Neuraminidase; influenza A/H3N2, A/H1N2, A/H1N1, and B viruses

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32-100μM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01-10nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations.[5]

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Oseltamivir carboxylate and peramivir were evaluated in combination for inhibition of virus yield in MDCK cell cultures using doses of 0.32 to 100 μM (Table 1). Oseltamivir carboxylate alone reduced virus yield by 4.4 log10 at 100 μM. Peramivir at 32 and 100 μM reduced virus yield by ≥ 5 log10 below the detection limit of the assay. Greater than 10-fold inhibition of virus titer from expected was found at three specific conditions, when 10 μM oseltamivir carboxylate was combined with either 3.2 or 10 μM peramivir, and using the combination of 3.2 μM of each inhibitor. A three-dimensional MacSynergy plot of the data showing values above and below expected are shown in Figure 1. A region of significant synergy was evident between 1 and 10 μM oseltamivir and 1 and 10 μM peramivir, giving a volume of synergy of 9.1. A region of minor antagonism occurred when 0.32 μM peramivir was combined with 3.2-32 μM oseltamivir carboxylate, for a calculated volume of antagonism of −1.7. The net effect across the entire surface was a volume of synergy of 7.4.[5]

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The effects of the combination of oseltamivir carboxylate and peramivir on neuraminidase activity are presented in Table 2. Minimal neuraminidase activity was evident in the presence of 10 nM oseltamivir carboxylate treatment or 1 to 10 nM peramivir treatment. The majority of the low-dose combinations (0.01 to 3.2 nM oseltamivir carboxylate combined with 0.01 to 0.32 nM peramivir) caused greater inhibition than either compound used alone. Higher concentrations of each inhibitor used in combination (0.32 to 10 nM) caused less inhibition than expected. This was in a region where peramivir alone was highly inhibitory to enzymatic activity, with not much potential for further inhibition by a drug combination. The three-dimensional MacSynergy plot of the data is shown in Figure 2. The percentages of increase or decrease for the combinations were small. The low-dose combination region had a volume of synergy of 86 (moderate synergy), whereas the high-dose combination region had a volume of antagonism of −65 (moderate antagonism) for a net effect across the entire surface of 21 (indifference).[5]

In mice infected with the H1N1pdm virus, oseltamivir (20 mg/kg/day) administered orally, twice daily for 10 days, prevents death [2]. In mice infected with H5N1 influenza virus, oseltamivir (10 mg/kg/day, oral gavage, 5 days) in combination with amantadine (HY-B0402) (15 or 30 mg/kg) has a more effective protective effect than monotherapy [3]. To stop mice from contracting the influenza virus, oseltamivir can also be administered in conjunction with ribavirin (HY-B0434), favipiravir (HY-14768), or peramivir (HY-17015A) [4][5].

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Infected mice were treated twice daily for 5 days starting 2h prior to virus challenge using drug doses of 0.05-0.4mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2mg/kg/day) compared to single drug treatments. The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.[5]

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Combination treatment with amantadine (15 or 30 mg/kg/day) and oseltamivir (10 mg/kg/day) provided greater protection (60% and 90%, respectively) against lethal infection with amantadine-sensitive H5N1 virus than did monotherapy. Moreover, spread of the virus to the brain was prevented by both combination regimens. The efficacy of the drug combinations against amantadine-resistant H5N1 virus was comparable to that of oseltamivir alone. Oseltamivir produced a dose-dependent effect against both recombinant H5N1 viruses (P < 0.05) but did not provide complete protection against lethal infection. Importantly, no mutations in the HA, NA and M2 proteins were detected when the two drugs were used in combination. Conclusions: Combination chemotherapy provided a survival advantage over single-agent treatment of mice inoculated with neurotropic H5N1 influenza virus. This strategy might be an option for the control of pandemic influenza viruses that are sensitive to amantadine. Combinations that include other drugs should be explored.[3]

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We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC(50) = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used.[4]

Viral neuraminidase inhibition assay[5]
The effects of compounds on viral neuraminidase activity were determined using a commercially available kit (NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit, Applied Biosystems, Foster City, CA) in 96-well solid white microplates following the Manufacturer's instructions and as has been reported (Smee et al., 2010). Compounds in half-log dilution increments were incubated with virus (as the source of neuraminidase). The amount of influenza A/NWS/33 (H1N1) virus in each microwell was approximately 500 cell culture infectious doses. Plates were pre-incubated for 10 min at 37°C prior to addition of chemiluminescent substrate. Following addition of substrate the plates were incubated for 30 min at 37°C. The neuraminidase activity was evaluated using a Centro LB 960 luminometer (Berthold Technologies, Oak Ridge, TN) for 0.5 sec immediately after addition of NA-Star® accelerator solution. Percentages of chemiluminescent counts at each compound concentration were based upon counts normalized to 100% under untreated conditions.

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NA enzyme inhibition assay.[4]
NA activity was determined by the method described by Potier et al. Briefly, H5N1 viruses and various concentrations of oseltamivir carboxylate or zanamivir were preincubated for 30 min at 37°C before the substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid was added. After 1 h, the reaction was terminated by adding 14 mM NaOH, and the fluorescence was quantitated with a Perkin-Elmer fluorimeter (model LS50B) with an excitation wavelength of 360 nm and an emission wavelength of 448 nm. The 50% inhibitory concentration (IC50) was defined as the concentration of NA inhibitor necessary to reduce the activity NA by 50% relative to that in a reaction mixture containing virus but no inhibitor.

Cell culture antiviral studies[5]
Antiviral activities of oseltamivir carboxylate and peramivir were determined in confluent cultures of MDCK cells. The assays were performed in 96-well microplates infected with approximately fifty 50% cell culture infectious doses (CCID50) of virus, by quantifying virus yield after three days in culture. The plates of samples were frozen at - 80°C. Medium from two microwells were later pooled and used to produce samples for titration. Virus yields at each inhibitor concentration were determined by titration of samples (in 10-fold dilution increments) on fresh monolayers of MDCK cells in 96-well microplates by endpoint dilution method (Reed and Muench, 1938) using four microwells per dilution. Microplates were examined at 3 and 6 days of infection for the presence or absence of viral cytopathology. Virus titers were expressed as log10 CCID50 per 0.1 ml.

Animal experiment design[5]
Female BALB/c mice (18-20 g) were anesthetized by i.p. injection of ketamine (100 mg/kg) followed by intranasal infection with a 50-μl suspension of influenza virus; the infection inoculation of approximately 104.5 CCID50/mouse equaled three 50% mouse lethal challenge doses (MLD50). Compounds were administered p.o. (oseltamivir) by gavage or i.m. (peramivir) twice a day at 12-hour intervals for 5 days starting 2 hours before virus challenge. Placebo-treated mice received both p.o. and i.m. treatments. Ten drug-treated infected mice and 10 placebo-treated controls were observed daily for death through 21 days. Mice that died during the treatment phase were excluded from the total count. Body weights were determined every other day.

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In this report, we extend the in vivo observations by comparing the efficacies of JNJ63623872 and oseltamivir in mice infected with influenza A/California/04/2009 (H1N1pdm) and A/Victoria/3/75 (H3N2) viruses. Animals received JNJ63623872 or oseltamivir orally twice daily for 10 days starting 2 h pre-infection. JNJ63623872 (2, 6, and 20 mg/kg/day) and oseltamivir (20 mg/kg/day) completely prevented death in the H1N1pdm virus infection. Weight loss at nadir was only 12% in mice receiving 2 mg/kg/day of JNJ63623872 compared to 23% and 32%, respectively, in oseltamivir-treated (20 mg/kg/day) and placebo groups. Lung hemorrhage scores, lung weights, and lung virus titers on day 6 were reduced in a dose-responsive manner by JNJ63623872 treatments, whereas oseltamivir treatments were not as effective. JNJ63623872 was less active against H3N2 virus infection, with more body weight loss occurring and only 30% survival at the 2-mg/kg/day dose. Lung scores, lung weights, and H3N2 viral titers in lungs of mice were reduced less by JNJ63623872 treatments compared to the H1N1pdm infection. Nevertheless, the 20-mg/kg/day dose of JNJ63623872 was more effective than oseltamivir (20 mg/kg/day) in improving body weight and reducing the severity of lung infection. JNJ63623872 appears to be an important new drug candidate to treat influenza A H1N1pdm and H3N2 virus infections.[2]

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BALB/c mice were treated by oral gavage for 5 days with amantadine (1.5, 15 or 30 mg/kg/day) and oseltamivir (1 or 10 mg/kg/day) separately or in combination. Mice were challenged 24 h after initiation of treatment with 10 mouse 50% lethal doses of either amantadine-sensitive (having S31 in the M2 protein) or amantadine-resistant (having N31 in the M2 protein) recombinant A/Vietnam/1203/04 (H5N1) virus.[3]

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Assessment of drug efficacy in vivo.[4]
Female 6-week-old BALB/c mice were anesthetized with isoflurane and were intranasally inoculated with 50 μl of 10-fold serial dilutions of A/Vietnam/1203/04 (H5N1) or A/Turkey/15/06 (H5N1) virus in phosphate-buffered saline (PBS). The 50% mouse lethal dose (MLD50) was calculated after a 21-day observation period. For the A/Vietnam/1203/04 (H5N1) and the A/Turkey/15/06 (H5N1) viruses, the MLD50s/ml were ∼1 PFU and 4 PFU, respectively. Groups of 15 mice each were then given oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day) or ribavirin (37.5, 55, or 75 mg/kg/day) by oral gavage twice daily for 8 days. In the combination treatment experiments, oseltamivir was coadministered with ribavirin on the same schedule. Virus-inoculated control mice received sterile PBS (placebo). The first drug dose was given 4 h before intranasal inoculation with 5 MLD50/mouse of A/Vietnam/1203/04 (H5N1) or 5 MLD50/mouse of A/Turkey/15/06 (H5N1) virus; these doses were equivalent to ∼4 and 20 PFU/mouse, respectively. Survival and weight change were observed; animals that showed signs of severe disease and weight loss of >25% were humanely killed. Three mice each in the experimental and the placebo groups were killed on day 3 after inoculation; and the lungs, brains, and spleens were removed, homogenized, and suspended in 1 ml of PBS. Virus from each organ was titrated by inoculation of embryonated chicken eggs with serial dilutions of the suspensions. The titers were calculated by the method of Reed and Muench (32) and are expressed as the mean log10 50% egg-infective dose (EID50)/ml ± standard deviation (SD). The limit of virus detection was 0.75 log10 EID50/ml. For calculation of the mean, samples with a virus titer of <0.75 log10 EID50/ml were assigned a value of 0.[4]

Absorption, Distribution and Excretion
Following oral administration of oseltamivir phosphate, oseltamivir is rapidly absorbed from the gastrointestinal tract and extensively converted to the active metabolite, oseltamivir carboxylate, primarily by hepatic esterases. At least 75% of the oral dose enters systemic circulation as the active metabolite. Prodrug exposure is less than 5% relative to the active metabolite. Plasma concentrations of both the prodrug and the active metabolite are dose-proportional and unaffected by co-administration with food. The pharmacokinetic parameters after twice-daily administration of 75 mg oseltamivir capsules are as follows: Cmax of oseltamivir and oseltamivir carboxylate are 65 ng/mL and 348 ng/mL, respectively; AUC (0-12h) are 112 ng·h/mL and 2719 ng·h/mL, respectively. After absorption, over 90% of oseltamivir is converted to oseltamivir carboxylate and ultimately eliminated entirely by renal excretion. Clinical studies have found that less than 20% of the oral radiolabeled dose is excreted in feces.
The steady-state average volume of distribution of oseltamivir carboxylate in the human body is approximately 23 to 26 liters, roughly equivalent to the volume of extracellular fluid. Because neuraminidase activity is located extracellularly, oseltamivir carboxylate can be distributed to all sites of influenza virus transmission.
The renal clearance of this drug (18.8 L/h) exceeds the glomerular filtration rate (7.5 L/h), indicating tubular secretion in addition to glomerular filtration.
Protein binding: Oseltamivir phosphate: moderate (42%). Oseltamivir carboxylate: very low (<3%).
The volume of distribution of oseltamivir carboxylate was 23 to 26 liters after intravenous administration to 24 subjects.
Oral oseltamivir phosphate is readily absorbed and then largely converted to its active form—oseltamivir carboxylate—by hepatic esterases. At least 75% of the oral dose enters the systemic circulation as oseltamivir carboxylate. Of the oral dose, less than 5% of oseltamivir phosphate enters the systemic circulation.
Excretion: Kidneys: Oseltamivir carboxylate is primarily excreted via the kidneys (>99%). Renal clearance (18.8 L/hr) exceeds glomerular filtration rate (7.5 L/hr), indicating tubular secretion. Feces: Fecal excretion is <20% following oral administration of the radiolabeled dose.
For more complete data on absorption, distribution, and excretion of oseltamivir (8 items), please visit the HSDB record page.

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Metabolism/Metabolites
Oseltamivir is primarily converted to its active metabolite, oseltamivir carboxylate, by esterases in the liver. Oseltamivir carboxylate is not further metabolized. Neither oseltamivir nor its carboxylate is a substrate or inhibitor of cytochrome P450 isoenzymes. Phase II conjugates of these two compounds have not been found in vivo.
Oseltamivir is extensively converted to oseltamivir carboxylate by esterases in the liver. Neither oseltamivir nor its carboxylate is a substrate or inhibitor of cytochrome P450 isoenzymes.
Biotransformation: Liver; oseltamivir ethyl ester prodrug undergoes extensive hydrolysis to its active stellate form—oseltamivir carboxylate.
Biological Half-Life

After oral administration, plasma oseltamivir concentrations in most subjects decrease within 1 to 3 hours, while plasma oseltamivir carboxylate concentrations decrease within 6 to 10 hours.
Elimination: Oseltamivir is eliminated within 1 to 3 hours, while oseltamivir carboxylate is eliminated within 6 to 10 hours.

Hepatotoxicity
In clinical trials of oseltamivir, 2% of subjects experienced elevated serum transaminases, but all patients were asymptomatic and experienced transient symptoms. No clinically significant liver injury with jaundice was reported. The rate of ALT elevation in the oseltamivir group was generally similar to that in the placebo or control groups. Since its approval in 1999, oseltamivir has been widely used during seasonal influenza outbreaks. A few cases of mild liver injury have been reported in patients treated with oseltamivir, but the relationship between these injuries and oseltamivir has not been fully established. There are currently no reports of acute liver failure or chronic liver disease caused by oseltamivir use. Furthermore, some influenza patients may experience elevated serum enzymes and even mild jaundice during the acute phase, regardless of whether they receive any treatment. Probability Score: D (Possibly a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Limited data suggest that oseltamivir and its active metabolites are rarely excreted into breast milk. The mother takes 150 mg daily, resulting in a low concentration of the drug in breast milk, which is not expected to have any adverse effects on breastfed infants. Infants older than 2 weeks can take oseltamivir directly at a much higher dose than that found in breast milk.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
The binding rate of the active oseltamivir carboxylate metabolite to human plasma proteins is approximately 3%, which is negligible; while the binding rate of oseltamivir to human plasma proteins is 42%, insufficient to cause significant displacement-based drug interactions.

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Interactions
Concomitant use with probenecid leads to an approximately two-fold increase in the concentration of the active metabolite, due to reduced secretion of active anion exchangers in the kidneys.
In vitro studies have shown that neither oseltamivir nor oseltamivir carboxylate causes significant drug interactions. Oseltamivir is a good substrate for P450 mixed-function oxidases or glucuronyl transferases. Cimetidine is a non-specific cytochrome P450 isoenzyme inhibitor that competes with basic or cationic drugs for renal tubular secretion, but has no effect on the plasma concentrations of oseltamivir or oseltamivir carboxylate. Co-administration with amoxicillin does not alter the plasma concentrations of either compound, indicating weaker competition for the anion secretion pathway.

[1]. Sensitivity of Influenza Viruses to Zanamivir and Oseltamivir: A Study Performed on Viruses Circulating in France Prior to the Introduction of Neuraminidase Inhibitors in Clinical Practice. Antiviral Res. 2005 Oct;68(1):43-8.

[2]. Activities of JNJ63623872 and oseltamivir against influenza A H1N1pdm and H3N2 virus infections in mice. Antiviral Res. 2016 Dec;136:45-50.

[3]. Amantadine-oseltamivir combination therapy for H5N1 influenza virus infection in mice. Antivir Ther. 2007;12(3):363-70.

[4]. Oseltamivir-ribavirin combination therapy for highly pathogenic H5N1 influenza virus infection in mice. Antimicrob Agents Chemother. 2008 Nov;52(11):3889-97.

[5]. Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice. Antiviral Res. 2010 Oct;88(1):38-44.

Therapeutic Uses

Antiviral drug; enzyme inhibitor
Currently, the U.S. Centers for Disease Control and Prevention (CDC) recommends the use of oseltamivir or zanamivir to treat swine influenza (H1N1) virus infection.
Drug: Antiviral drug; an orally effective inhibitor of influenza virus neuraminidase; converted in vivo to the active acidic metabolite GS-4071.
Oseltamivir is indicated for the treatment of uncomplicated acute infections caused by influenza A virus in children aged 1 year and older with symptoms presenting no more than 2 days prior. /Included on U.S. product label/
For more complete data on the therapeutic uses of oseltamivir (8 types), please visit the HSDB record page.
Drug Warnings

The swine influenza (H1N1) virus contains a unique combination of gene segments that have not been previously reported in swine or human influenza viruses in the United States or elsewhere. The H1N1 virus is resistant to amantadine and ribavirin, but not to oseltamivir or zanamivir.
Adverse reactions occurring in ≥1% of adults, and at a higher rate than in the placebo group, include nausea, vomiting, bronchitis, insomnia, and dizziness. The most common adverse reaction is nausea (with or without vomiting), which usually occurs after the first dose and resolves within 1–2 days, but leads to discontinuation in less than 1% of adults. Adverse reactions occurring in 1% or more of children, and at a higher rate than in the placebo group, include vomiting, abdominal pain, epistaxis, ear problems, and conjunctivitis. Unlike amantadine and ribavirin, neuraminidase inhibitors (such as oseltamivir) do not appear to have adverse effects on the central nervous system.
FDA Pregnancy Risk Classification: C/Risk cannot be ruled out. There is a lack of adequate, well-controlled clinical studies, and animal studies have not shown any risk to the fetus. Use of this drug during pregnancy may cause harm to the fetus; however, the potential benefits may outweigh the potential risks. /
Severe bacterial infections may begin with flu-like symptoms or may coexist with or occur as a complication of influenza. Oseltamivir has not been shown to prevent such complications. For more complete data on drug warnings (7 in total) for oseltamivir, please visit the HSDB record page.

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Pharmacodynamics
Post-marketing reports indicate that patients with influenza treated with oseltamivir have experienced delirium and behavioral abnormalities, leading to injury and, in some cases, death. Because these events are reported voluntarily in clinical practice, their frequency cannot be estimated, but based on the efficacy of oseltamivir, these events appear to be uncommon. These events are primarily seen in pediatric patients and typically have a rapid onset and resolution. The association between oseltamivir and these events has not been established. Influenza can be accompanied by a variety of neurological and behavioral symptoms, including hallucinations, delirium, and behavioral abnormalities, which can even lead to death in some cases. These events can occur in cases of encephalitis or encephalopathy, but can also occur without apparent serious illness.

C16H28N2O4

312.40

312.204

C, 61.51; H, 9.03; N, 8.97; O, 20.48

196618-13-0

Oseltamivir-d3;1093851-61-6;Oseltamivir acid;187227-45-8; 204255-11-8 (phosphate); Oseltamivir-d3-1;Oseltamivir-d5;1093851-63-8;Oseltamivir-d3 hydrochloride; 196618-13-0; 204255-09-4 (HCl)

65028

White to off-white solid powder

1.2±0.1 g/cm3

445.4±55.0 °C at 760 mmHg

109 °C

223.2±31.5 °C

0.0±2.4 mmHg at 25°C

1.529

2.52

2

5

8

22

418

3

CCC(CC)O[C@@H]1C=C(C[C@@H]([C@H]1NC(=O)C)N)C(=O)OCC

VSZGPKBBMSAYNT-RRFJBIMHSA-N

InChI=1S/C16H28N2O4/c1-5-12(6-2)22-14-9-11(16(20)21-7-3)8-13(17)15(14)18-10(4)19/h9,12-15H,5-8,17H2,1-4H3,(H,18,19)/t13-,14+,15+/m0/s1

ethyl (3R,4R,5S)-4-acetamido-5-amino-3-pentan-3-yloxycyclohexene-1-carboxylate

Tamvir; oseltamivir; Tamiflu-Free; (-)-oseltamivir; GS-4104; GS 4104; oseltamivirum;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (320.10 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)