Patient Experiences with 100 mg Sildenafil Strength

The petri dishes were placed on an orbital shaker at 70 rpm at 37 °C and 5 % CO2.

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Prigione, D. Brunetti, A.d.S., O.P., W.J.H.K., P.L., A.S., F.E., E.B., M. Schuelke, and C.R.R. A. Prigione and M.

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Schuelke filed patent applications for sildenafil in the treatment of CIV and CV defects and obtained Orphan Drug Designation (ODD) for sildenafil in Leigh syndrome from the Committee for Orphan Medicinal Products (COMP) of the European Medicines Agency (EMA) (EU/3/23/2831). MT-ATP6 mutant iPSC lines were previously derived from three patients with m.9185T>C variant (ATP61_1: HHUi001-A; ATP6_2: HHUi002-A; ATP6_3: HHUi003-C), one patient with m.8993T>C variant (ATP6_4: MDCi008-A), two patients with m.8993T>G variant (ATP6_5: MDCi009-A; ATP6_6: MDCi010-A), and one patient with m.9176T>G variant (ATP6_7: BIHi267-B) (Figure S10H).27,33,34,35 Genotyping of the lines was carried out by Sanger sequencing (Figure S1A). SURF1 mutant iPSC isogenic lines (SURF1_1: HMGUi001-A-64) and NDUFS4 mutant iPSC isogenic lines (NDUFS4_1: HVRDi004-B-6; NDUFS4_2: HVRDi004-B-7) were previously derived (Figure S10H).25,89 Among healthy control iPSCs (Figure S10H), CTRL_1, (HMGUi001-A), CTRL_2 (HHUUKDi009-A), CTRL_4 (BIHi269-B), CTRL_7 (BIHi266-A), and CTRL_9 (UMGi014-C) were previously described,27,33,128,129 CTRL_3 (CRMi003-A)126 was purchased from RUCDR Infinite Biologics, CTRL_5 (HVRDi004-B, PGP1)127 was purchased from Synthego, CTRL_6 (IUFi004-A, iPS12)130 was purchased from Cell Applications, and CTRL_8 (WISCi004-B)131 was purchased from WiCell. We cultured all iPSC lines on Matrigel (Corning)-coated plates using StemMACS iPS-Brew XF medium, supplemented with MycoZap (Lonza). CTRL_8 and ATP6_2 iPSCs were cultured in mTeSR Plus Medium (STEMCELL Technologies) prior to BCEC differentiation. On day 35, CDMII was replaced with cortical differentiation medium III (CDMIII) that in addition contains 10 % FBS (Gibco) and heparin (Merck).

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CDMIII was changed every two to four days, depending on the colour of the medium.

Storage Condition	Details	Temperature Range
Cool, Dry Place	Keep away from light and moisture	15-30°C
Keep Out of Reach of Children	To prevent accidental ingestion	-
Packaging	Original tightly sealed containers	-

From day 70 on, organoids were kept in cortical differentiation medium IV (CDMIV) that additionally was supplemented with 1 % B27 with vitamin A.

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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.

Prior authorization

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. 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). 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.

• Side effects at high doses like 100g can include vision impairment and hearing loss.
• Sildenafil can cause headaches, dizziness, or temporary vision changes.
• Patients taking medications for blood pressure should consult their doctor beforehand.
• The drug's effect lasts around 4-6 hours, but duration varies.
• Misuse or overdose increases the risk of serious health complications.

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.

• Sildenafil 100g is rarely prescribed and considered an experimental or off-label dose.
• Like all medications, it should only be used as directed by a healthcare professional.
• Overdose signs include severe hypotension, vision loss, or chest pain.
• Use with caution in patients with liver or kidney impairment.
• Avoid alcohol consumption when using sildenafil, especially at high doses.

Cells were counted with CytoSmart Cell Counter (Greiner Bio-One) and 2.75 million cells were seeded in 1.5 ml per AggreWell.

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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. Male KO animals were compared to sex and age-matched WT animals.

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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). 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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Information on sex and age of the individuals from which the iPSCs were derived is provided in Figure S10H. We cultivated all iPSCs in a humidified atmosphere of 5 % CO2 at 37 °C and 5 % oxygen. iPSCs were split at 70-80 % confluence with 0.5 μM EDTA (Invitrogen) in 1 x PBA (Gibco). We added 10 μM ROCK inhibitor (Enzo Biochem Inc) after splitting to promote survival. Cultures were routinely monitored for mycoplasma contamination by PCR using nine primers to amplify the six most common mycoplasma strains (Table S5).

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The positive control and internal contro were kindly provided sildenafil citrate tablets 50mg by Dr. Cord Uphoff (DZMS, Germany). We monitored the identity of iPSCs using STR analysis, which was performed by the Forensic Department of the University Hospital Düsseldorf (Dr. Petra Böhme); 21 microsatellite loci were amplified using PCR and tagged products were analyzed using GeneMapper ID v.3.2.1 (Applied Biosystems). 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). On day 1, spheroids were transferred to a petri dish using a cut P1000 tip and placed on a shaker.

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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 sildenafil tables 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).

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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. 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). 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).

• Storage of sildenafil should be in a cool, dry place away from direct sunlight.
• Common side effects include dizziness, upset stomach, or vision changes.
• The dosage of sildenafil should be tailored to individual health needs.
• Sildenafil can interact with other medications, affecting its safety and efficacy.
• Always seek medical guidance before using sildenafil, especially at high doses.

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).

• Sildenafil 100g refers to a high dosage of the medication used to treat erectile dysfunction.
• This dosage is significantly higher than the common 50mg or 25mg tablets.
• Always consult a healthcare provider before using sildenafil in such high amounts.
• Overuse can cause severe side effects like dangerous drops in blood pressure.
• Sildenafil 100g is typically used only under strict medical supervision for specific cases.

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.

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Prigione; writing – review & editing, A. Prigione, O.P., M. Schuelke, D.-F.D., E.B., and C.R.R. ; supervision, A. Prigione, O.P., C.R.R., I.S., M.J.W.A.-H., E.M., S.J., G.U., S.P., W.J.H.K., A.S., F.E., E.B., M.

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Schuelke, L.C., V.C., S.J., A.d.S., and D. Brunetti; visualization, A. Zink, A.W., O.P., A. Prigione, M.-T.H., G.P., S.H., and M. Schuelke; funding acquisition, A.

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