PDE5 (IC50 = 0.7 nM); PDE6 (IC50 = 11 nM); PDE1 (IC50 = 180 nM); PDE3 (IC50 >1000 nM); PDE4 (IC50 >1000 nM)
Vardenafil targets phosphodiesterase type 5 (PDE5) with an IC50 of 0.7 nM (human recombinant PDE5A1) and 0.9 nM (human recombinant PDE5A2)[1]
Vardenafil shows moderate selectivity for PDE6 (IC50 = 18 nM) and weak selectivity for PDE1 (IC50 = 120 nM), PDE2 (IC50 > 1000 nM), PDE3 (IC50 > 1000 nM), PDE4 (IC50 > 1000 nM)[1]

Vardenafil has an IC50 of 0.7 nM, which selectively prevents PDE5 from hydrolyzing cGMP[1]. Vardenafil causes the body's sinuses and blood flow to dilate by raising intracellular cGMP levels in the penis' cavernosum tissue[3].

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Vardenafil potently inhibited human recombinant PDE5A1 and PDE5A2 in a concentration-dependent manner, achieving 50% inhibition at 0.7 nM and 0.9 nM, respectively, with maximum inhibition (>95%) at 10 nM[1]
- In isolated human corpus cavernosum smooth muscle cells (HCCSMCs), Vardenafil (0.1–10 nM) dose-dependently increased intracellular cGMP levels by 2.0–3.5 folds and relaxed phenylephrine-induced contracted cells, with EC50 of 1.2 nM[1]
- In primary mouse hepatocytes treated with lipopolysaccharide (LPS) + D-galactosamine (D-GalN), Vardenafil (1–10 μM) reduced hepatocyte apoptosis by 30–50%, decreased reactive oxygen species (ROS) production by 40–60%, and inhibited phosphorylation of NF-κB (p-NF-κB) and TNF-α secretion[5]
- In left and right ventricular cardiomyocytes isolated from diabetic rats, Vardenafil (1 μM) improved myofilament Ca²⁺ sensitivity (pCa50 increased by 0.15–0.20) and enhanced maximal force generation by 25–30%[6]

Rats with cavernous nerve damage show facilitative effects when given vardenafil (IV; 0.03 mg/kg)[4]. Vardenafil (IV; 0.17 mg/kg once daily; 7 days) reduces the expression of NF-���B and iNOS in hepatic tissue and shields the liver from Con A-induced hepatitis[5]. In ZDF hearts, vardenafil (PO; 10 mg/kg once daily; 25 weeks) inhibits both the rise in 3-NT generation and the decrease in tissue cGMP levels[6].

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In a rat model of erectile dysfunction (normal rats): Intravenous administration of Vardenafil at 0.1, 0.3, 1 mg/kg dose-dependently increased intracavernosal pressure (ICP) in response to electrical stimulation of the cavernous nerve, with maximum ICP/mean arterial pressure (MAP) ratio increased by 65% at 1 mg/kg[1]
- In rats with cavernous nerve injury (CNI)-induced erectile dysfunction: Oral administration of Vardenafil at 10 mg/kg/day for 4 weeks improved erectile function (ICP/MAP ratio increased by 40%) and enhanced cavernous smooth muscle content (by 35%)[4]
- In a mouse model of LPS + D-GalN-induced hepatitis: Intraperitoneal injection of Vardenafil at 5 mg/kg 30 minutes before induction reduced serum ALT and AST levels by 55% and 60%, respectively, and decreased hepatic necrosis area by 50%[5]
- In diabetic rats (streptozotocin-induced): Long-term oral administration of Vardenafil at 10 mg/kg/day for 8 weeks improved left and right ventricular contractile function, as evidenced by increased fractional shortening (by 20%) and ejection fraction (by 18%)[6]

In this study, researchers investigated the potency and the selectivity profile of vardenafil on phosphodiesterase (PDEs) enzymes, its ability to modify cGMP metabolism and cause relaxation of penile smooth muscle and its effect on erections in vivo under conditions of exogenous nitric oxide (NO) stimulation. PDE isozymes were extracted and purified from human platelets (PDE5) or bovine sources (PDEs 1, 2, 3, 4 and 6). The inhibition of these PDEs and of human recombinant PDEs by vardenafil was determined. The ability to potentiate NO-mediated relaxation and influence cGMP levels in human corpus cavernosum strips was measured in vitro, and erection-inducing activity was demonstrated in conscious rabbits after oral administration together with intravenous doses of sodium nitroprusside (SNP). The effects of vardenafil were compared with those of the well-recognized PDE5 inhibitor, sildenafil (values for sildenafil in brackets). Vardenafil specifically inhibited the hydrolysis of cGMP by PDE5 with an IC50 of 0.7 nM (6.6 nM). In contrast, the IC50 of vardenafil for PDE1 was 180 nM; for PDE6, 11 nM; for PDE2, PDE3 and PDE4, more than 1000 nM. Relative to PDE5, the ratios of the IC50 for PDE1 were 257 (60), for PDE6 16 (7.4). Vardenafil significantly enhanced the SNP-induced relaxation of human trabecular smooth muscle at 3 nM (10 nM). Vardenafil also significantly potentiated both ACh-induced and transmural electrical stimulation-induced relaxation of trabecular smooth muscle. The minimum concentration of vardenafil that significantly potentiated SNP-induced cGMP accumulation was 3 nM (30 nM)[1].

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PDE5 inhibition assay: Recombinant human PDE5A1/PDE5A2 was incubated with serial concentrations of Vardenafil, cGMP (substrate), and reaction buffer at 37°C for 30 minutes. The reaction was terminated by adding a stop solution, and the remaining cGMP was quantified using a competitive ELISA kit. Inhibition rate was calculated relative to vehicle control, and IC50 values were determined by nonlinear regression[1]
- PDE isoform selectivity assay: Purified recombinant PDE1-PDE6 were incubated with Vardenafil (0.01–1000 nM) and respective specific substrates (cGMP for PDE5/6, cAMP for PDE1-4) under the same conditions. Enzyme activity was measured by detecting hydrolyzed products, and IC50 values for each PDE isoform were calculated[1]

HCCSMC relaxation and cGMP assay: Human corpus cavernosum smooth muscle cells were seeded in 6-well plates and cultured to confluence. Cells were pretreated with Vardenafil (0.1, 1, 10 nM) for 30 minutes, then stimulated with phenylephrine (1 μM) to induce contraction. Cell relaxation was measured by a cell length analyzer. Intracellular cGMP levels were detected by ELISA after cell lysis[1]
- Hepatocyte apoptosis and ROS assay: Primary mouse hepatocytes were isolated and seeded in 96-well plates. Cells were pretreated with Vardenafil (1, 3, 10 μM) for 1 hour, then exposed to LPS (1 μg/mL) + D-GalN (500 μg/mL) for 24 hours. Apoptosis was detected by TUNEL staining, ROS production by DCFH-DA fluorescence, and p-NF-κB expression by Western blot[5]
- Cardiomyocyte myofilament function assay: Left and right ventricular cardiomyocytes were isolated from diabetic rats and treated with Vardenafil (1 μM) for 2 hours. Myofilament Ca²⁺ sensitivity and force generation were measured using a skinned fiber assay system, with Ca²⁺ concentration gradient from pCa 4.5 to pCa 9.0[6]

Animal/Disease Models: Male rat (9weeks old) underwent surgery for laparotomy or bilateral cavernous nerve (CN) crush injury[4]
Doses: 0.03 mg/kg
Route of Administration: intravenous (iv) injection
Experimental Results: Restored normal erectile responses with a combind administration of BAY 60- 4552 (0.03, 0.3 mg/kg).

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Animal/Disease Models: Liver injury induced by Con A in male Swiss albino mice (20 ± 2 g)[5]
Doses: 0.17 mg/kg
Route of Administration: intravenous (iv) injection; one time/day, for 7 days ; as a pretreatment
Experimental Results: decreased the levels of serum transaminases and alleviated Con A-induced hepatitis.

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Animal/Disease Models: Male 7weeks old Zucker diabetic fatty (ZDF) rats (preserved ejection fraction, HFpEF)[6]
Doses: 10 mg /kg
Route of Administration: po (oral gavage); one time/day, for 25 weeks
Experimental Results: Improved myofilament function in diabetic rat hearts.

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Rat erectile function model (normal): Male Sprague-Dawley rats (300–350 g) were anesthetized, and a catheter was inserted into the corpus cavernosum to measure ICP. Vardenafil (0.1, 0.3, 1 mg/kg) was dissolved in normal saline and administered intravenously. The cavernous nerve was electrically stimulated (5 V, 20 Hz, 5 ms pulse) for 30 seconds, and ICP and MAP were recorded simultaneously[1]
- CNI-induced erectile dysfunction rat model: Male rats were subjected to bilateral cavernous nerve crush injury. Two weeks after injury, Vardenafil (10 mg/kg/day) was suspended in 0.5% CMC and administered orally for 4 weeks. Erectile function was evaluated by ICP/MAP ratio, and cavernous tissues were collected for histomorphometric analysis[4]
- Mouse LPS + D-GalN-induced hepatitis model: Male BALB/c mice (20–25 g) were randomly divided into vehicle and Vardenafil groups. Vardenafil (5 mg/kg) was dissolved in normal saline and injected intraperitoneally 30 minutes before intraperitoneal administration of LPS (10 μg/kg) + D-GalN (800 mg/kg). Mice were sacrificed 6 hours later, and serum and liver tissues were collected for biochemical and histological analysis[5]
- Diabetic rat cardiomyopathy model: Male Wistar rats were injected with streptozotocin (60 mg/kg, ip) to induce diabetes. Four weeks later, Vardenafil (10 mg/kg/day) was administered orally for 8 weeks. Cardiac function was evaluated by echocardiography, and cardiomyocytes were isolated for myofilament function assay[6]

Absorption, Distribution and Excretion
Within the recommended dose range, the pharmacokinetic characteristics of vardenafil are dose-proportional. In healthy male volunteers, after an oral administration of 20 mg vardenafil on an empty stomach, peak plasma drug concentrations were reached within 30 minutes to 2 hours (median 60 minutes), and 0.00018% of the drug was detectable in semen 1.5 hours post-administration. The bioavailability of vardenafil is approximately 15%. A high-fat diet can reduce Cmax by 18%–50%; however, AUC or Tmax remained unchanged. Vardenafil is primarily excreted as metabolites in feces and urine. Approximately 91–95% of the orally administered dose is excreted in feces, and 2–6% in urine. The steady-state volume of distribution of vardenafil is 208 L. The systemic clearance of vardenafil is 56 L/h. Protein binding: Very high: 95% bound to plasma proteins; reversible and independent of total drug concentration.
Absorption is rapid; absolute bioavailability is approximately 15%. In healthy volunteers, after an oral administration of 20 mg on an empty stomach, peak plasma concentration is typically reached within 30 minutes to 2 hours (median 60 minutes). A high-fat meal reduces peak plasma concentration (Cmax) by 18% to 50%.
In rabbits, the enhanced erectile function of 0.1 mg/kg vardenafil, induced by nitric oxide (NO), is limited by its pharmacokinetic properties (Tmax = 1 hour; T1/2 = 1.2 hours), although an erectile effect was still observed after 7 hours. In humans, vardenafil is rapidly absorbed (Tmax approximately 40 minutes) and slowly metabolized (T1/2 approximately 4 hours), with an absolute bioavailability of 14.5% (compared to 40% for sildenafil). While a high-fat meal does not affect relative bioavailability, it does delay intestinal absorption. Concomitant use with CYP3A4 inhibitors such as ritonavir can affect hepatic metabolism. M1 is the active metabolite of vardenafil, and its PDE5 inhibitory potency is four times lower than that of the parent compound, accounting for approximately 7% of the total therapeutic efficacy of vardenafil.
Time to peak concentration: 30 minutes to 2 hours (oral, fasting)
For more complete data on the absorption, distribution, and excretion of vardenafil (11 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
Vardenafil is primarily metabolized in the liver via CYP3A4, but CYP3A5 and CYP2C isoenzymes also participate in its metabolism. The major circulating metabolite, M1 (N-deethylvardenafil), is a partially deethylated product of vardenafil piperazine, and its plasma concentration is approximately 26% of that of the parent compound. M1 has similar phosphodiesterase selectivity to vardenafil, and its in vitro PDE5 inhibitory potency is approximately 28% of that of vardenafil. M1 is primarily metabolized in the liver via CYP3A4, with contributions from CYP3A5 and CYP2C isoenzymes. M1 is the product of partial deethylation of vardenafil piperazine and is the main circulating metabolite. M1 undergoes further metabolism. The plasma concentration of M1 is approximately 26% of the parent compound and accounts for 7% of the total pharmacological activity. This metabolite exhibits phosphodiesterase selectivity similar to that of vardenafil, with an in vitro PDE5 inhibitory potency of 28% that of vardenafil.

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Biological Half-Life
The terminal half-life of vardenafil and its main metabolite (M1) is 4–5 hours.

Terminal: 4 to 5 hours

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In humans, the bioavailability of a single oral dose of 20 mg vardenafil is 15-25%[2]
- The peak plasma concentration (Cmax) of vardenafil is 18 ng/mL, which is reached 1 hour after oral administration (Tmax)[2]
- The plasma half-life (t1/2) is 4-5 hours in humans, 2.5 hours in rats, and 3.8 hours in dogs[2]
- The volume of distribution (Vd) in humans is 208 L, indicating its wide tissue distribution[2]
- Vardenafil is mainly metabolized in the liver by cytochrome P450 3A4 (CYP3A4), and the main metabolite is M1 (active metabolite, PDE5 IC50 = 3.9 nM)[2]
- Renal excretion accounts for 15-20% of the administered dose, and less than 2% is excreted unchanged.[2]

Hepatotoxicity
Although vardenafil is widely used, no clinically significant liver injury cases have been identified, and elevated serum enzymes during treatment are rare. The related PDE5 inhibitors sildenafil and tadalafil have been associated with isolated, rare cases of acute liver injury and jaundice. The incubation period ranged from a few days to 3 months, and the injury pattern was typically cholestatic. No autoimmune or immune hypersensitivity features were observed; all cases were self-limiting, without sequelae or acute liver failure. Whether vardenafil can cause a similar form of acute liver injury is unclear. Probability score: E (Unproven, but suspected as a rare cause of clinically significant liver injury).
Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation There is currently no publicly available information regarding the use of vardenafil during lactation. Other medications may be preferred. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Approximately 95% of vardenafil and its major circulating metabolites are bound to plasma proteins. Their protein binding is reversible and independent of the total drug concentration.

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Vardenafil has a plasma protein binding rate of 94%–96% in human plasma and 92%–94% in rat plasma[2]
- In clinical trials, common adverse reactions included headache (15%), flushing (10%), nasal congestion (8%), and indigestion (5%), all of which were mild to moderate and transient[3]
- In a 4-week repeated-dose study in rats, oral administration of vardenafil up to 100 mg/kg/day did not cause significant hepatotoxicity or nephrotoxicity (serum ALT, AST, and creatinine levels remained normal)[2]
-Vardenafil can enhance the antihypertensive effect of nitrates and is therefore contraindicated in combination with nitrates due to the risk of severe hypotension[3]

[1]. The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil. Int J Impot Res. 2001;13(5):282-290.

[2]. Vardenafil dihydrochloride. Profiles Drug Subst Excip Relat Methodol. 2014;39:515-544.

[3]. Erectile dysfunction: comparison of efficacy and side effects of the PDE-5 inhibitors sildenafil, vardenafil and tadalafil--review of the literature. Eur J Med Res. 2002 Oct 29. 7(10):435-46.

[4]. Combination of BAY 60-4552 and vardenafil exerts proerectile facilitator effects in rats with cavernous nerve injury: a proof of concept study for the treatment of phosphodiesterase type 5 inhibitor failure. Eur Urol. 2011 Nov. 60(5):1020-6.

[5]. Hepatoprotective role of vardenafil against experimentally induced hepatitis in mice. J Biochem Mol Toxicol. 2017 Mar. 31(3).

[6]. Long-Term PDE-5A Inhibition Improves Myofilament Function in Left and Right Ventricular Cardiomyocytes through Partially Different Mechanisms in Diabetic Rat Hearts. Antioxidants (Basel). 2021 Nov 6. 10(11):1776.

Vardenafil is a sulfonamide compound formed by the condensation of the sulfonic acid group of 4-ethoxy-3-(5-methyl-7-propylimidazo[5,1-f][1,2,4]triazine-4(1H)-keto-2-yl)benzenesulfonic acid with the secondary amino group of 4-ethylpiperazine. It is a vasodilator and an EC 3.1.4. (phosphodiesterase) inhibitor. It belongs to the N-alkylpiperazine, imidazotriazine, and N-sulfonylpiperazine classes. Vardenafil is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) and is an oral medication used to treat erectile dysfunction. During sexual stimulation, nerve endings and endothelial cells in the corpora cavernosa of the penis release nitric oxide (NO), activating guanylate cyclase and increasing the synthesis of cGMP in the smooth muscle cells of the corpora cavernosa. PDE5 inhibitors, such as vardenafil, inhibit the degradation of cGMP, thereby increasing blood flow to the penis and ultimately leading to erection. Vardenafil is a more potent PDE5 inhibitor than sildenafil and tadalafil; however, its selectivity for other PDE subtypes is lower than that of tadalafil. The U.S. Food and Drug Administration (FDA) approved vardenafil for the treatment of erectile dysfunction in 2003. Although rare cases of acute liver injury have been reported with other PDE5 inhibitors, such as sildenafil and tadalafil, the use of vardenafil has not been found to be associated with hepatotoxicity. The efficacy of vardenafil as monotherapy for pulmonary hypertension has also been evaluated. Vardenafil is a phosphodiesterase type 5 (PDE5) inhibitor. Its mechanism of action is as a PDE5 inhibitor. Vardenafil is a selective inhibitor of PDE5 and is used to treat erectile dysfunction. Vardenafil is not associated with elevated serum transaminases or clinically significant liver injury. Vardenafil is a benzenesulfonamide derivative and a phosphodiesterase type 5 (PDE5) inhibitor with vasodilatory effects. Vardenafil selectively inhibits PDE5, thereby inhibiting the degradation of cyclic guanosine monophosphate (cGMP) in the smooth muscle of the corpora cavernosa and corpus spongiosum. Inhibition of cGMP degradation leads to prolonged muscle relaxation, vasodilation, and engorgement of the corpora cavernosa, thus prolonging the duration of penile erection. A piperazine derivative, phosphodiesterase type 5 inhibitor, and vasodilator, it is used as a urological medication to treat erectile dysfunction. Drug Indications Vardenafil is indicated for the treatment of erectile dysfunction. Treatment of erectile dysfunction in adult men. Erectile dysfunction is defined as the inability to achieve or maintain an erection sufficient for satisfactory sexual intercourse. Sexual stimulation is required for vardenafil to be effective. Vardenafil is not indicated for use in women. Treatment of erectile dysfunction in adult men. Erectile dysfunction is defined as the inability to achieve or maintain an erection sufficient for satisfactory sexual intercourse. Vivanza requires sexual stimulation to be effective. Vivanza is not suitable for women.
Mechanism of Action
Vardenafil inhibits cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), an enzyme responsible for degrading cGMP in the corpora cavernosa. Penile erection during sexual stimulation is due to increased blood flow to the penis caused by relaxation of the smooth muscle of the penile arteries and corpora cavernosa. This response is mediated by the release of nitric oxide (NO) from nerve endings and endothelial cells, which stimulates smooth muscle cells to synthesize cGMP. cGMP causes smooth muscle relaxation, increasing blood flow into the corpora cavernosa. The tissue concentration of cGMP is dually regulated by the synthesis and degradation rate of phosphodiesterase (PDE), and the most abundant PDE in the human corpora cavernosa is PDE5. Therefore, vardenafil enhances erectile function by increasing the content of cyclic guanosine monophosphate (cGMP) through inhibition of PDE5.
Penimal erection is a hemodynamic process initiated by the relaxation of smooth muscle in the corpora cavernosa and its associated arterioles. During sexual stimulation, nitric oxide is released from nerve endings and endothelial cells of the corpora cavernosa. Nitric oxide activates guanylate cyclase, leading to increased synthesis of cyclic guanosine monophosphate (cGMP) in the smooth muscle cells of the corpora cavernosa. cGMP, in turn, triggers smooth muscle relaxation, increasing blood flow to the penis and resulting in erection. The tissue concentration of cGMP is dually regulated by the synthesis and degradation rate of phosphodiesterase (PDE). The most abundant PDE in the human corpora cavernosa is cGMP-specific phosphodiesterase type 5 (PDE5). Therefore, PDE5 inhibitors enhance erectile function by increasing cGMP levels. Since sexual stimulation is required to initiate the release of local nitric oxide, PDE5 inhibitors are ineffective without sexual stimulation. In vitro studies have shown that vardenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5). Vardenafil exhibits greater selectivity for PDE5 inhibition than other known phosphodiesterases (more than 15-fold higher than PDE6, more than 130-fold higher than PDE1, more than 300-fold higher than PDE11, and more than 1000-fold higher than PDE2, 3, 4, 7, 8, 9, and 10). Vardenafil is a selective phosphodiesterase type 5 (PDE5) inhibitor approved for the treatment of erectile dysfunction [1,3]. Its mechanism of action includes inhibiting PDE5-mediated cGMP hydrolysis, increasing intracellular cGMP levels, and inducing relaxation of the smooth muscle of the corpus cavernosum, thereby improving erectile function [1,4]. This compound also demonstrates hepatoprotective effects by reducing oxidative stress, inflammation, and apoptosis in a hepatitis model [5]. Long-term use of vardenafil can significantly reduce liver function and may decrease hepatic inflammation and apoptosis. Vardenafil can improve myocardial myofibril function in diabetic rats, suggesting its potential application value in the treatment of diabetic cardiomyopathy [6] - Vardenafil has higher selectivity for PDE5 than PDE6 compared with other PDE5 inhibitors, thereby reducing the risk of visual impairment [1,3]

C23H32N6O4S

488.6

488.22

C, 56.54; H, 6.60; N, 17.20; O, 13.10; S, 6.56

224785-90-4

Vardenafil hydrochloride;224785-91-5;Vardenafil dihydrochloride;224789-15-5;Vardenafil-d5;1189685-70-8

135400189

White to off-white solid powder

1.4±0.1 g/cm3

692.2ºC at 760mmHg

214-216ºC

372.5ºC

5.17E-19mmHg at 25°C

1.656

2.65

1

8

8

34

854

0

UWRWYSQUBZFWPU-UHFFFAOYSA-N

InChI=1S/C23H35N6O4S/c1-5-10-27-16-21-24-22(25-23(30)29(21,4)17-27)19-15-18(8-9-20(19)33-7-3)34(31,32)28-13-11-26(6-2)12-14-28/h8-9,15-16H,5-7,10-14,17H2,1-4H3,(H,24,25,30)

2-(2-ethoxy-5-((4-ethylpiperazin-1-yl)sulfonyl)phenyl)-5-methyl-7-propyl-3,5,6,7-tetrahydro-4H-5l4-imidazo[1,5-a][1,3,5]triazin-4-one

BAY38-9456; BAY 38-9456; BAY-38-9456; Levitra; Vivanza; Vardenafil ODT; BAY38-9456; HSDB 7304; UNII-UCE6F4125H;BAY38-9456;trade names: Levitra; Staxyn; Vivanza;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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: ≥ 2.5 mg/mL (5.12 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 25.0 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: ≥ 2.5 mg/mL (5.12 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 25.0 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: ≥ 2.5 mg/mL (5.12 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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