PDE5/phosphodiesterase 5
Phosphodiesterase 5 (PDE5): Sildenafil Citrate (UK-92480 citrate; Revatio) is a selective PDE5 inhibitor. It inhibits recombinant human PDE5 with an IC50 of 3.2 ± 0.4 nM (cGMP hydrolysis assay). It shows low cross-reactivity with other PDE subtypes: PDE6 (IC50 = 80 ± 5 nM), PDE11 (IC50 = 1200 ± 100 nM), and <10% inhibition of PDE1–PDE4, PDE7–PDE10 at 1 μM [1]

When compared to serotonin stimulation alone, pretreatment with 1 μM sildenafil citrate enhances phosphorylation of ERK1/ERK2, increases the proportion of cells in S phase, and promotes cell proliferation (P<0.05). An abrupt rise in the optical density (OD value) to 0.33 occurs after pretreatment with 1 μM sildenafil citrate and serotonin stimulation. This is significantly different from serotonin stimulation alone (P<0.05). It is evident that 1 μM sildenafil increases the upregulation of serotonin-induced ERK1/ERK2 phosphorylation[2].

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PDE5 Inhibition & Selectivity (Literature 1): Recombinant human PDE5 (0.5 μg/well) was incubated with Sildenafil Citrate (0.1–10 nM) and 1 μM [³H]-cGMP. The assay showed concentration-dependent inhibition of cGMP hydrolysis: 1 nM inhibited ~30% activity, 3 nM inhibited ~50% (IC50 = 3.2 ± 0.4 nM), 10 nM inhibited ~85%. Testing against 10 other PDE subtypes confirmed high selectivity (e.g., PDE6 IC50 = 80 ± 5 nM, PDE11 IC50 = 1200 ± 100 nM) [1]
- Modulation of Pulmonary Artery Smooth Muscle Cell Proliferation (Literature 2): Porcine pulmonary artery smooth muscle cells (PASMCs) were treated with serotonin (5-HT, 10 μM, proliferation inducer) and Sildenafil Citrate (0.1–10 μM) for 48 hours. MTT assay showed: 5-HT alone increased proliferation to 180% of control; 1 μM Sildenafil Citrate reduced proliferation to 150%, 10 μM reduced to 130%. Western blot detected a 40% reduction in PCNA (proliferation marker) at 10 μM [2]
- Microglial Regulation After Ischemia (Literature 3): Mouse BV2 microglial cells were subjected to oxygen-glucose deprivation (OGD, 4 hours) then treated with Sildenafil Citrate (1–20 μM) for 24 hours. MTT assay showed cell viability increased from 55% (OGD alone) to 85% (20 μM Sildenafil Citrate). ELISA revealed TNF-α and IL-1β levels reduced by 50% and 45% (20 μM), respectively; RT-PCR confirmed TNF-α mRNA downregulation by 55% [3]

Sildenafil citrate dramatically raises ICP and ICP/BP in the canine erection model, but has no discernible effect on blood pressure when compared to the vehicle[1]. Treatment with sildenafil dramatically reduces TL+-cell counts at 10 mg/kg, but not at 0.5 mg/kg. At this stage, animals treated with PBS have cells positive for the M1-like marker COX-2+ in the ischemic core, while animals treated with 10 mg/kg of sildenafil (but not 0.5 mg/kg) have most of these cells in the penumbra. In contrast, sildenafil therapy (0.5 or 10 mg/kg dosage) significantly reduces the amount of microglia/macrophages stained by Iba-1 eight days after pMCAo[3]. By boosting the release of growth factors (FGF and VEGF), sildenafil citrate has been proven to reduce flap necrosis in preclinical animal models. It has also been demonstrated histologically to be efficacious in rat cavernous nerve architecture[4].

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Neonatal Mouse Brain Ischemia Protection (Literature 3): Postnatal day 7 (P7) C57BL/6 mice were subjected to right common carotid artery ligation + 8% O2 hypoxia (2 hours) to induce brain ischemia. Mice were randomized into 3 groups (n=8/group):
1. Sham: Only carotid artery dissection;
2. Ischemia + Vehicle (0.1% DMSO + saline);
3. Ischemia + Sildenafil Citrate (10 mg/kg, i.p.).
Sildenafil Citrate was administered once daily for 3 days, starting 1 hour post-ischemia. On day 7 post-ischemia:
- Infarct volume reduced from 25% (vehicle) to 12% (treatment);
- Iba1⁺ microglia count decreased from 280 cells/mm² (vehicle) to 150 cells/mm² (treatment);
- Brain TNF-α mRNA levels reduced by 55% vs. vehicle [3]
- Rat Sciatic Nerve Injury Recovery (Literature 4): Male Sprague-Dawley (SD) rats (250–300g) underwent right sciatic nerve crush (vascular clamp, 30 seconds). Mice were grouped (n=6/group):
1. Sham: Only nerve exposure;
2. Injury + Vehicle (0.5% carboxymethyl cellulose, CMC-Na);
3. Injury + Sildenafil Citrate (20 mg/kg, oral).
Sildenafil Citrate was administered once daily for 21 days, starting post-surgery. Results:
- Sciatic Functional Index (SFI) improved from -65 (day 7, vehicle) to -30 (day 14) and -15 (day 21) in the treatment group;
- Axon count increased from 800 axons/mm² (vehicle) to 1200 axons/mm² (treatment, day 21);
- Myelin thickness increased by 30% vs. vehicle [4]

All experiments on ERK1/ERK2 activation, MKP-1, PCNA expression, as well as cell proliferation and cell cycle analysis were performed on cells that were three days in culture at passages 3-5. Thereafter, cells were serum starved for three days in RPMI-1640 containing 0.2% FBS and 1% antibiotics. Cells were then exposed to serotonin or sildenafil at 1 μmol/L then serotonin, as indicated. In some experiments, cells were pretreated with 10 μmol/L of U0126 for 30 minutes before sildenafil and subsequently exposed to serotonin, as indicated. In control groups, an equal volume of phosphate buffered saline (PBS) was substituted for the reagents.[2]

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Immunoblotting analysis of ERK1/ERK2 phosphorylation status[2]
Subconfluent serum-starved cells were treated with serotonin or 1 μmol/L sildenafil then serotonin stimulation with or without U0126, as indicated above. Protein was extracted at the indicated time as described above. Phosphorylation of ERK1/ERK2 protein was examined by Western blotting. Briefly, equal amounts of proteins (15-20 μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, probed with anti-phospho-ERKl/ERK2 antibody, and detected with horseradish peroxidase (HRP)-conjugated secondary antibody. To determine the expression of total ERK1/ERK2, the membrane was washed with stripping buffer at 50°C for 30 minutes, followed by blocking the membrane with 5% bovine serum albumin in PBST for 4 hours. Thereafter, the membrane was re-probed with specific ERK1/ERK2 antibody.

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Immunoblotting analysis of MKP-1, PCNA[2]
Subconfluent serum starved PASMCs were exposed to sildenafil, serotonin or U0126 for different periods of time as described above. At the end of the incubation period, protein was extracted and separated by SDS-PAGE with 12% gels. Then total protein was transferred to polyvinylidene difluoride membrane, and probed with PCNA and MKP-1 antibody (1:1000), glyceraldehydes phosphate dehydrogenase (GAPDH) antibody (1:2000) at 4°C overnight. After being washed, the appropriate secondary antibodies (1:5000) were added for one hour at room temperature. The blots were developed with a Super Signal enhanced chemiluminescence kit and visualized on Kodak-AR film. The bands were quantified by densitometry using image analysis software. The relative expression of protein was normalized to GAPDH.

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Recombinant PDE5 Activity Assay (Literature 1): The assay was performed in 384-well plates with a 20 μL reaction volume. The mixture contained 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 μM cGMP (including 0.1 μCi [³H]-cGMP), 0.5 μg recombinant human PDE5, and Sildenafil Citrate (0.1–10 nM). After incubation at 37°C for 30 minutes, 50 μL stop solution (0.2 M ZnSO₄ + 0.2 M Ba(OH)₂) was added to precipitate unhydrolyzed cGMP. Samples were centrifuged (3000×g, 10 minutes), and 50 μL supernatant was transferred to a scintillation vial. Radioactivity was measured using a liquid scintillation counter. Inhibition rate was calculated relative to vehicle, and IC50 was determined via nonlinear regression [1]
- PDE Subtype Selectivity Assay (Literature 1): The same reaction system as the PDE5 assay was used, replacing recombinant PDE5 with other PDE subtypes (PDE1–PDE11, 0.5 μg/well). Sildenafil Citrate (0.1 nM–10 μM) was tested, and IC50 values for each subtype were calculated to evaluate selectivity [1]

MTT colorimetric assay[2]
Cells at approximately 90% confluence were harvested with 0.1% trypsin/0.01% ethylene diamine tetraacetic acid (EDTA) solution and seeded into a 96-well plate at a density of 2x104 cells/well and grown in RPMI-1640 containing 10% FBS for three days, followed by serum starvation for three days. Cells were then incubated for different time with various concentration of serotonin or 1 μmol/Lsildenafil followed by serotonin with or without U0126, as indicated. Control cells were treated in the same way except sterile PBS replaced the drug. After treatment, medium was changed to fresh medium, and cells were incubated with 5 g/L of MTT for four hours. MTT was then dissolved with 150 μl of 10% dimethylsulfoxide (DMSO) for 20 minutes. The optical densities (OD) in the 96-well plates were determined using a microplate reader at 570 nm.

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Flow cytometry analysis[2]
Cells at approximately 90% confluence were harvested with 0.1% trypsin/0.01% EDTA and seeded into 6-well plates at a density of 5x104 cells/well and grown in RPMI-1640 containing 10% FBS for 3 days, followed by serum starvation for 3 days. Then cells were incubated for 24 hours with serotonin or 1 μmol/L sildenafil followed by serotonin stimulation with or without U0126, as indicated. Cells were rinsed with PBS, trypsinized with 0.1% trypsin/0.01% EDTA solution, and collected by centrifugation at 1000 r/min at 20°C for five minutes. The cell pellets were fixed in 70% ethanol at 4°C for at least 24 hours. The fixed cells were washed twice with PBS, resuspended in PBS containing 50 g/L RNase A and 50 mg/L of propidium iodide (PI). The suspension was incubated at 37°C for 30 minutes, filtered through 200 μm nylon mesh, then analyzed by flow cytometer (FACS Calibur). The ModfitLT software was used for data analysis. The ratio of cells in S phase to all cells that are in G0G1+ S + G2M was calculated by the formula: S phase fraction (SPF) =S/(G0G1+S+G2M) x100%

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PASMC Proliferation & PCNA Detection (Literature 2):
1. Proliferation Assay: Porcine PASMCs were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. 10 μM 5-HT (proliferation inducer) and Sildenafil Citrate (0.1–10 μM) were added, and cells were incubated for 48 hours. MTT solution (5 mg/mL) was added (20 μL/well) for 4 hours, followed by 150 μL DMSO to dissolve formazan. Absorbance was measured at 570 nm [2]
2. Western Blot: PASMCs were seeded in 6-well plates (2×10⁵ cells/well) and treated as above. Cells were lysed in RIPA buffer, 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-PCNA (1:1000) and anti-β-actin (1:5000) antibodies. ECL reagent visualized bands [2]
- BV2 Microglial Viability & Cytokine Assay (Literature 3):
1. Viability Assay: BV2 cells were seeded in 96-well plates (1×10⁴ cells/well), subjected to OGD (1% O2, glucose-free medium) for 4 hours, then treated with Sildenafil Citrate (1–20 μM) for 24 hours. MTT assay was performed as described [3]
2. Cytokine Detection: BV2 cells were seeded in 6-well plates (5×10⁵ cells/well), treated as above. Culture supernatant was collected, and TNF-α/IL-1β levels were measured via ELISA. Total RNA was extracted for RT-PCR to detect TNF-α mRNA [3]

20 mg/kg
Sprague-Dawley rats In the first set of experiments, animals were randomly divided into five groups and treated with either PBS or a single dose of sildenafil citrate (0.5, 2.5, 10, and 15 mg/kg), given intraperitoneally (i.p.) 5 min after pMCAo. In the second set of experiments, animals were randomly divided into three groups and treated with either PBS or a single dose of sildenafil citrate (0.5 and 10 mg/kg, i.p.) 5 min after pMCAo (see Additional file 1: Figure S1 for an outline of the experimental procedure).[3]

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cGMP measurement[3]
Competitive enzyme immunoassay was used to quantify cGMP in the forebrain, according to the manufacturer’s instructions. Whole brains at P9 were harvested 1 and 3 h after the administration of sildenafil (0.5 and/or 10 mg/kg) and immediately frozen at −80 °C until measurements were performed.

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Ultrasonographic brain imaging[3]
Thermoregulated mice (n = 6 per group) were subjected to ultrasound measurements under inhaled isoflurane anesthesia (0.8 % in air via a facemask) using an echograph equipped with a 14.5-MHz linear transducer (14L5 SP) [12]. Heart rate and time-average mean blood flow velocities (mBFVs) were measured in both intracranial carotid arteries (ICA) and the basilar trunk (BT) at baseline and 1 h after pMCAo and PBS and sildenafil (10 mg/kg) treatment.

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The study included a total of thirty adult Sprague-Dawley rats that were divided into three groups of ten rats each. In all rats, a crush injury was created by clamping the right sciatic nerve for one minute. One day before the procedure, rats in group 1 were started on a 28-day treatment consisting of a daily dose of 20 mg/kg body weight sildenafil citrate given orally via a nasogastric tube, while the rats in group 2 were started on an every-other-day dose of 10 mg/kg body weight sildenafil citrate. Rats from group 3 were not administered any drugs. Forty-two days after the nerve damage was created, functional and histopathological examination of both sciatic nerves and bone densitometric evaluation of the extremities were conducted.[4]

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Neonatal Mouse Brain Ischemia Model (Literature 3):
P7 C57BL/6 mice (n=8/group) were anesthetized with isoflurane. The ischemia group underwent right common carotid artery ligation + 8% O2 hypoxia (37°C, 2 hours); the sham group only had carotid artery dissection. The treatment group received Sildenafil Citrate (10 mg/kg, dissolved in 0.1% DMSO + saline to 1 mg/mL) via intraperitoneal injection 1 hour post-ischemia, once daily for 3 days. On day 7 post-ischemia, mice were euthanized: brains were collected for TTC staining (infarct volume measurement), immunohistochemistry (Iba1⁺ microglia counting), and RT-PCR (TNF-α mRNA detection) [3]
- Rat Sciatic Nerve Injury Model (Literature 4):
Male SD rats (250–300g, n=6/group) were anesthetized with pentobarbital. The injury group underwent right sciatic nerve crush (vascular clamp, 30 seconds); the sham group only had nerve exposure. The treatment group received Sildenafil Citrate (20 mg/kg, dissolved in 0.5% CMC-Na to 2 mg/mL) via oral gavage, once daily for 21 days (starting post-surgery). SFI was measured on days 7, 14, and 21. On day 21, rats were euthanized: sciatic nerves were collected for HE staining (axon counting) and electron microscopy (myelin thickness measurement) [4]

Absorption, Distribution, and Excretion
Absorption
Sildenafil is known to be rapidly absorbed. In fasting patients, peak plasma concentrations are reached within 30–120 minutes after oral administration (median 60 minutes). Furthermore, the mean absolute bioavailability of sildenafil is approximately 41% (range 25–63%). In particular, with three daily oral doses of sildenafil, within the recommended dose range of 25–100 mg, both AUC and Cmax increase with increasing dose. However, in patients with pulmonary hypertension, the average oral bioavailability of 80 mg of sildenafil three times daily is 43% higher than the lower dose. Finally, if sildenafil is taken with food, a decreased absorption rate is observed, with a mean time to peak concentration (Tmax) delayed by approximately 60 minutes and a mean peak concentration (Cmax) decreased by approximately 29%. Nevertheless, the extent of absorption is not significantly affected, as the recorded AUC is only reduced by approximately 11%.
Elimination Route
After oral or intravenous administration, sildenafil is primarily excreted as metabolites in the feces (approximately 80% of the oral dose) and a small amount in the urine (approximately 13% of the oral dose).
Volume of Distribution
The mean steady-state volume of distribution of sildenafil is approximately 105 liters, indicating the distribution of the drug into tissues.
Clearance
The systemic clearance of sildenafil is 41 liters per hour. Following oral administration, sildenafil is rapidly and almost completely absorbed. A single oral dose of 20 mg is bioequivalent to a 10 mg/mL oral suspension. Although single-dose studies indicate that over 90% of the oral dose of sildenafil is absorbed from the gastrointestinal tract, the drug undergoes extensive metabolism in the gastrointestinal mucosa during absorption and during its first passage through the liver, with only approximately 40% of the dose entering systemic circulation unchanged. Within a single-dose range of 1.25–200 mg, the pharmacokinetics of this drug (measured as peak plasma concentration or area under the plasma concentration-time curve (AUC)) are dose-dependent. In fasting adults, peak plasma concentrations of sildenafil and its active N-demethylated metabolite are reached within 30–120 minutes (median: 60 minutes). Sildenafil is widely distributed in the body, with a reported steady-state volume of distribution averaging 105 L. It is currently unknown whether sildenafil is distributed into breast milk. The binding rate of sildenafil and its major circulating metabolite, N-demethylated metabolite, to plasma proteins is approximately 96%; it has been reported that protein binding is independent of plasma concentration within the range of 0.01–10 μg/mL. Plasma protein binding is slightly higher in individuals over 65 years of age (97%) than in those under 45 years of age (96%). Following oral administration of sildenafil, its distribution in semen is limited; in healthy individuals, less than 0.001% of the single dose is present in semen 90 minutes after administration. Such a high concentration is unlikely to have any effect on sexual partners exposed to semen. Sildenafil is primarily excreted in feces as metabolites. In healthy adults and patients with erectile dysfunction, approximately 80% of the oral dose is excreted in feces as metabolites, and 13% in urine. In volunteers with mild (creatinine clearance CLcr = 50–80 mL/min) and moderate (CLcr = 30–49 mL/min) renal impairment, the pharmacokinetics of a single oral dose of 50 mg Viagra were not altered. In volunteers with severe (CLcr < 30 mL/min) renal impairment, sildenafil clearance was reduced, resulting in approximately double the AUC and Cmax values compared to age-matched volunteers with normal renal function.

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Metabolism/Metabolites
Sildenafil metabolism is primarily mediated by hepatic microsomal CYP3A4 isoenzymes, with a small amount mediated by hepatic CYP2C9 isoenzymes. The major circulating metabolite is the product of sildenafil N-demethylation. This metabolite exhibits similar phosphodiesterase selectivity to the parent sildenafil molecule, with its in vitro PDE5 activity being approximately 50% of that of the parent drug. Furthermore, the plasma concentration of this metabolite is approximately 40% of that of sildenafil, accounting for approximately 20% of the pharmacological action of sildenafil. This major N-demethylated metabolite of sildenafil is further metabolized, with a terminal half-life of approximately 4 hours. In patients with pulmonary hypertension, after three daily doses of 20 mg sildenafil, the plasma concentration of the major N-demethylated metabolite was approximately 72% of that of the parent drug sildenafil, thus accounting for approximately 36% of the overall pharmacological effect of sildenafil. Sildenafil is primarily cleared via hepatic microsomal isoenzymes CYP3A4 (major pathway) and CYP2C9 (minor pathway). The major circulating metabolite produced after N-demethylation of sildenafil is further metabolized by itself. The phosphodiesterase (PDE) selectivity of this metabolite is similar to that of sildenafil, with an in vitro inhibitory potency against phosphodiesterase type 5 (PDE-5) of approximately 50% that of the parent drug. The plasma concentration of this metabolite is approximately 40% of that of sildenafil, thus accounting for approximately 20% of the pharmacological effect of sildenafil. Pharmacokinetics were studied in mice, rats, rabbits, dogs, and humans after single intravenous and/or oral administration of sildenafil or ¹⁴C-sildenafil (Viagra), respectively. In all species, the five major metabolic pathways are piperazine N-demethylation, pyrazole N-demethylation, loss of two carbon segments on the piperazine ring (N,N'-deethylation), oxidation of the piperazine ring, and aliphatic hydroxylation. Other metabolites are produced by combinations of these pathways. Sildenafil is the major component detected in human plasma. After oral administration, the AUC(∞) of piperazine N-demethylated metabolites and piperazine N,N'-deethylated metabolites are 55% and 27% of the parent compound, respectively. PMID: 10219969
Sildenafil is primarily excreted in feces as metabolites. In healthy adults and patients with erectile dysfunction, approximately 80% of the oral dose is excreted in feces as metabolites, and 13% in urine. In feces, N-dealkylated, hydroxylated, N-demethylated, and N-dealkyl/demethylated metabolites of sildenafil account for approximately 22%, 13%, 3%, and 3% of total fecal excretion, respectively. In healthy individuals, sildenafil is primarily excreted in the urine as a hydroxylated metabolite, accounting for approximately 41% of total urinary excretion. Sprague Dawley rats (n=10 per group, 10 males and 10 females) were administered sildenafil at doses of 10, 45, or 200 mg/kg/day via gavage for one month. Plasma concentrations of sildenafil were higher in female rats than in male rats, while the concentration of the metabolite UK-103,320 was higher in male rats than in female rats. Therefore, female rats were primarily exposed to the parent drug, while male rats were exposed to almost equal amounts of both the drug and the metabolite. These data suggest that N-demethylation of sildenafil to UK-103,320 is an important biotransformation pathway for sildenafil in male rats. Concentrations of UK-103,340 were typically below the limit of detection (30 ng/mL). .../Excerpt from table/
Sildenafil appears to be completely metabolized in the liver to up to 16 metabolites, most of which account for only a small fraction of the dose; the parent drug is barely detectable in urine or feces after oral or intravenous administration. Sildenafil is primarily metabolized via hepatic cytochrome P-450 (CYP) microsomal isoenzymes 3A4 (major pathway) and 2C9 (minor pathway), and potent CYP3A4 inhibitors significantly reduce sildenafil clearance. The hepatic metabolism of sildenafil is complex, typically involving N,N-deethylation (ring-opening) or N-demethylation of the piperazine ring and aliphatic hydroxylation; the drug and its metabolites do not appear to undergo conjugation reactions. The N-demethylated metabolite is the major circulating metabolite, with phosphodiesterase selectivity similar to that of sildenafil, and approximately 50% of the PDE5 potency of the parent drug in vitro. The N-demethylated metabolite is further metabolized to the N-dealkylated (N,N-deethylated) metabolite. The drug also undergoes N-dealkylation and N-demethylation of the piperazine ring.

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Biological Half-Life
The terminal half-life of sildenafil is approximately 3 to 5 hours. After oral administration of sildenafil, plasma concentrations decrease in a biphasic manner, with a terminal elimination half-life of approximately 4 hours (range: 3–5 hours). High clearance in rodents is the main determinant of the short elimination half-life (0.4–1.3 hours), while moderate clearance in dogs and humans results in longer half-lives (6.1 hours and 3.7 hours, respectively).

Toxicity Overview
Identification and Uses: Sildenafil is a white to off-white crystalline powder available in film-coated tablets, oral suspensions, and injections. Sildenafil is a phosphodiesterase-5 (PDE-5) inhibitor. It is used to treat erectile dysfunction and pulmonary arterial hypertension (PAH) in adults to improve exercise capacity and delay clinical deterioration. Human Exposure and Toxicity: Generally, overdose of sildenafil may exacerbate common adverse reactions. In studies of single doses of up to 800 mg sildenafil in healthy subjects, the types of adverse events observed (e.g., decreased blood pressure, syncope, and prolonged erection) were similar to those observed at lower doses, but at an increased incidence. Serious adverse reactions have also been reported at therapeutic doses, including sudden hearing loss or impairment, sudden loss of vision in one or both eyes, and erections lasting longer than 4 hours or priapism (painful erections lasting longer than 6 hours). Post-marketing reports indicate a time-related correlation between sildenafil treatment for erectile dysfunction and serious cardiovascular, cerebrovascular, and vascular events, including myocardial infarction, sudden death, ventricular arrhythmias, cerebral hemorrhage, transient ischemic attack, hypertension, subarachnoid hemorrhage, and pulmonary hemorrhage. Most (but not all) of these patients had pre-existing cardiovascular risk factors. Therefore, it is impossible to determine whether these events are directly related to sildenafil, sexual activity, underlying cardiovascular disease, a combination of these factors, or other factors. Sildenafil is not recommended for children. In a long-term trial in children with pulmonary arterial hypertension (PAH), increased sildenafil doses were observed to lead to increased mortality. Pulmonary vasodilators (such as sildenafil) may significantly worsen cardiovascular conditions in patients with pulmonary venous occlusive disease. Sildenafil significantly enhances the vasodilatory effects of organic nitrates and nitrites. This drug did not show chromosome breakage in vitro in human lymphocyte assays. Animal studies: Death occurred in rats after oral administration of 1000 mg/kg and 500 mg/kg doses, and in mice after oral administration of 1000 mg/kg dose. Female rats were more affected than male rats. Acute sildenafil treatment stimulated testosterone production in adult male rats. No fertility impairment was observed in female rats administered up to 60 mg/kg daily for 36 consecutive days and in male rats for 102 consecutive days. However, in another study, male rats were given sildenafil citrate (0.06 mg/0.05 mL) by gavage and allowed to mate. Fertilization rate and embryo number were assessed after treatment. In mating of males given sildenafil citrate, the fertilization rate was significantly reduced on day 1 (approximately 33%). On days 2–4, the number of embryos developing in the treatment group was significantly lower than in the control group. The cleavage rate of these embryos also showed a decreasing trend, but this did not reach statistical significance. During organogenesis, rats and rabbits receiving doses up to 200 mg/kg/day showed no evidence of teratogenicity, embryotoxicity, or fetal toxicity. In another study, adult male rabbits were administered sildenafil up to 9 mg/kg/day for four consecutive weeks to investigate testicular histological changes caused by drug overdose. Abnormalities in the seminiferous tubule germinal epithelium included spermatocyte nuclear pyknosis, spermatocyte degeneration and shedding, spermatocyte giant cells, and spermatogenesis arrest. Furthermore, increased interstitial cells, tubular degeneration, and interstitial thickening were observed. These histological findings suggest that long-term overdose of sildenafil leads to significant morphological and histological changes in the testes, potentially leading to complete spermatogenesis arrest. Oral administration of sildenafil to rats and mice for up to two years showed no carcinogenicity. Sildenafil did not show mutagenicity in in vitro bacterial and Chinese hamster ovary cell assays. The drug also did not show chromosomal breakage in in vivo mouse micronucleus assays. Sildenafil is primarily cleared via the hepatic microsomal isoenzymes CYP3A4 (major pathway) and CYP2C9 (minor pathway). Its main circulating metabolite is the N-demethylated form of sildenafil, which itself is further metabolized. The phosphodiesterase (PDE) selectivity of this metabolite is similar to that of sildenafil, exhibiting approximately 50% of the in vitro inhibitory potency against phosphodiesterase type 5 (PDE-5) compared to the parent drug. The plasma concentration of this metabolite is approximately 40% of that of sildenafil, thus accounting for approximately 20% of the pharmacological action of sildenafil.

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Hepatotoxicity
At least five cases of acute liver injury have been reported in connection with sildenafil use, but no cases of acute liver failure have been reported. Due to the intermittent and sometimes undocumented use of sildenafil, the latency period in most reports is unclear, but appears to be between 1 and 8 weeks. Elevated serum enzymes vary in pattern from hepatocellular to cholestatic, and sometimes evolve from one type to another. The most compelling case was mild cholestatic or “mixed” hepatitis occurring within 1 to 3 months of starting sildenafil. No immune hypersensitivity features or autoantibodies were observed. Cases with acute onset and elevated serum transaminase levels following sildenafil use have been reported, exhibiting some characteristics of ischemic injury. In other cases, the injury pattern suggested the use of anabolic steroids. In two cases, re-exposure to sildenafil did not lead to relapse. Therefore, the hepatotoxicity of sildenafil is not entirely convincing, and even if present, should be extremely rare. Probability score: C (likely a rare cause of clinically significant liver injury). Effects during pregnancy and lactation

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◉ Overview of medication use during lactation
Limited data suggest that the amount of sildenafil and its active metabolites excreted in breast milk is very small. The amount ingested by the infant is far below the doses used to treat infants, and no adverse effects are expected on breastfed infants.
◉ Effects on breastfed infants
A 23-year-old woman with congenital heart disease and pulmonary hypertension received sildenafil and bosentan during pregnancy at an unknown dosage. She continued to take these medications and warfarin postpartum. Her baby was delivered by cesarean section at 30 weeks of gestation, weighing 1.41 kg. According to the author, she breastfed in the neonatal intensive care unit for 11 weeks with “good results,” but the baby died at 26 weeks of gestation from respiratory syncytial virus infection. [3]
A woman who was breastfeeding a 21-month-old baby was taking 20 mg sildenafil three times a day and 125 mg bosentan twice a day to treat pulmonary hypertension. These medications were started more than 6 months postpartum. The mother did not report any possible adverse reactions, serious health problems, or hospitalizations in the baby from birth to 651 days postpartum (the baby was partially breastfed). [2]
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Interactions
Sildenafil and other phosphodiesterase type 5 (PDE) inhibitors (e.g., tadalafil, vardenafil) can significantly enhance the vasodilatory effects of organic nitrates and nitrites (e.g., nitroglycerin, isosorbide dinitrate) (e.g., sildenafil can reduce systolic blood pressure by more than 25 mmHg), and may lead to life-threatening hypotension and/or hemodynamic disturbances. Nitrates and nitrites promote the production of cyclic guanosine monophosphate (cGMP) by stimulating guanylate cyclase, while phosphodiesterase type 5 (PDE 5) inhibitors (e.g., sildenafil, tadalafil, vardenafil) reduce cGMP degradation by inhibiting PDE 5, resulting in increased cGMP accumulation and producing more significant smooth muscle relaxation and vasodilation effects than PDE 5 inhibitors or nitrates/nitrites alone. This interaction can occur with any organic nitrate, nitrite, or nitric oxide donor (e.g., sodium nitroprusside), regardless of its primary hemodynamic site.

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Protein Binding
Sildenafil and its primary circulating metabolite, N-demethylated metabolite, are typically observed to bind to plasma proteins at an estimated rate of approximately 96%. However, protein binding of sildenafil has been determined to be independent of total drug concentration.

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In vitro cytotoxicity (References 2, 3):
- After treatment with sildenafil citrate (0.1–20 μM) for 48 hours, PASMC cell survival rate >90% (MTT method) [2]
- After treatment with sildenafil citrate (1–20 μM) for 24 hours, BV2 cell survival rate >85% (MTT method) [3]
- In vivo safety (References 3, 4):
- Newborn mice (P7) treated with sildenafil citrate (10 mg/kg, 3 days) showed no significant changes in body weight (6.1 ± 0.2 g vs. 6.2 ± 0.3 g, solvent control group) or serum biochemical indicators (ALT, AST, BUN) [3]
- SD rats treated with sildenafil citrate (20 mg/kg, 21 days) showed normal weight gain (305 ± 12 g vs. 310 ± 15 g, control group), and no histopathological abnormalities were observed in liver and kidney tissues [4]

[1]. The Selectivity and Potency of the New PDE5 Inhibitor TPN729MA. J Sex Med. 2013 Nov;10(11):2790-7.

[2]. Sildenafil potentiates the proliferative effect of porcine pulmonary artery smooth muscle cells induced by serotonin in vitro. Chin Med J (Engl). 2011 Sep;124(17):2733-40.

[3]. Sildenafil, a cyclic GMP phosphodiesterase inhibitor, induces microglial modulation after focal ischemia in the neonatal mouse brain. J Neuroinflammation. 2016 Apr 28;13(1):95.

[4]. The Effect of Sildenafil on Recuperation from Sciatic Nerve Injury in Rats. Balkan Med J. 2016 Mar;33(2):204-11.

Sildenafil citrate is the citrate salt of sildenafil. It is a vasodilator and an EC 3.1.4.35 (3',5'-cyclic guanosine monophosphate phosphodiesterase) inhibitor. It contains sildenafil. Sildenafil citrate is the citrate form of sildenafil, a highly bioavailable pyrazolopyrimidinone derivative with a structure related to zaprostrobin, possessing vasodilatory and potential anti-inflammatory activity. After oral administration, sildenafil selectively targets and inhibits cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), thereby inhibiting PDE5-mediated degradation of cGMP in smooth muscle and increasing cGMP availability. This leads to relaxation and elongation of the smooth muscle of the corpus cavernosum, resulting in vasodilation, increased blood flow, and prolonged penile erection. In pulmonary vascular smooth muscle, the increase in cGMP leads to smooth muscle relaxation, dilation of the pulmonary vascular bed, thereby relieving pulmonary hypertension and increasing pulmonary blood flow. In addition, sildenafil can reduce airway inflammation and mucus secretion. A phosphodiesterase type 5 inhibitor; a vasodilator and urinary tract medication used to treat erectile dysfunction and primary pulmonary hypertension. See also: Sildenafil (containing the active ingredient).

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Indications Adults: For the treatment of adult patients with pulmonary hypertension in WHO functional class II and III to improve exercise capacity. Proven effective in primary pulmonary hypertension and connective tissue disease-related pulmonary hypertension. Children: For the treatment of children aged 1 to 17 years with pulmonary hypertension. Proven to improve exercise capacity or pulmonary hemodynamics in primary pulmonary hypertension and pulmonary hypertension associated with congenital heart disease (see Section 5.1). Adults: For the treatment of adult patients with pulmonary hypertension in WHO functional class II and III to improve exercise capacity. Proven to improve exercise capacity in primary pulmonary hypertension and connective tissue disease-related pulmonary hypertension. Pediatric population: Treatment of children aged 1 to 17 years with pulmonary hypertension. It has been shown to be effective in improving exercise capacity or pulmonary hemodynamics in primary pulmonary hypertension and pulmonary hypertension associated with congenital heart disease. Mechanism of action: Sildenafil citrate inhibits PDE5, prevents cGMP hydrolysis, and increases intracellular cGMP levels. Elevated cGMP levels can activate protein kinase G (PKG), thereby regulating smooth muscle relaxation, inhibiting cell proliferation, and reducing the production of pro-inflammatory cytokines [1,2,3]
- Therapeutic potential:
- Pulmonary hypertension: Inhibition of 5-HT-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) suggests its potential for pulmonary artery remodeling [2]
- Cerebral ischemia: Reduction of microglial activation and infarct volume suggests its neuroprotective effect [3]
- Peripheral nerve injury: Promotion of axonal regeneration and functional recovery supports its application in neurorepair [4]
- Selectivity advantage (Reference 1): High selectivity for PDE5 (rather than PDE6/PDE11) minimizes off-target effects (e.g., visual impairment caused by PDE6 inhibition) [1]

C22H30N6O4S.C6H8O7

666.7

666.231

C, 50.44; H, 5.75; N, 12.61; O, 26.40; S, 4.81

171599-83-0

Sildenafil;139755-83-2;Sildenafil citrate-d8;1215071-03-6; 171599-83-0 (citrate); 252951-59-0 (nitrate)

135413523

White to off-white solid powder

1.447g/cm3

672.4ºC at 760 mmHg

187-189ºC

360.5ºC

0mmHg at 25°C

1.683

1.319

5

15

12

46

1070

0

S(C1C([H])=C([H])C(=C(C2=NC3C(C([H])([H])C([H])([H])C([H])([H])[H])=NN(C([H])([H])[H])C=3C(N2[H])=O)C=1[H])OC([H])([H])C([H])([H])[H])(N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])(=O)=O.O([H])C(C(=O)O[H])(C([H])([H])C(=O)O[H])C([H])([H])C(=O)O[H]

DEIYFTQMQPDXOT-UHFFFAOYSA-N

InChI=1S/C22H30N6O4S.C6H8O7/c1-5-7-17-19-20(27(4)25-17)22(29)24-21(23-19)16-14-15(8-9-18(16)32-6-2)33(30,31)28-12-10-26(3)11-13-28;7-3(8)1-6(13,5(11)12)2-4(9)10/h8-9,14H,5-7,10-13H2,1-4H3,(H,23,24,29);13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)

5-(2-ethoxy-5-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1,4-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one 2-hydroxypropane-1,2,3-tricarboxylate

UK-92480 citrate; UK 92480 citrate; Sildenafil Citrate; UK92480 citrate; UK 92480-10; 171599-83-0; Revatio; VIAGRA; Caverta; Sildenafil (citrate); Sildenafil citrate [USAN]; LIQREV; UK-92,480-10. Trade names: Revatio.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 5 mg/mL (7.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (7.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (7.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)