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
Oseltamivir is readily absorbed from the gastrointestinal tract after oral administration of oseltamivir phosphate and is extensively converted by predominantly hepatic esterases to the active metabolite oseltamivir carboxylate. At least 75 % of an oral dose reaches the systemic circulation as the active metabolite. Exposure to the pro-drug is less than 5 % relative to the active metabolite. Plasma concentrations of both pro-drug and active metabolite are proportional to dose and are unaffected by co-administration with food. Pharmacokinetic parameters following twice daily dosing of oseltamivir 75mg capsules are as follows: Cmax of oseltamivir and oseltamivir carboxylate were found to be 65ng/mL and 348ng/mL, respectively, while AUC (0-12h) of oseltamivir and oseltamivir carboxylate were found to be 112ng·h/mL and 2719ng·h/mL, respectively.
Following absorption, oseltamivir is more than 90 % eliminated through conversion to oseltamivir carboxylate and subsequent elimination entirely through renal excretion. During clinical studies, less than 20 % of oral radiolabelled dose was found to be eliminated in faeces.
The mean volume of distribution at steady state of the oseltamivir carboxylate ranges approximately between 23 and 26 liters in humans, a volume that is roughly equivalent to extracellular body fluid. Since neuraminidase activity is extracellular, oseltamivir carboxylate distributes to all sites of influenza virus spread.
Renal clearance (18.8 l/h) of the drug exceeds glomerular filtration rate (7.5 l/h) indicating that tubular secretion occurs in addition to glomerular filtration.
Protein binding: Oseltamivir phosphate: Moderate (42%). Oseltamivir carboxylate: Very low < 3%.
Oseltamivir carboxylate: Volume of distribution is 23 to 26 liters following intravenous administration in 24 subjects.
Oral oseltamivir phosphate is readily absorbed then extensively converted to oseltamivir carboxylate, the active form, predominantly by hepatic esterases. At least 75% of an oral dose reaches the systemic circulation as oseltamivir carboxylate. Less than 5% of an oral dose reaches the systemic circulation as oseltamivir phosphate.
Elimination: Renal: Oseltamivir carboxylate is extensively eliminated by renal excretion (> 99%). Renal clearance (18.8 L/hr) exceeds glomerular filtration rate (7.5 L/hr), indicating that tubular secretion occurs. Fecal: Elimination of an oral radiolabeled dose in < 20% in the feces.
For more Absorption, Distribution and Excretion (Complete) data for OSELTAMIVIR (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
Oseltamivir is extensively converted to the active metabolite, oseltamivir carboxylate, by esterases located predominantly in the liver. Oseltamivir carboxylate is not further metabolized. Neither oseltamivir nor oseltamivir carboxylate is a substrate for, or inhibitor of, cytochrome P450 isoforms. No phase 2 conjugates of either compound have been identified in vivo.
Oseltamivir is extensively converted to oseltamivir carboxylate by esterases located predominantly in the liver. Neither oseltamivir nor oseltamivir carboxylate is a substrate for, or inhibitor of, cytochrome p450 isoforms.
Biotransformation: Hepatic; oseltamivir, ethyl ester prodrug, undergoes extensive hydrolysis to the active aster form, oseltamivir carboxylate.

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Biological Half-Life
Plasma concentrations of oseltamivir declined with a half-life of 1 to 3 hours in most subjects after oral administration, although plasma concentrations of oseltamivir carboxylate declined with a half-life of 6 to 10 hours in most subjects after oral administration.
Elimination: 1 to 3 hours for oseltamivir and 6 to 10 hours for oseltamivir carboxylate.

Hepatotoxicity
In clinical trials of oseltamivir, serum aminotransferase elevations occurred in 2% of treated subjects, but were asymptomatic and transient in all and there were no reports of clinically apparent liver injury with jaundice. The rates of ALT elevations with oseltamivir were generally similar to those treated with placebo or with a comparative agents. Since its approval in 1999, oseltamivir has been widely used during influenza seasonal outbreaks. There have been a few, isolated reports of mild liver injury in patients receiving oseltamivir, but the relationship of the injury with oseltamivir has not always been very convincingly shown. There have been no reports of acute liver failure or chronic liver disease attributed to oseltamivir use. Furthermore, a proportion of patients with influenza have serum enzyme elevations and even mild jaundice during the acute illness, independent of any therapy.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited data indicate that oseltamivir and its active metabolite are poorly excreted into breastmilk. Maternal dosages of 150 mg daily produce low levels in milk and would not be expected to cause any adverse effects in breastfed infants. Infants over 2 weeks of age can receive oseltamivir directly in doses much larger than those in breastmilk.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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

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Interactions
Concomitant administration /with probenecid/ results in an approximate two-fold increase in the active metabolite due to a decrease in active anionic tubular secretion in the kidney.
In vitro studies demonstrate that neither oseltamivir nor oseltamivir carboxylate is a good substrate for P450 mixed-function oxidases or for glucuronyl transferases. Cimetidine, a non-specific inhibitor of cytochrome P450 isoforms and competitor for renal tubular secretion of basic or cationic drugs, has no effect on plasma levels of oseltamivir or oseltamivir carboxylate.
Coadministration with amoxicillin does not alter plasma levels of either compound, indicating that competition for the anionic secretion pathway is weak.

[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 Agents; Enzyme Inhibitors
At this time, CDC recommends the use of oseltamivir or zanamivir for the treatment of infection with swine influenza (H1N1) viruses.
MEDICATION: Antiviral; Orally active inhibitor of influenza virus neuraminidase; converted in vivo to the active acid metabolite, GS-4071.
Oseltamivir is indicated for the treatment of uncomplicated acute infection caused by influenza A virus in patients older than 1 year of age who have been symptomatic for no more than 2 days. /Included in US product labeling/
For more Therapeutic Uses (Complete) data for OSELTAMIVIR (8 total), please visit the HSDB record page.

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Drug Warnings
Swine influenza (H1N1) viruses contain a unique combination of gene segments that have not been reported previously among swine or human influenza viruses in the US or elsewhere. The H1N1 viruses are resistant to amantadine and rimantadine but not to oseltamivir or zanamivir.
Adverse effects occurring in 1% or more of adults and at an incidence greater than that with placebo include nausea, vomiting, bronchitis, insomnia, and vertigo. Nausea, with or without vomiting, was most common, usually occurring after the initial dose and resolving within 1-2 days, but resulting in drug discontinuance in less than 1% of adults. Adverse effects occurring in 1% or more of children and at an incidence greater than with placebo include vomiting, abdominal pain, epistaxis, otic disorder, and conjunctivitis. Unlike amantadine and rimantadine, neuraminidase inhibitors like oseltamivir do not appear to adversely affect the CNS.
FDA Pregnancy Risk Category: C /RISK CANNOT BE RULED OUT. Adequate, well controlled human studies are lacking, and animal studies have shown risk to the fetus or are lacking as well. There is a chance of fetal harm if the drug is given during pregnancy; but the potential benefits may outweigh the potential risk./
Serious bacterial infections may begin with influenza-like symptoms or may coexist with or occur as complications during the course of influenza. /Oseltamivir/ has not been shown to prevent such complications.
For more Drug Warnings (Complete) data for OSELTAMIVIR (7 total), please visit the HSDB record page.

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Pharmacodynamics
There have been postmarketing reports of delirium and abnormal behavior leading to injury, and in some cases resulting in fatal outcomes, in patients with influenza who were receiving oseltamivir. Because these events were reported voluntarily during clinical practice, estimates of frequency cannot be made but they appear to be uncommon based on oseltamivir. These events were reported primarily among pediatric patients and often had an abrupt onset and rapid resolution. The contribution of oseltamivir to these events has not been established. Influenza can be associated with a variety of neurologic and behavioral symptoms that can include events such as hallucinations, delirium, and abnormal behavior, in some cases resulting in fatal outcomes. These events may occur in the setting of encephalitis or encephalopathy but can occur without obvious severe disease.

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.)