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Vardenafil

Manufactured by Merck Group
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Sourced in Germany, United States, Canada, Sao Tome and Principe, United Kingdom, Italy

Vardenafil is a pharmaceutical compound developed by Merck Group. It functions as a phosphodiesterase type 5 (PDE5) inhibitor, which aids in the regulation of blood flow. The core function of Vardenafil is to facilitate the management of specific medical conditions, as determined by licensed healthcare professionals.

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Lab products found in correlation

Vardenafil, by GE Healthcare (2 mentions) Superdex 200 10 300 gl column, by GE Healthcare (2 mentions) Rolipram, by Merck Group (1 mentions) Egm 2 endothelial cell growth medium 2 bulletkit, by Lonza (1 mentions) Adenosine 3 5 cyclic monophosphate sodium salt monohydrate, by Merck Group (1 mentions) Guanosine 3 5 cyclic monophosphate sodium salt, by Merck Group (1 mentions) Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions) 3 isobutyl 1 methylxanthine, by Merck Group (1 mentions) Tris base, by Merck Group (1 mentions) Polybrene, by Merck Group (1 mentions) Smooth muscle cell growth supplement, by ScienCell (1 mentions) Vardenafil, by Selleck Chemicals (1 mentions) Anti s6k, by Cell Signaling Technology (1 mentions) Anti phospho akt ser473 9271, by Cell Signaling Technology (1 mentions) Fibronectin, by Merck Group (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti phospho s6k thr389, by Cell Signaling Technology (1 mentions) Nintedanib, by Cayman Chemical (1 mentions) Anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) P smad3, by Cell Signaling Technology (1 mentions)

10 protocols using vardenafil

1

Evaluating PDE Inhibitors for Brain Effects

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We administered two selective PDE inhibitors: PDE5 inhibitor vardenafil (kindly donated by BAYER, Wuppertal, Germany) and PDE4 inhibitor rolipram (Sigma–Aldrich, Zwijndrecht, the Netherlands). Both inhibitors were previously shown to cross the blood–brain barrier (Akkerman et al., 2016 ). Both PDE inhibitors were dissolved in the same vehicle (98% methyl cellulose tylose solution (0.5%) and 2% Tween80) and were administered in a volume of 4 mL/kg. The drugs were given i.p. at a dose of 0.3 mg/kg for vardenafil and 0.03 mg/kg for rolipram. Dosages, injection volumes and time of injection are based on extensive previous experience of the lab with the current drugs (Bollen et al., 2014 ; Izquierdo et al., 2002 (link); Rutten et al., 2007 (link)). The solutions were prepared freshly each testing day.
Argyrousi E.K., Heckman P.R., van Hagen B.T., Muysers H., van Goethem N.P, & Prickaerts J. (2019). Pro-cognitive effect of upregulating cyclic guanosine monophosphate signalling during memory acquisition or early consolidation is mediated by increased AMPA receptor trafficking. Journal of Psychopharmacology (Oxford, England), 34(1), 103-114.
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2

GPCR-Mediated cGMP Signaling Assay

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1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), 3-isobutyl-1-methylxanthine (IBMX), adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, C-type natriuretic peptide (CNP), diethylamine NONOate diethylammonium salt (DEA), forskolin, guanosine 3′,5′-cyclic monophosphate sodium salt, Polybrene, Tris base, and vardenafil, were from Sigma-Aldrich (Burlington, MA, USA). Dulbecco’s modified Eagle medium (DMEM), Medium 199 (M199), L-glutamine, and penicillin–streptomycin (P/S) were from Gibco (Waltham, MA, USA). Foetal bovine serum (FBS) was from Scientifix (Melbourne, VIC, Australia). EGM-2 endothelial cell growth medium-2 BulletKit was from Lonza (Basel, Switzerland). Smooth muscle cell growth supplement was from ScienCell (Carlsbad, CA, USA). Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) was from ChemSupply (Adelaide, SA, Australia). Coelenterazine h was from Nanolight (Pinetop, AZ, USA).
Valkovic A.L., Kocan M., Hoare B., Marshall S., Scott D.J, & Bathgate R.A. (2022). A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells. International Journal of Molecular Sciences, 23(3), 1908.
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3

Purification of Gα-1D4 Complex with PDE6

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1D4-tagged recombinant GαT·GTP was washed with buffer containing 20 mM Tris pH 8.0 and 5 mM MgCl2 on a 10kD MWCO concentrator to remove DTT and then mixed with the 1D4 antibody (University of British Columbia, CA) in a 7:1 molar ratio and incubated on ice for 30 min. The mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl2 to purify the 2:1 GαT·GTP-1D4 complex. PDE6 was washed with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl2 on a 100kD MWCO concentrator to remove DTT and mixed with an equal molar amount of the purified 2:1 GαT·GTP-1D4 complex together with 10 μM of vardenafil (Sigma-Aldrich). The mixture was incubated on ice for 30 min and purified by gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, 5 mM MgCl2 and 1 μM vardenafil. Peak fractions were pooled and concentrated with a 100kD MWCO concentrator to ~10 mg/mL.
Gao Y., Eskici G., Ramachandran S., Poitevin F., Seven A.B., Panova O., Skiniotis G, & Cerione R.A. (2020). Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6. Molecular cell, 80(2), 237-245.e4.
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4

Fibrosis Signaling Pathway Protocols

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PDE5-I vardenafil was obtained from Sequoia Research Products Ltd. (vardenafil citrate), Sigma Aldrich, or Selleckchem (vardenafil HCl). Nintedanib was obtained from Cayman Chemical. TGF-β1 was obtained from R&D Systems. The antibodies used in this study were as follows: fibronectin (Sigma Aldrich, F3648, St. Louis, MO, USA), CTGF (Santa Cruz Biotechnology, sc-14939, Dallas, TX, USA), αSMA (Sigma, A2547), p-SMAD3 (Cell Signaling 95205, Danvers, MA, USA), total SMAD3 (Abcam AB 28379), and GAPDH (Millipore MAB374). The secondary antibodies used for fibronectin ELISA included anti-rabbit IgG peroxidase (Sigma Aldrich, A0545) and anti-rabbit IgG-HRP (SantaCruz Biotechnology sc-2004). Additional antibodies for TGF-β1 signaling included the following: anti-mouse serpin E1/PAI-1 antibody (AF3828; R&D systems), anti-CTGF (sc-14939; SCBT), anti-αSMA (A2547; Sigma), anti-fibronectin (F3648, Sigma), anti-GAPDH (AB2302; Millipore, Burlington, MA, USA), anti-phospho-SMAD3 and anti-phospho-SMAD2 antibodies generated in our laboratory, anti-SMAD2 (ab63576; Abcam, Cambridge, UK), anti-SMAD3 (ab28379; Abcam), anti-phospho-AKT (Ser473; 9271, Cell Signaling), anti-phospho-AKT (T308; 4056, Cell Signaling), anti-phospho S6K (Thr389, 9234, Cell Signaling), anti-Akt (9272, Cell Signaling), and anti-S6K (9202, Cell Signaling).
Bourne MH J.r., Kottom T.J., Hebrink D.M., Choudhury M., Leof E.B, & Limper A.H. (2021). Vardenafil Activity in Lung Fibrosis and In Vitro Synergy with Nintedanib. Cells, 10(12), 3502.
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5

Detecting ABCB1 Expression and Function

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To detect the percentage of ABCB1 expressing cells, 300,000-500,000 cells were incubated with 4E3 (anti-ABCB1; 1:20; Abcam) for 30 minutes at 37°C in fluorescent activated cell sorting (FACS) buffer (high glucose DMEM plus 5% BSA). The cells were then washed twice with FACS buffer and incubated with Alexa Fluor® 647 goat anti-mouse IgG (1: 1000; Invitrogen) for 30 minutes on ice in the dark. As a control, cells were stained with secondary Ab alone (Alexa Fluor 647; Invitrogen). Any unbound Ab was removed by washing the labelled cells twice with FACS buffer. To investigate ABCB1 function, cells were incubated with Rhodamine 123 (Rh123, Sigma; ABCB1 flourescent substrate), with and without ABCB1 inhibitors verapamil (VPL, Sigma), and vardenafil (TRC-Canada) at concentrations given in figure legends. Fluorescence was detected on a Cytomics FC500 flow cytometer (Beckman Coulter) and analysed using WinMDI version 2.8. Data presented are mean ± SEM of three independent experiments.
Othman R.T., Kimishi I., Bradshaw T.D., Storer L.C., Korshunov A., Pfister S.M., Grundy R.G., Kerr I.D, & Coyle B. (2014). Overcoming multiple drug resistance mechanisms in medulloblastoma. Acta Neuropathologica Communications, 2, 57.
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6

Preparation and Storage of Enterotoxins

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Synthetic heat-stable enterotoxin (BA Chem, Bubendorf, Switzerland) was resuspended in 100 mM acetic acid, 0.1% BSA, and frozen in single-use aliquots. LT and LT-E112K were reconstituted in water and stored at 4°C. IBMX, MK-571, vardenafil, forskolin, and buffer components were acquired from Sigma (St. Louis, Missouri). Tissue culture media was obtained from ThermoFisher Scientific (Waltham, Massachusetts)
Foulke-Abel J., Yu H., Sunuwar L., Lin R., Fleckenstein J.M., Kaper J.B, & Donowitz M. (2020). Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection. Gut Microbes, 12(1), 1752125.
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7

Lipid Bilayer Characterization Protocols

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Nonactin, phloretin, vardenafil, RH 421, tetracaine hydrochloride, methyl-β-cyclodextrin, KCl, HEPES, pentane, ethanol, DMSO, sorbitol, and polymyxin B (PMB) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, United Kingdom). Solutions of 0.1 M KCl were buffered using HEPES-KOH at pH 7.4. The 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DOPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1’,3’-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (sodium salt) (TOCL), 1,2-diphytanoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DPhPG), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), Di[3-deoxy-D-manno-octulosonyl]-lipid A (ammonium salt) (Kdo2-Lipid A), detoxified lipid A from Salmonella minnesota R595 (Lipid A), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC), oleic acid, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine (Rh-DPPE) lipids were obtained from Avanti Polar Lipids®. The chemical structures of the lipids used in the study are presented in Figure 1.
All experiments were performed at room temperature (25 °C).
Zakharova A.A., Efimova S.S, & Ostroumova O.S. (2022). Lipid Microenvironment Modulates the Pore-Forming Ability of Polymyxin B. Antibiotics, 11(10), 1445.
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8

High-Throughput Compound Screening for Drug Discovery

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Two collections of compounds assembled at the NCATS were screened A collection of 44,420 diverse small molecule drugs; and the Mechanism Interrogation PlatE 4.0 collection of 386 pharmacologically active small molecules (22 (link)). The active compounds were re-purchased for repeat and secondary assays. Milciclib (catalog #PHA-848125), MK-0752 (#HY-10974) and PP-121 (#HY-10372) were purchased from MedChem Express. Ouabain (#1076) and AMG-51 (#SYN-111) were purchased from Fisher Scientific. Vardenafil (#Y0001647) and Oridonin (#O9639) were purchased from Sigma-Aldrich. Peruvoside (# P227570) was purchased from Toronto Research Chemicals. NCGC00117362 (#D233–0871), NCGC00111761 (#C686–0165), NCGC00117328 (#D233–0834), NCGC00117505 (#D244–0327), NCGC00117477 (#D244–0252), NCGC00117166 (#D233–0497), NCGC00115018 (#D053–0260), NGC00117330 (#D233–0835) and NCGC00117364 (#D233–0885) were purchased from ChemDiv. Collagen type I (rat-tail), and fibronectin (human) were purchased from BD Biosciences. Rabbit anti-phospho m-TOR against Ser2448 (#2971), rabbit anti-mTOR (#2972), rabbit anti-phospho Rb against Ser807/811 (#8516, clone D20B12), mouse anti-Rb (#9309), mouse anti-Cdk6 (#3136), lysis buffer (#9803), and 3x Blue Loading Buffer (#56036) were purchased from Cell Signaling Technology. Mouse anti-Cdk1/Cdk2 (#sc-53219) was purchased from Santa Cruz Biotechnology Inc.
Kenny H.A., Lal-Nag M., Shen M., Kara B., Nahotko D.A., Wroblewski K., Fazal S., Chen S., Chiang C.Y., Chen Y.J., Brimacombe K.R., Marugan J., Ferrer M, & Lengyel E. (2019). Quantitative high-throughput screening using an organotypic model identifies compounds that inhibit ovarian cancer metastasis. Molecular cancer therapeutics, 19(1), 52-62.
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9

Neuro-2a Cell Culture with Vardenafil and Pitstop2

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Mouse Neuro-2a (N2a) cells were grown in 50% Dulbecco modified Eagle’s medium (DMEM), 50% OptiMEM, with 0.1 mM non-essential amino acids, 1% penicillin-streptomycin mixture, and 5% fetal bovine serum. Vardenafil and Pitstop2 (Sigma-Aldrich, Italy) were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C until use.
Caudano F., Montalto G., Passalacqua M., Pronzato M.A., Fedele E, & Ricciarelli R. (2020). cGMP favors the interaction between APP and BACE1 by inhibiting Rab5 GTPase activity. Scientific Reports, 10, 1358.
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10

Purification of GαT·GTP-1D4 Complex and PDE6 Interaction

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1D4-tagged recombinant GαT•GTP was washed with buffer containing 20 mM Tris pH 8.0 and 5 mM MgCl2 on a 10kD MWCO concentrator to remove DTT and then mixed with the 1D4 antibody (University of British Columbia, CA) in a 7:1 molar ratio and incubated on ice for 30 min. The mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl2 to purify the 2:1 GαT•GTP-1D4 complex. PDE6 was washed with buffer containing 20 mM Tris pH 8.0, and 5 mM MgCl2 on a 100kD MWCO concentrator to remove DTT and mixed with an equal molar amount of the purified 2:1 GαT•GTP-1D4 complex together with 10 μM of vardenafil (Sigma-Aldrich).
The mixture was incubated on ice for 30 min and purified by gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8.0, 5 mM MgCl2 and 1 μM vardenafil. Peak fractions were pooled and concentrated with a 100kD MWCO concentrator to ~10 mg/mL.
Gao Y., Eskici G., Ramachandran S., Poitevin F., Seven A.B., Panova O., Skiniotis G., & Cerione R.A. (2020). Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6.
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