How Sildenafil Works in the Body
The use of iPSCs in pseudoanonymized manner was approved by the local ethic committees of Charité-Universitätsmedizin Berlin (EA2/131/13 and EA2/107/14) and Heinrich Heine University Düsseldorf (Study number 2020-967_5). Germline Ndufs4 KO mice were obtained from the University of Washington.132 All mice were on the C57/BL6/J background and were fed the regular diet from Harlan Teklad.132 Sildenafil citrate treatment was initiated soon after weaning and genotyping, between day 25-30 of age.
- Sildenafil may cause temporary visual disturbances; report any persistent changes to a doctor.
- It should not be used with certain medications due to the risk of interactions.
- Some users misuse sildenafil for enhancing athletic performance, which is unsafe.
- Correct dosing ensures effectiveness while minimizing side effects.
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Treatment studies were carried out in 16 female mice and 14 male mice. Mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University, USA (M023M236). Domestic pigs of large white background were used to study SURF1 KO. Male KO animals were compared to sex and age-matched WT animals. 7 SURF1 KO male animals were treated with sildenafil citrate starting immediately after birth. On the first day after birth, pigs were fed reconstituted milk (NeoPig Rescue, Provimi, 10-12 ml every hour) via gastric tube. By the second day, WT piglets and almost all sildenafil-treated SURF1 KO piglets began drinking milk independently from an automatic feeder connected to the incubator. At day 30 after birth, pigs were transferred to the breeding cages and gradually switched from reconstituted milk to pelleted feed. Experiments in pigs were carried out according to the ARRIVE guidelines and approved by the Local Ethics Committee of Avantea, in accordance with the Italian Law (D.Lgs 26/2014) and EU directive 2010/63/EU regulating animal experimentation and approved by relevant authorities (Ministry of Health project # 367/2022-PR). Big-conductance calcium-activated potassium (BKCa) KO human U87 glioblastoma lines with stably deletion of the KCNMA1 gene, encoding for BKCa,80 were kindly provided by Prof. Adam Szewczyk (Nencki Institute, Warsaw). WT and BKCa KO glioblastoma (U-87 MG) cells were grown in DMEM medium (Thermo Fisher Scientific) with 10 % fetal bovine serum (FBS) (Gibco) 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C.80 The off-label individual basis treatment with sildenafil citrate (Revatio®) in LS patients carrying MT-ATP6 variants was registered on ClinicalTrials.gov (ID: NCT06967831). The treatment was performed as “individual basis treatment", also known as “named-patient basis (NPB) therapy”, which refers to pre-authorization access to unauthorized medicines requested by doctors for specific patients with serious unmet needs. In this treatment, the physicians carry an individual responsibility and there is no requirement of formal approval by EMA or a local IRB. This process is governed by national legislation within each EU Member State, under Article 5(1) of Directive 2001/83/EC.
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In this treatment, the physicians carry an individual responsibility and there is no requirement of formal approval by EMA or a local IRB. This process is governed by national legislation within each EU Member State, under Article 5(1) of Directive 2001/83/EC. Patients were of Caucasian ethnicity and included both male and female individuals (according to sex assigned at birth). Details of patient features and clinical outcomes are reported in Tables 1, S3, and S4. We obtained written informed consent to use patient material and health data from the patients and their guardians according to the Declaration of Helsinki.
Does sildenafil interact with other medicines (drug interactions)?
On day 6, we transferred the suspended cells onto Matrigel-coated well plates using: the same media without SB-431542 and dorsomorphin, but with the addition of 150 μM ascorbic acid (Sigma-Aldrich). We maintained NPCs on this media and used them for experiments between passage 7 and passage 30. Differentiation into neuronal cultures containing dopaminergic neurons was performed as previously described.25 Briefly, starting from NPCs at low confluence (10-30 %), we switched to media containing Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, with the addition of 200 mM vitamin C, 100 ng/ml FGF8 (R&D Systems) and 1 mM PMA. After 8 days, we used the media Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 0.5 mM PMA, 500 mM cAMP (STEMCELL Technologies), 10 ng/mL BDNF (MACS Miltenyi), 10 ng/ml GDNF (MACS Miltenyi) and 1ng/mL TGF-beta 3 (MACS Miltenyi). After ten days, the medium was changed to Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 500 mM cAMP, 10 ng/ml BDNF, 10 ng/ml GDNF, and 1 ng/ml TGF-beta 3.
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Cortical brain organoids were generated following two protocols (Figure S7A). The first protocol was based on our modifications62 of a previous publication.134 Briefly, on day 0, iPSCs at 80 % confluence were seeded into low-attachment U-bottom 96-well plates (Corning) to induce neurosphere formation. iPSCs were washed with DPBS (Gibco) and detached with Accutase (Gibco) for 5 min at 37 °C. Cell pellets were resuspended in cortical differentiation medium I (CDMI) consisting of Glasgow-MEM, 20 % Knockout Serum Replacement, MEM-NEAA, sodium pyruvate, 2-mercaptoethanol and penicillin-streptomycin (all from Gibco). The supplemented seeding suspension was distributed to low attachment U-bottom 96-well plates by adding 100 μl per well and incubated at 37 °C and 5 % CO2. Patients were of Caucasian ethnicity and included both male and female individuals (according to sex assigned at birth). Details of patient features and clinical outcomes are reported in Tables 1, S3, and S4. We obtained written informed consent to use patient material and health data from the patients and their guardians according to the Declaration of Helsinki.
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On day 6, we transferred the suspended cells onto Matrigel-coated well plates using: the same media without SB-431542 and dorsomorphin, but with the addition of 150 μM ascorbic acid (Sigma-Aldrich). We maintained NPCs on this media and used them for experiments between passage 7 and passage 30.
- Sildenafil was initially developed to treat hypertension and angina.
- The typical prescribed dose is up to 100mg, but only under medical supervision.
- Recreational misuse of high doses can result in serious health issues.
- Educate yourself about the potential risks and proper use of sildenafil.
- The drug works best when taken about 30-60 minutes before activity.
Differentiation into neuronal cultures containing dopaminergic neurons was performed as previously described.25 Briefly, starting from NPCs at low confluence (10-30 %), we switched to media containing Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, with the addition of 200 mM vitamin C, 100 ng/ml FGF8 (R&D Systems) and 1 mM PMA. After 8 days, we used the media Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 0.5 mM PMA, 500 mM cAMP (STEMCELL Technologies), 10 ng/mL BDNF (MACS Miltenyi), 10 ng/ml GDNF (MACS Miltenyi) and 1ng/mL TGF-beta 3 (MACS Miltenyi). After ten days, the medium was changed to Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 500 mM cAMP, 10 ng/ml BDNF, 10 ng/ml GDNF, and 1 ng/ml TGF-beta 3. Cortical brain organoids were generated following two protocols (Figure S7A). The first protocol was based on our modifications62 of a previous publication.134 Briefly, on day 0, iPSCs at 80 % confluence were seeded into low-attachment U-bottom 96-well plates (Corning) to induce neurosphere formation. iPSCs were washed with DPBS (Gibco) and detached with Accutase (Gibco) for 5 min at 37 °C. Cell pellets were resuspended in cortical differentiation medium I (CDMI) consisting of Glasgow-MEM, 20 % Knockout Serum Replacement, MEM-NEAA, sodium pyruvate, 2-mercaptoethanol and penicillin-streptomycin (all from Gibco). The supplemented seeding suspension was distributed to low attachment U-bottom 96-well plates by adding 100 μl per well and incubated at 37 °C and 5 % CO2.
| Side Effect | Severity | Notes |
|---|---|---|
| Headache | Common | Usually mild |
| Flushing | Common | Transient |
| Dizziness | Common | Particularly when standing up quickly |
| Vision disturbances | Less common | Temporary, includes blue tint or blurred vision |
| Priapism | Rare | Needs urgent medical attention |
On day 18, the developing organoids were transferred to 100 mm petri dishes filled with cortical differentiation medium II (CDMII) consisting of DMEM/ F12, 1 % GlutaMAX, 1 % N2 supplement, 1 % chemically defined lipid concentrate and penicillin-streptomycin (Gibco). The petri dishes were placed on an orbital shaker at 70 rpm at 37 °C and 5 % CO2. On day 35, CDMII was replaced with cortical differentiation medium III (CDMIII) that in addition contains 10 % FBS (Gibco) and heparin (Merck). CDMIII was changed every two to four days, depending on the sildenafil price ireland colour of the medium. From day 70 on, organoids were kept in cortical differentiation medium IV (CDMIV) that additionally was supplemented with 1 % B27 with vitamin A. The second protocol was based on a different publication.63 For this, AggreWell plates (STEMCELL Technologies) were prepared by incubation with Anti-Adherence Rinsing Solution (STEMCELL Technologies) for 15 min. iPSCs were detached from the 6-well plates using Accutase as described above. The cell suspension was pelleted at 200 x g for 4 min and resuspended in iPS Brew (Miltenyi Biotec) with 10 μM ROCK inhibitor. Cells were counted with CytoSmart Cell Counter (Greiner Bio-One) and 2.75 million cells were seeded in 1.5 ml per AggreWell. AggreWell plates were centrifuged at 100 x g for 3 min and left at 37 °C and 5 % CO2 for 24 h. On day 1, spheroids were transferred to a petri dish using a cut P1000 tip and placed on a shaker. Until day 6, ES medium was changed daily. Neurospheres were fed with Neural Differentiation Medium (NDM) consisting of Neurobasal A medium (Gibco), GlutaMAX, penicillin-streptomycin, MycoZAP and 2 % B27 without vitamin A (Gibco). From day 6-15, medium was changed daily and supplemented with 20 ng/ml EGF (R&D Systems) and 20 ng/ml FGF2 (R&D Systems). For long-term maturation after day 46, NDM medium was prepared with 2 % B27 with vitamin A, and organoids were fed according to their needs. Images of brain organoids were acquired using the Eclipse Ts2 light microscope (Nikon), and at later growth stages, when the organoids were larger, using a DMS1000 microscope (Leica). The size of the organoids was determined by area calculation using ImageJ. For MMP quantification, we applied a high-content analysis (HCA)-based live-cell detection assay that we previously established.31 Briefly, we seeded NPCs onto black-wall, clear-bottom 96-well plates (SCREENSTAR, Greiner) pre-coated with Geltrex (Thermo Fisher Scientific) at a density of 1.5 x 105 cells/cm2, and incubated them in NPC medium overnight at 37 °C, 5 % CO2. The next day, NPCs were treated with either dimethylsulfoxide (DMSO) or sildenafil dilution series in 100 μl and incubated for 16 h at 37°C, 5% CO2. After 30 min incubation, cells were washed with 100 μl NPC medium without phenol red.
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Images of brain organoids were acquired using the Eclipse Ts2 light microscope (Nikon), and at later growth stages, when the organoids were larger, using a DMS1000 microscope (Leica). The size of the organoids was determined by area calculation using ImageJ. For MMP quantification, we applied a high-content analysis (HCA)-based live-cell detection assay that we previously established.31 Briefly, we seeded NPCs onto black-wall, clear-bottom 96-well plates (SCREENSTAR, Greiner) pre-coated with Geltrex (Thermo Fisher Scientific) at a density of 1.5 x 105 cells/cm2, and incubated them in NPC medium overnight at 37 °C, 5 % CO2. The next day, NPCs were treated with either dimethylsulfoxide (DMSO) or sildenafil dilution series in 100 μl and incubated for 16 h at 37°C, 5% CO2. After 30 min incubation, cells were washed with 100 μl NPC medium without phenol red.
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NPCs were kept in 100 μl medium (w/o phenol red) for the duration of the assay. For confirming MMP depolarizing effect, we used the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) in combination with the mitochondrial complex III inhibitor antimycin A (AA).31 Imaging was performed with an Operetta CLS high content imaging system (Revvity) with 20 x water objective in confocal mode. Imaging time did not exceed 30 min. TMRM and Hoechst 33342 intensity within the cells was calculated using Columbus software (Revvity, version 2.9.0). To assess the effect of KT5823 on MMP in presence and absence of sildenafil, NPCs were seeded, incubated as described above, and pre-treated for 1 h with either DMSO alone or 1 μM KT5823 dissolved in DMSO.
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After the incubation time, 10 μM sildenafil dissolved in DMSO was added on top and incubated for 16 h at 37°C, 5% CO2. The final DMSO concentration was adjusted to 0.3 % for each condition. On the day of the assay, NPCs were live-stained and imaged. TMRM and Hoechst 33342 intensity within the cells was analyzed with CellProfiler (version 4.2.5). MMP values are presented as the TMRM intensity in a mutant or treated sample over the TMRM intensity in the control sample (%). NPCs were kept in 100 μl medium (w/o phenol red) for the duration of the assay. For confirming MMP depolarizing effect, we used the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) in combination with the mitochondrial complex III inhibitor antimycin A (AA).31 Imaging was performed with an Operetta CLS high content imaging system (Revvity) with 20 x water objective in confocal mode. Imaging time did not exceed 30 min. TMRM and Hoechst 33342 intensity within the cells was calculated using Columbus software (Revvity, version 2.9.0). To assess the effect of KT5823 on MMP in presence and absence of sildenafil, NPCs were seeded, incubated as described above, and pre-treated for 1 h with either DMSO alone or 1 μM KT5823 dissolved in DMSO.
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After the incubation time, 10 μM sildenafil dissolved in DMSO was added on top and incubated for 16 h at 37°C, 5% CO2. The final DMSO concentration was adjusted to 0.3 % for each condition.
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The use of iPSCs in pseudoanonymized manner was approved by the local ethic committees of Charité-Universitätsmedizin Berlin (EA2/131/13 and EA2/107/14) and Heinrich Heine University Düsseldorf (Study number 2020-967_5). Germline Ndufs4 KO mice were obtained from the University of Washington.132 All mice were on the C57/BL6/J background and were fed the regular diet from Harlan Teklad.132 Sildenafil citrate treatment was initiated soon after weaning and genotyping, between day 25-30 of age. Treatment studies were carried out in 16 female mice and 14 male mice. Mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University, USA (M023M236). Domestic pigs of large white background were used to study SURF1 KO.
ERK and calcineurin were activated in the RV as well as in the LV, and were inhibited by sildenafil treatment
Male KO animals were compared to sex and age-matched WT animals. 7 SURF1 KO male animals were treated with sildenafil citrate starting immediately after birth. On the first day after birth, pigs were fed reconstituted milk (NeoPig Rescue, Provimi, 10-12 ml every hour) via gastric tube. By the second day, WT piglets and almost all sildenafil-treated SURF1 KO piglets began drinking milk independently from an automatic feeder connected to the incubator. At day 30 after birth, pigs were transferred to the breeding cages and gradually switched from reconstituted milk to pelleted feed.
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Experiments in pigs were carried out according to the ARRIVE guidelines and approved by the Local Ethics Committee of Avantea, in accordance with the Italian Law (D.Lgs 26/2014) and EU directive 2010/63/EU regulating animal experimentation and approved by relevant authorities (Ministry of Health project # 367/2022-PR). Big-conductance calcium-activated potassium (BKCa) KO human U87 glioblastoma lines with stably deletion of the KCNMA1 gene, encoding for BKCa,80 were kindly provided by Prof. Adam Szewczyk (Nencki Institute, Warsaw). WT and BKCa KO glioblastoma (U-87 MG) cells were grown in DMEM medium (Thermo Fisher Scientific) with 10 % fetal bovine serum (FBS) (Gibco) 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C.80 The off-label individual basis treatment with sildenafil citrate (Revatio®) in LS patients carrying MT-ATP6 variants was registered on ClinicalTrials.gov (ID: NCT06967831). The treatment was performed as “individual basis treatment", also known as “named-patient basis (NPB) therapy”, which refers to pre-authorization access to unauthorized medicines requested by doctors for specific patients with serious unmet needs. On the day of the assay, NPCs were live-stained and imaged. TMRM and Hoechst 33342 intensity within the cells was analyzed with CellProfiler (version 4.2.5). MMP values are presented as the TMRM intensity in a mutant or treated sample over the TMRM intensity in the control sample (%).
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Cells were harvested and resuspended in culture medium at approximately 1x106 cells/ml. TMRM probe (20 nM final concentration) was added and incubated for 30 min at 37 °C and 5 % CO2. After incubation, cells were washed once in 1 ml of culture medium, then resuspended in 300 μl of DPBS and analyzed on the flow cytometer. The TMRM-emitted fluorescence was detected by emission filters appropriate for R- phycoerythrin “PE” (585 nm ± 42 nm). An appropriate gating strategy was applied to select only live and single cells. Cells were harvested and resuspended in culture medium at approximately 1x106 cells/ml. TMRM probe (20 nM final concentration) was added and incubated for 30 min at 37 °C and 5 % CO2. After incubation, cells were washed once in 1 ml of culture medium, then resuspended in 300 μl of DPBS and analyzed on the flow cytometer.
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On day 18, the developing organoids were transferred to 100 mm petri dishes filled with cortical differentiation medium II (CDMII) consisting of DMEM/ F12, 1 % GlutaMAX, 1 % N2 supplement, 1 % chemically defined lipid concentrate and penicillin-streptomycin (Gibco). The petri dishes were placed on an orbital shaker at 70 rpm at 37 °C and 5 % CO2. On day 35, CDMII was replaced with cortical differentiation medium III (CDMIII) that in addition contains 10 % FBS (Gibco) and heparin (Merck). CDMIII was changed every two to four days, depending on the sildenafil price ireland colour of the medium. From day 70 on, organoids were kept in cortical differentiation medium IV (CDMIV) that additionally was supplemented with 1 % B27 with vitamin A.
What should I know about sildenafil before taking it?
The second protocol was based on a different publication.63 For this, AggreWell plates (STEMCELL Technologies) were prepared by incubation with Anti-Adherence Rinsing Solution (STEMCELL Technologies) for 15 min. iPSCs were detached from the 6-well plates using Accutase as described above. The cell suspension was pelleted at 200 x g for 4 min and resuspended in iPS Brew (Miltenyi Biotec) with 10 μM ROCK inhibitor. Cells were counted with CytoSmart Cell Counter (Greiner Bio-One) and 2.75 million cells were seeded in 1.5 ml per AggreWell. AggreWell plates were centrifuged at 100 x g for 3 min and left at 37 °C and 5 % CO2 for 24 h.
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On day 1, spheroids were transferred to a petri dish using a cut P1000 tip and placed on a shaker. Until day 6, ES medium was changed daily. Neurospheres were fed with Neural Differentiation Medium (NDM) consisting of Neurobasal A medium (Gibco), GlutaMAX, penicillin-streptomycin, MycoZAP and 2 % B27 without vitamin A (Gibco). From day 6-15, medium was changed daily and supplemented with 20 ng/ml EGF (R&D Systems) and 20 ng/ml FGF2 (R&D Systems). For long-term maturation after day 46, NDM medium was prepared with 2 % B27 with vitamin A, and organoids were fed according to their needs. The TMRM-emitted fluorescence was detected by emission filters appropriate for R- phycoerythrin “PE” (585 nm ± 42 nm).
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Data were analyzed with FlowJo software (version 7.6); the median fluorescence intensity (MFI) for each sample was calculated and normalized on the relative unstained sample. TMRM intensity was quantified in at least 20,000 cells per sample; at least three biological replicates were analyzed. An appropriate gating strategy was applied to select only live and single cells. Data were analyzed with FlowJo software (version 7.6); the median fluorescence intensity (MFI) for each sample was calculated and normalized on the relative unstained sample. TMRM intensity was quantified in at least 20,000 cells per sample; at least three biological replicates were analyzed.