Methods and compositions for treating flaviviruses, pestiviruses and hepacivirus
A compound, an independent technology, applied in the direction of antiviral agents, active ingredients of carbohydrates, sugar derivatives, etc.
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Embodiment 1
[0295] Phosphorylation assay for the conversion of nucleosides to activated triphosphates
[0296] To determine the cellular metabolism of compounds, HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD) and made at 225 cm 2 Grow in tissue culture flasks in minimal essential medium supplemented with non-essential amino acids, 1% penicillin-streptomycin. The medium was refreshed every three days and the cells were subcultured weekly. After stripping the adherent monolayer of cells by exposing to 30 mL of insulin-EDTA for 10 min and successively washing three times with medium, confluent HepG2 cells were incubated at 2.5 × 10 6 Cells / well density were seeded in 6-well plates and exposed to 10 μM [ 3 H] labeled active compound (500 dpm / pmol). Keep cells at 37 °C and 5% CO 2 in the atmosphere. At selected time points, cells were washed three times with ice-cold phosphate-buffered saline (PBS). Intracellular active compounds and their respective ...
Embodiment 2
[0298] Bioavailability assay in macaques
[0299] Within one week before the start of the study, rhesus monkeys were surgically implanted with chronic venous catheters and subcutaneous venous access ports (VAP) to facilitate blood collection, and underwent physical examination, including histological and serum chemistry evaluation, and recorded body weight. Each monkey (total of six) received approximately 250 μCi by intravenous bolus (3 monkeys, IV), or by oral gavage (3 monkeys, PO) 3 H activity, wherein the dose level of the active compound per dose is 10 mg / kg, and the dose concentration is 5 mg / mL. Each dosing syringe was weighed prior to dosing to determine by weight the amount of dosage form administered. At indicated intervals (approximately 18-0 hours pre-dose, 0-4, 4-8, and 8-12 hours post-dose), urine samples were collected via capture plates and processed. Collected similarly via chronic venous catheter and VAP, or if chronic venous catheter approach is not possi...
Embodiment 3
[0301]Myelotoxicity Assay
[0302] Human bone marrow cells were collected from normal healthy volunteers according to the aforementioned Sommadossi J-P, Carlisle R. "Toxicity of 3'-azido-3'-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoieticprogenitor cells in vitro"Antimicrobial Agent sand Chemotherapy 1987;31:452-454; and Sommadossi J-P, Schinazi RF, Chu CK, Xie M-Y."Comparison of cytotoxicity of the(-)-and(+)-enantiomer of 2′,3′- Mononuclear populations were isolated by Fico-Pak gradient centrifugation as described in dideoxy-3'-thiacytidine inorganic human bone marrow progenitor cells" Biochemical Pharmacology 1992;44:1921-1925. CFU-GM and BFU-E medium assays were performed using double layer soft agar or methylcellulose methods. Drugs were diluted in tissue culture medium and filtered. at 37°C and 5% CO 2 After 14 to 18 days in a humid air atmosphere, colonies larger than 50 cells were counted using an inverse microscope. Results...
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