The following immunosuppressive drugs were used to suppress the immune system of the animals: Mycophenolate mofetil (MMF) powder for infusion (20mg/kg) (CellCept, Roche, Woerden, The Netherlands), tacrolimus concentrate for infusion (5 mg/ml) (Prograft, Astellas Pharma BV, Leiderdorp, The Netherlands) and oral solution of prednisolone sodium phosphate (5 mg/ml) (Hospital Pharmacy, UMCN St Radboud, Nijmegen, The Netherlands). This regimen is similar to the immunosuppressive treatment of patients undergoing solid organ transplantation. To prevent opportunistic infections, all animals received an antibiotic prophylaxis of amoxicillin supplemented with oral suspension of clavulanic acid (250 mg and 62.5 mg per 5 mL, respectively) (Pharmachemie BV, Haarlem, The Netherlands). Oseltamivir phosphate (OSP; 10mg/Kg) was added to the regimes of ferrets receiving antiviral therapy and was provided by Hoffman-La Roche LtD. (Tamiflu, Basel, Switzerland). Preparation of the drugs was done as previous described [5 (link)].
Oseltamivir phosphate
Manufactured by Roche
Sourced in Switzerland, China
Oseltamivir phosphate is a synthetic antiviral medication used as the active ingredient in the treatment of influenza. It functions as a neuraminidase inhibitor, which helps prevent the spread of the influenza virus within the human body.
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Lab products found in correlation
Amantadine, by Merck Group (2 mentions)
Oseltamivir carboxylate, by Santa Cruz Biotechnology (2 mentions)
Tamiflu, by Roche (1 mentions)
Cellcept, by Roche (1 mentions)
Wheat germ agglutinin (wga), by Merck Group (1 mentions)
Gallic acid, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Minimum essential medium (mem), by Fujifilm (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Favipiravir, by BOC Sciences (1 mentions)
Oseltamivir carboxylate, by Roche (1 mentions)
Benzbromarone, by Tokyo Chemical Industry (1 mentions)
Np gtx125989, by GeneTex (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Amantadine hydrochloride, by Merck Group (1 mentions)
Ab128193, by Abcam (1 mentions)
Influenza neuraminidase inhibitor detection kit, by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
10 protocols using oseltamivir phosphate
1
Immunosuppressive Regimen for Transplant Models
2
Oseltamivir Efficacy Against Influenza in Ferrets
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Ferrets (n = 4 in each group; source: outbred from multiple breeders) were anaesthetized [50:50 mix of Ketamine (100 mg/mL): Ilium Xylazil (Xyalazine; 20 mg/mL)] and infected by intranasal inoculation with 105 TCID50 (median tissue culture infectious dose) of MDCK (Madin-Darby canine kidney (MDCK; ATCC CCL-34)-propagated A/Perth/265/2009 A(H1N1)pdm09 influenza virus (2.0 x 106 TCID50/mL). For oseltamivir treatment, ferrets were orally given 5 mg/kg oseltamivir phosphate (kindly provided by Hoffmann-La Roche Ltd., Basel, Switzerland) two hours prior to infection and then twice daily post-infection. Body temperature, weight and nasal washes of all ferrets were collected at day 2 post-infection. Virus titer, cell concentration and protein concentration of nasal washes were measured as described [38 (link)]. Oseltamivir treated and untreated ferrets were nasal washed on day 2 post-infection to determine viral load, cell count and nasal wash protein concentration. Ferrets were housed individually in high efficiency particulate air filtered cages with ad libtum to food, water and enrichment toys. Ferrets were sacrificed by intramuscular injection of anaesthesia [50:50 mix of Ketamine (100 mg/mL): Ilium Xylazil (Xyalazine; 20 mg/mL)] followed by an overdose of pentobarbitone sodium (Lethabarb; 0.5 mL/kg).
3
Binding Affinities of Lectins to Influenza Viral Proteins
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The fucoidan KW extracted from Kjellmaniella crassifolia was provided by School of Medicine and Pharmacy, Ocean University of China. The NA proteins of subtype H1N1 (A/California/04/2009) (11058-VNAHC) and H3N2 (A/Babol/36/2005) (40017-VNAHC) were purchased from Sino Biological Inc. (Beijing, China). FITC-labeled lectins (concanavalin A (ConA), peanut agglutinin (PNA), ulex europaeus I (UEA-I), and wheat germ agglutinin (WGA)) were purchased from Sigma (St. Louis, MO, USA). FITC-labeled maackia amurensis agglutinin I (MAAI) was purchased from Vector Laboratories (Burlingame, CA, USA). Ribavirin (50 mg/mL) was obtained from LuKang Cisen (Jining, China). Oseltamivir carboxylate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Oseltamivir phosphate was obtained from Roche (Shanghai, China). Amantadine was purchased from Sigma (USA).
4
Influenza Virus Inhibition Assay
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Madin Darby Canine Kidney (MDCK) cells, a kind gift from Dr. Kyosuke Nagata (Tsukuba University, Japan), were maintained in minimum essential medium (MEM) purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), and supplemented with 5% fetal bovine serum from Life Technologies (Scoresby, Australia), 100 units/mL penicillin, and 100 µg/mL streptomycin (Nacalai Tesque Inc, Kyoto, Japan) at 37 °C in 5% CO 2 . Influenza viruses A/WSN/33 (H1N1), A/Puerto Rico/8/34 (H1N1), A/Virginia/ATCC2/2009 (H1N1), A/Aichi/2/68 (H3N2), and B/Lee/40 were prepared as described. 25 Oseltamivir phosphate (F. Hoffmann-La Roche, Basel, Switzerland) and zanamivir (LTK laboratories, St. Paul, MN, USA) were dissolved in phosphate-buffered saline (PBS) and dimethyl sulfoxide (DMSO), respectively. Amantadine (Sigma Aldrich, Tokyo, Japan) was dissolved in water and gallic acid (Nacalai Tesque) in ethanol. Resveratrol (Merck Millipore, Billerica, MA, USA) was dissolved in DMSO. Peanut skin samples were obtained as previously described, 26 and are summarized in Table 1.
5
Antiviral drug preparation protocols
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Oseltamivir-phosphate (Tamiflu) was purchased from a local pharmacy and oseltamivir-carboxylate was synthesized by Hoffmann-La Roche (Basel, Switzerland). Both compounds were suspended in sterile water. Favipiravir was purchased from BOC Sciences (Shirley, NY, USA) and prepared in sterile water supplemented with 74.6 mg/mL of meglumine excipient. Baloxavir marboxil (HY-109025A) was purchased from MedChem Express (Princeton, NJ, USA) and prepared with 0.5% methylcellulose.
6
Preparation and Characterization of SFXF Granules
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Oseltamivir Phosphate was purchased from the F. Hoffmann-La Roche Ltd. (Basel, Swiss) (no. J20040058). SFXF granule was manufactured and provided by Beijing Tcmages Pharmaceutical Co., Ltd. SFXF consists of lonicera japonica (10 g), forsythia (10 g), dyers woad leaf (10 g), great burdock achene (10 g), periostracum cicadae (8 g), thunberg fritillary bulb (10 g), scutellaria (10 g), radix asteris (15 g), almond (10 g), platycodon grandiflorum (10 g), glycyrrhiza uralensis (6 g), and radix isatidis (10 g). One hundred grams of granules was dissolved in 500 mL water and kept at 4°C over night. The autoclaved herbal juice was then concentrated by continuous freeze-drying operation for 72 hours until the solvent was completely removed. These granules were kept in airtight containers at −70°C until further use.
7
Antiviral Screening of Compound Library
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Hit compounds were purchased from Namiki Syoji Co. Ltd (Tokyo, Japan) and dissolved in 100% dimethyl sulfoxide (DMSO). Benzbromarone was purchased from Tokyo Chemical Industry Co. Ltd (Tokyo, Japan) and dissolved in 100% DMSO. Oseltamivir phosphate purchased from F. Hoffmann-La Roche Ltd (Basel, Switzerland) was dissolved in phosphate-buffered saline (PBS) at a concentration of 10 mM. All of the compounds were maintained at −30 °C until use. Before performing the experiments, the compounds were diluted with Eagle’s minimum essential medium (MEM) supplemented with 1% 100× vitamin solution (MEM vitamin). HeLa cells were obtained from Dr Takujiro Homma (Nagasaki University, Japan) and maintained in Dulbecco’s modified Eagle’s medium (Sigma Aldrich, St Louis, MO) containing 10% fetal bovine serum (FBS). MDCK cells kindly donated by Dr Kyosuke Nagata (Tsukuba University, Japan) were grown in MEM supplemented with 5% FBS. These cells were maintained at 37 °C in an atmosphere of 5% CO2. Influenza viruses were prepared as described previously48 (link) and stored at −80 °C. Anti-HA (GTX127357), -M1 (GTX125928), -PA (GTX118991), -PB1 (GTX125923) and -NP (GTX125989) antibodies were purchased from GeneTex, Inc. (Irvine, CA). Anti-actin (A5060) antibody was purchased from Sigma Aldrich.
8
Antiviral Compounds: Preparation and Storage
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Oseltamivir phosphate (F. Hoffmann-La Roche, Basel, Switzerland) was dissolved in phosphate-buffered saline (PBS) at a concentration of 10 mM. Amantadine hydrochloride (Sigma Aldrich, Tokyo, Japan) was dissolved in ultra-pure water at 300 mM. Sennoside A and Sennoside B were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 30 mM. All of the compounds were maintained at −30°C until use. Before performing the experiments, the compounds were diluted with Eagle's minimum essential medium (MEM) supplemented with 1% 100 × vitamin solution (MEM vitamin). Madin-Darby canine kidney (MDCK) cells, kindly donated by Dr. Kyosuke Nagata (Tsukuba University, Japan), were grown in MEM supplemented with 5% fetal bovine serum (FBS). These cells were maintained at 37°C in an atmosphere of 5% CO2. Influenza viruses, except for A/California/7/2009 (H1N1), were prepared as described previously [16 (link)] and stored at −80°C. Allantoic fluid from embryonated eggs of A/California/7/2009 (H1N1) was obtained from Dr. Hiroshi Kido (Tokushima University) and stored at −80°C.
9
Influenza Inhibition by Patchouli Alcohol
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Patchouli alcohol (PA) (with purity > 98%) was purchased from TargetMol (Shanghai, China). Dulbecco’s Modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Excell (Suzhou, China). Mouse anti-influenza A virus NP antibody and alkaline phosphatase (AP)-labeled secondary antibodies were obtained from Santa Cruz Biotechnology (USA). DyLight 649 conjugated secondary antibody was obtained from Abbkine (California, USA). Ribavirin injection (50 mg/ml) was purchased from LuKang Cisen (Jining, China). Oseltamivir carboxylate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Oseltamivir phosphate was obtained from Roche (Shanghai, China). The influenza neuraminidase inhibitor detection kit was purchased from Beyotime (Shanghai, China). The anti-NP protein was provided by Abcam (ab128193), and other antibody, such as anti-phosphorylated PI3K(4228 s), Akt (9271 s), mTOR (5536 s), ERK1/2 (4370 s), and NF-κB (3033 s) antibodies, or anti-GAPDH (2118 s) and α-tubulin antibodies (2125 s), were obtained from Cell Signaling Technology (Danvers, USA).
10
Formulation and Reconstitution of UV-4 Compound
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The active ingredient UV-4 was formulated for all studies in acidified water or in the form of the hydrochloride salt (N-9-methoxynonyl-deoxynojirimycin-HCl, aka UV-4B), which has a molecular weight approximately 11.4% larger than UV-4 base (ex.100 mg/ml of UV-4 formulated as 111.4 mg/ml of UV-4B). All drug concentrations henceforward are referred as the UV-4 base. UV-4 and oseltamivir phosphate (Roche) were reconstituted in sterile water for all in vivo studies.
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