosum of the penis, sildenafil enables smooth muscle relaxation and penile erection in patients with ED.

The most common adverse events associated with sildenafil treatment are headache and flushing, 5 which reflect its modest vasodilating properties.

6 Oral sildenafil (100 mg) produces transient decreases in systolic (8 to 10 mm Hg) and diastolic (3 to 6 mm Hg) blood pressure in healthy male volunteers. 5 Peak decreases are observed 1 h after dosing and coincide with peak plasma concentrations of drug. These changes in blood pressure generally return to pretreatment values by 4 to 8 h after dosing in healthy men.

6 This post hoc analysis assessed the efficacy and safety of sildenafil in men with ED who were receiving concomitant treatment with antihypertensive agents.

Efficacy data were analyzed post hoc for groups of patients enrolled in 10 of 18 randomized, double-blind, placebo-controlled studies of sildenafil in the treatment of ED of various causes. In these 10 studies that

assessed

efficacy with the same outcome measure, a total of 3414 patients, 1218 (36%) of whom were taking concomitant antihypertensive medication, were randomized to sildenafil (5 to 200 mg) or placebo for 6 weeks to 6 months. The patients enrolled were 18 years of age or older with a clinical diagnosis of ED of at least 6 months’ duration and in a stable relationship with a female partner for at least 6 months. Patients were excluded if they had penile anatomic defects, a recent (within the previous 6 months) history of stroke, myocardial infarction, or life-threatening arrhythmia, a resting blood pressure 170/110 mm Hg, a known history of retinitis pigmentosa, any significant concomitant medical condition that would impair participation in the study, or were receiving regular treatment with nitrates or anticoagulants.

All patients provided written informed consent before enrollment. The patients were instructed to take study

medication

approximately 1 h before sexual activity but not more than one dose daily.

Safety data were analyzed for 3975 patients from 18 randomized, double-blind, placebo-controlled studies lasting from 6 weeks to 6 months, which included the 10 studies analyzed for efficacy data.

In the 18 studies, of the 4274 patients randomized to sildenafil (5 to 200 mg) or placebo, 2881 ( n = 1837 sildenafil, n = 1044 placebo) were not taking any antihypertensive medication, 1094 ( n = 704 sildenafil, n = 390 placebo) were taking concomitant antihypertensive medication classified as a diuretic, ?-blocker, ? 1 -blocker, angiotensin converting enzyme inhibitor, or calcium channel blocker, and 346 ( n = 217 sildenafil, n = 129 placebo) were taking two or more of these classes of antihypertensive agent during the studies.

The inclusion and exclusion criteria for the 18 studies were the same as mentioned

above

for the 10 studies.

Efficacy was assessed for patients in the sildenafil and placebo groups who were taking antihypertensive medication or who were not taking any antihypertensive agent using end-of-treatment responses to question 3 (ability to achieve an erection) and question 4 (ability to maintain an erection) of the International Index of Erectile Function (IIEF) 7 and responses to a global efficacy question (“Did the treatment improve your erections?”).

The responses to the two IIEF questions were graded on a scale of 1 (almost never or never) to 5 (almost always or always), with a score of 0 indicating no attempt at sexual intercourse.

Physical examinations and standard laboratory tests were performed throughout the studies. In all studies, investigators recorded all observed and patient-reported adverse events and classified the relationship of the adverse event to the study medication as definitely related, uncertain, or not related. A treatment-related adverse event was defined as any event classified as definitely related, of uncertain relation, or of unspecified relation to the study medication. Adverse

events

were analyzed for the 2881 patients not taking any antihypertensive medication and for the 1094 patients taking one or more of the following classes of antihypertensive agent: diuretic, ?-blocker, ? 1 -blocker, angiotensin converting enzyme inhibitor, or calcium channel blocker.

The mean responses to the two questions of the IIEF for each of the treatment groups were calculated and the treatment effect was analyzed using an analysis of covariance model, as described previously.

4 Comparisons between mean responses for patients taking versus not taking antihypertensive agents were analyzed using an analysis of covariance model that accounted for the treatment effect.

The responses to the global efficacy question were analyzed using a logistic regression model. 4 Intention to treat analyses were performed on all variables, and all statistical tests were two-sided and evaluated at the 5% significance level.

In the 10 double-blind, placebo-controlled studies in which efficacy was assessed, the mean age of the patients was 58 years for those taking antihypertensive medication and 55 years for those not taking any antihypertensive agent. The mean

duration

of ED in the two groups was 4.7 years and 5.2 years, respectively.

The etiology of ED was 67% organic, 11% psychogenic, and 22% mixed for the patients taking concomitant antihypertensive medication compared with 56% organic, 17% psychogenic, and 27% mixed for those not taking an antihypertensive agent.

The characteristics of the patients randomized to treatment in the 18 double-blind, placebo-controlled studies from which safety data were collected were similar to those described above.

The mean end of treatment scores for question 3 (achieving an erection) and question 4 (maintaining an erection) of the IIEF were significantly higher for the sildenafil group than for the placebo group for both patients taking and those not taking antihypertensive agents ( P values Table 1). Furthermore, the incidences of the most common adverse events (eg, headache, flushing, and dyspepsia) also were not affected by whether the patients receiving sildenafil were taking antihypertensive medication. Combined incidence (%) of treatment-related adverse events reported in 18 double-blind placebo-controlled studies of patients with ed receiving sildenafil (Sild) or placebo (Pbo) who were taking or not taking different classes of antihypertensive medication * Sildenafil Can Affect Innate and Adaptive Immune System in Both Experimental Animals and Patients. 1 Department of Clinical Immunology, Institute of Transplantology, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland. Sildenafil, a type 5 phosphodiesterase inhibitor (PDE5-I), is primarily used for treating erectile dysfunction. Sildenafil inhibits the degradation of cyclic guanosine monophosphate (cGMP) by competing with cGMP for binding site of PDE5. cGMP is a secondary messenger activating protein kinases and a common regulator of ion channel conductance, glycogenolysis, and cellular apoptosis.

PDE5 inhibitors (PDE-Is) found application in cardiology, nephrology, urology, dermatology, oncology, and gynecology. Positive result of sildenafil treatment is closely connected with its immunomodulatory effects.

Sildenafil influences angiogenesis, platelet activation, proliferation of regulatory T cells, and production of proinflammatory cytokines and autoantibodies.

Sildenafil action in humans and animals appears to be different.

Surprisingly, it also acts differently in males and females organisms. Although the immunomodulatory effects of PDE5 inhibitors appear to be promising, none of them reached the point of being tested in clinical trials. Data on the influence of selective PDE5-Is on the human immune system are limited. The main objective of this review is to discuss the immunomodulatory effects of sildenafil in both patients and experimental animals. This is the first review of

the

current state of knowledge about the effects of sildenafil on the immune system. Phosphodiesterases (PDEs) are critical components in the cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) and the cyclic guanosine monophosphate/protein kinase G (cGMP-PKG) signaling pathways.

Phosphodiesterases decompose the cyclic nucleotides, cAMP and cGMP, to inactivate 5?-AMP and 5?-GMP, respectively.

PDE activity is tightly coupled with the activity of adenyl cyclases (AC) and guanyl (GC) which synthesize these cyclic nucleotides: cAMP and cGMP.

The cyclic nucleotide-dependent protein kinases (

PKA

and PKG) control the functional cellular responses such as intracellular calcium level, inflammation, cell proliferation, and transcription. Therefore, cAMP and cGMP are considered as potential new therapeutic targets [1, 2]. The inhibition of action of PDE results in increased level of cyclic nucleotides. Several PDEs have been identified and characterized based on their molecular sequence, kinetics, regulation, and pharmacological characteristics.

These enzymes were divided into 11 families (PDE1–PDE11) [3, 4]. PDEs 1, 2, 3, 10, and 11 have affinity to cAMP and cGMP and PDEs 4, 7, and 8 hydrolyze cAMP only, while PDEs 5, 6, and 9 exhibit specificity for cGMP.

Increased activity of PDEs has been implicated in a number of clinical conditions including erectile dysfunction (PDE5), cardiovascular diseases (PDEs 3, 4, and 5), pulmonary inflammatory conditions (PDE4), autoimmune diseases (PDEs 3 4, 5, and 7), and cognition and memory disorders (PDEs 1, 2, 3, 4, 7, 9, and 10 A) [5–7]. All PDEs are composed of 3 main domains: the

catalytic

core (highly conserved), the regulatory N-terminus, and the carboxyl C-terminus.

N-terminal domains are responsible for regulation and subcellular localization of PDEs.

They contain domains involved in

ligand

binding, PDE oligomerization, kinase recognition, and phosphorylation. The N-terminal domains are flanked by the catalytic core.

The catalytic c-domain of PDEs possesses costco sildenafil coupon an active pocket composed of 3 helical subdomains: the N-terminal cyclic-fold region, the linker region, and the C-terminal helical bundle.

A deep

hydrophobic

pocket is formed at the interface of the three subdomains. The hydrophobic pocket is composed of four subsites: a metal binding site (M), a core pocket (Q pocket), hydrophobic pocket (H pocket), and a lid-region (L region).

At the bottom site is a metal binding domain which probably contains zinc or magnesium ions. Stabilization of the enzyme structure and activation of hydroxide are the presumable roles of metal ions [7, 8]. Substrate specificity of PDEs was suggested to depend on the ability to rotate of one glutamine which forms hydrophobic bonds

with

cAMP or cGMP [9, 10].

The fixed glutamine orientation allows to bind only one substrate [11]. that Gln-817 is critical for cGMP, sildenafil, and vardenafil affinity. The analogous atoms to substituents at N-1 and C-6 in cGMP form a bidentate H-bond with Gln 817 [11].

PDE5 costco sildenafil coupon was first identified in rat lung tissue, followed by numerous other tissues. The enzyme was first purified and cloned in 1980 [12]. Chemically, sildenafil is a 5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]-pyrimidin-7-one. The crystallographic examination of PDE5A shows that the PDE Q pocket accommodates the pyrazolopyrimidinone group of sildenafil. The ethoxyphenyl group of sildenafil fits into the hydrophobic H pocket. The L region of PDE5A surrounds the methylpiperazine group of sildenafil [3, 7].

Orally dosed sildenafil has an expected onset of action of 30 min, with estimated maximum effect at 1 h and a total duration of effect of 4–6 h. In blood, approximately 96% of sildenafil is protein-bound with the pick serum concentration ( ) of 440 ng/mL being reached at median time of 60 min following administration of a 100 mg oral dose [13]. Sildenafil increases cellular cGMP levels through competition for the phosphodiesterase binding site with cGMP in that way inhibiting degradation of cGMP to GMP.

cGMP through protein kinase G (PKG) plays an important role in the regulation of the activity of various cell populations including immune cells.

PKG is abundant in all smooth muscle cells and platelets, and its low levels occur in renal cells, fibroblasts, leukocytes, and neuronal cells.

Biological substrates for PKG include inositol, triphosphate receptor (IP 3 R), G-proteins, dopamine- and cAMP-regulated phosphoprotein (DARP-32), and phospholipase C. PKG promotes the opening of calcium-activated potassium channels and activates Ca 2+ influx, leading to cell hyperpolarization and relaxation [14]. The level of cGMP is also regulated by nitric oxide (NO). NO exerts multiple modulating effects on inflammation and plays a key role in the regulation of the immune responses.

Both

the

inducible nitric oxide synthase (iNOS) and the PDEs enzymes are expressed in numerous cell types including macrophages, dendritic cells, T cells, and neutrophils [15, 16]. NO is generated by three nitric

oxide

synthase (NOS) isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS).

The nitric oxide is the main activator of soluble guanylate cyclase (sGC), an enzyme which synthesizes guanosine 3?5?-cyclic monophosphate (cGMP).

Inhibition of cGMP catabolism by selective PDE5-Is increases NO levels by increasing the ratio of nitrite to nitrate and by stimulating transcription of mRNA for iNOS, as well as eNOS [17].

In addition, nitric oxide increases in tissues and cells the level of heme oxygenase HO-1 which oxidatively degrades heme into equimolar amounts of CO, biliverdin, and iron.

Carbon monoxide activates sGC and elevates cGMP in target tissues, which dilates blood vessels. Through that stimulation, sildenafil may serve a critical growth of cellular cGMP levels [18].

[17] demonstrated that sildenafil stimulates the expression of HO-1 and iNOS in vascular smooth muscle cells (SMCs) via the reactive oxygen species-nuclear erythroid 2-related factor 2 (ROS-Nrf2) and sGC-cGMP pathways, respectively [17]. Transcription factor Nrf2 is responsible for the induction of phase II enzymes of xenobiotics metabolism.

It activates transcription of genes encoding cytoprotective proteins which deactivates electrophiles metabolites and reactive oxygen species and stabilizes the redox potential of the cell.

Nrf2 controls antioxidant defense genes including HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1), gluthatione reductase (GR), and gluthatione peroxidase (GPx) [18, 19]. [20] indicate direct effect of sildenafil on cells.

Sildenafil treatment of astrocytes prevents and also restores effect of LPS on actin filaments.

Changes in actin stress fibres is a result of overactivation of Ca 2+ signaling by LPS.

proposed that sildenafil directly influences cell integrity through ankyrin B, a protein which is associated with the cytoskeleton, and interacts with Na + /K + -ATP-ase and IP 3 R connecting the pump to the Ca 2+ responses from internal cell stores and to the integrity of the cytoskeleton.

These data show that cGMP is involved in Na + /K + -ATP-ase activity. Sildenafil acting through Na + /K + -ATP-ase could change Ca 2+ waves and stop the inflammatory process. The action of sildenafil is amplified through these mechanisms.

The Effect of Sildenafil on the Immune System of Healthy Experimental Animals.

There is some evidence that immunomodulatory properties of sildenafil are gender-specific.

In healthy mice, a tendency to decrease the percentage of CD4 + cells and to increase the percentage of CD8 + T cells

was

demonstrated in males but not females treated with sildenafil [21]. [21] observed that the levels of T effector memory cells and T central memory cells were decreased in males and increased in females. Naive T cell levels were increased in males, while CD8 + (T central memory) cells were decreased.

Interestingly, there was no effect on lymphocyte regulatory T cells (Treg) or natural killer T-cells (

NKT

) in the entire gender-mixed population. The percentage of the activated NK cells and T conventional cells was increased in the female population and decreased in males. Sildenafil treatment did not affect the percentage of dendritic cells in the whole mouse population. SC in healthy mice led to a decrease in percentage of these cells in the spleen of female mice. Moreover, sildenafil was found to diminish serum levels of IL-6 in mice and demonstrated a tendency to increase IL-2.

No influence of sildenafil on serum levels of IL-10,

IL-1

? or vascular endothelial grow factor (VEGF) was observed. Sildenafil revealed a considerable immunosuppressive effect in male as opposed to female mice [21]. The differences in immunological effects of the drug between females and males could be explained by endocrine and genetic differences between the sexes [22].

It should be noted that sildenafil in this study was used in large doses—20 mg/kg for 21 days. Ex vivo studies of the effects of sildenafil on isolated splenocytes from healthy mice confirmed a lack of influence on the cytotoxicity of mononuclear cells (CD8 + , NKT, and NK cells).

SC did not affect either maturation or activation of dendritic cells (DC). But with respect to naive/memory phenotype costco sildenafil coupon of T cells sildenafil reduced the amount of CD8 + cells in whole population. Sildenafil at the concentration of 7.5 ? M after 24 h of culture increased the percentage of CD4 + T cells and reduced the percentage of B cells [21]. Szczypka and Obminska-Mrukowicz [23] investigated the effects of sildenafil on thymocytes, splenocytes, and T cells isolated from mice lymph nodes.

Oral administration of sildenafil at a dose 1 mg/kg temporarily decreased the percentage of CD4 + CD8 + thymocytes and increased CD8 + cells. This effect was only observed after the fifth dose of the drug administrated at 24 h intervals. Among lymphocytes isolated from the mesenteric lymph nodes, the percentage of CD19 + cells decreased and a rise in the percentage of CD3 + cells was noted 72 h after the last dose of the drug. In another study researchers used the same protocol of administration of SC to investigate cytokine concentration in animals serum.

Sildenafil decreased IL-2 level after 72 h from a single administration of 1 mg/kg and after 12 h enhanced temporarily the level of IL-5 (from 7 to 13 pg/mL) [24]. Szczypka and Obmiska-Mrukowicz [25] showed that sildenafil increased the production of IL-1 ? and nitric oxide (NO) by peritoneal macrophages ex vivo, increased the percentage of phagocytosing granulocytes, and decreased the percentage of phagocytosing monocytes. The Effect of Sildenafil on the Experimental Animals Immune System in Pathological Conditions. PDE5 is present in glial cells and neurons [26, 27]. [29] reported that immunomodulatory therapies can be particularly costco sildenafil coupon beneficial in the treatment of multiple sclerosis (MS), a disease in which the

immune

system reacts against the central nervous system (CNS) antigens, initiating a detrimental inflammatory cascade which leads to demyelination and axonal degeneration. [30], in their research on experimental autoimmune encephalomyelitis (EAE) as a model of MS, found that administration of sildenafil to mice (10 mg/kg, equivalent to a dose of 57 mg/day for a 70-kg human) for 7 days, at the onset of clinical symptoms, resulted in reduced cellular infiltration in spinal cord and white matter. Sildenafil treatment increased the number of cells expressing the forkhead box p3 (Foxp3) transcription factor, as well as the expression of mRNA of granzyme B (GrB) cluster proteins.

CD4 + and CD8 + Treg cells are known to act by granzymes in the inhibition of T effectors cells and antigen presenting cells (APC). It

was

shown that GrB protein was upregulated in Treg cells [30]. Moreover, production of IL-2, IFN- ? , and IL-4 in cultures of splenocytes isolated from sildenafil-treated mice was reduced.

A tendency to reduce the level of TNF- ? and IL-17 was also observed. Sildenafil did not change the level of inhibitory cytokine IL-10.

In vitro proliferation of splenocytes from control group mice after stimulation with phytohemagglutinin (PHA) and exposure to sildenafil from 0.1 to 10 ? M/mL was decreased in the SC concentration-dependent manner [30]. Examination of sildenafil-treated EAE mice brains showed that infiltration of inflammatory cells to the brain was reduced due to a lower expression of the intracellular adhesion molecule-1 (ICAM-1).



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