Influenza virus neuraminidase (IC50 = 2 nM) ; influenza A/H3N2, A/H1N2, A/H1N1, and B viruses

Oseltamivir appears to be effective against influenza B and A/H1N1 viruses (mean B IC50 value: 13 nM; mean H1N1 IC50 value: 1.34 nM), but it is more effective against A/H1N2 and A/H3N2 viruses (average H3N2 IC50 value: 0.67 nM; average H1N2 IC50 value: 0.9 nM)[3]. The IC50 of RWJ-270201 (about 0.34 nM) in an influenza A virus neuraminidase inhibition experiment was similar to that of oseltamivir acid (0.45 nM). The B virus isolate RWJ-270201 has an IC50 of 1.36 nM, which is less than oseltamivir carboxylate (8.5 nM) and similar to zanamivir (2.7 nM) [4].

The antiviral activity of oseltamivir acid (0.1, 1, or 10 mg/kg/day) on the Vietnam/1203/04 (VN1203/04) virus is dose-dependent. It is administered twice daily by borderline gavage. Fifty percent of the mice were protected by a 5-day treatment schedule of 10 mg/kg/day; the delayed death of the treated group suggests that the virus was still replicating after the treatment was stopped. Rationale: Doses of 1 and 10 mg/kg/day resulted in 60% and 80% morbidity, respectively, and significantly decreased viral titers in organs [5].

GS 4071 (oseltamivir carboxylate) is a potent carbocyclic transition-state analog inhibitor of influenza virus neuraminidase with activity against both influenza A and B viruses in vitro. GS 4116, the guanidino analog of GS 4071, is a 10-fold more potent inhibitor of influenza virus replication in tissue culture than GS 4071. In this study we determined the oral bioavailabilities of GS 4071, GS 4116, and their respective ethyl ester prodrugs in rats. Both parent compounds and the prodrug of the guanidino analog exhibited poor oral bioavailability (2 to 4%) and low peak concentrations in plasma (Cmaxs; Cmax <0.06 microg/ml). In contrast, GS 4104, the ethyl ester prodrug of GS 4071, exhibited good oral bioavailability (35%) as GS 4071 and high Cmaxs of GS 4071 (Cmax = 0.47 microg/ml) which are 150 times the concentration necessary to inhibit influenza virus neuraminidase activity by 90%. The bioavailability of GS 4104 as GS 4071 was also determined in mice (30%), ferrets (11%), and dogs (73%). The plasma of all four species exhibited high, sustained concentrations of GS 4071 such that at 12 h postdosing the concentrations of GS 4071 in plasma exceeded those necessary to inhibit influenza virus neuraminidase activity by 90%. These results demonstrate that GS 4104 is an orally bioavailable prodrug of GS 4071 in animals and that it has the potential to be an oral agent for the prevention and treatment of influenza A and B virus infections in humans.[1]

RWJ-270201 is a novel cyclopentane inhibitor of influenza A and B virus neuraminidases (NAs). We compared the ability of RWJ-270201 to inhibit NA activity of clinical influenza isolates and viruses with defined resistance mutations with that of zanamivir and oseltamivir carboxylate. In NA inhibition assays with influenza A viruses, the median 50% inhibitory concentration (IC(50)) of RWJ-270201 (approximately 0.34 nM) was comparable to that of oseltamivir carboxylate (0.45 nM) but lower than that of zanamivir (0.95 nM). For influenza B virus isolates, the IC(50) of RWJ-270201 (1.36 nM) was comparable to that of zanamivir (2.7 nM) and less than that of oseltamivir carboxylate (8.5 nM). A zanamivir-resistant variant bearing a Glu119-to-Gly (Glu119-->Gly) or Glu119-->Ala substitution in an NA (N2) remained susceptible to RWJ-270201 and oseltamivir carboxylate. However, a zanamivir-selected variant with an Arg292-->Lys substitution in an NA (N2) showed a moderate level of resistance to RWJ-270201 (IC(50) = 30 nM) and zanamivir (IC(50) = 20 nM) and a high level of resistance to oseltamivir carboxylate (IC(50) > 3,000 nM). The zanamivir-resistant influenza B virus variant bearing an Arg152-->Lys substitution was resistant to each NA inhibitor (IC(50) = 100 to 750 nM). The oseltamivir-selected variant (N1) with the His274-->Tyr substitution exhibited resistance to oseltamivir carboxylate (IC(50) = 400 nM) and to RWJ-270201 (IC(50) = 40 nM) but retained full susceptibility to zanamivir (IC(50) = 1.5 nM). Thus, drug-resistant variants with substitutions in framework residues 119 or 274 can retain susceptibility to other NA inhibitors, whereas replacement of functional residue 152 or 292 leads to variable levels of cross-resistance. We conclude that RWJ-270201 is a potent inhibitor of NAs of wild-type and some zanamivir-resistant or oseltamivir-resistant influenza A and B virus variants[4].

Distribution of oseltamivir and oseltamivir carboxylate (OC) to the CNS and brain of rats.[6]
Several studies were performed to characterize the pharmacokinetics of oseltamivir and OC in the plasma, cerebrospinal fluid (CSF), and brain of Sprague-Dawley rats following single-dose bolus administration of oseltamivir (intravenous [i.v.] and oral) and OC (i.v.). In the i.v. studies, nonfasted adult rats (two groups of 35 animals for each test substance) received a dose of 30 mg/kg body weight of either oseltamivir or OC in aqueous solution with sodium chloride (0.9%; pH 4.0) via slow injection into the tail vein over 20 to 30 s. In both i.v. studies, pharmacokinetic sampling took place at 5 min and at 0.25, 0.5, 1, 2, 4, and 8 h postdose (four or five rats/time point). In the oral study, rats received oseltamivir phosphate by oral gavage at a dose of 1,000 mg/kg free base, and sampling was performed at 1, 2, 4, 6, and 8 h postdose (four rats/time point). Rats were terminally anesthetized using isoflurane (5% in oxygen) at each scheduled time point (at 5 min and at 0.25, 0.5, 1, 2, 4, and 8 h postdose for i.v. studies and at 1, 2, 4, 6, and 8 h postdose for oral studies), and approximately 0.5 ml of blood and as much CSF as possible were collected via puncture of the heart and cysterna magna, respectively. To investigate the effect of residual blood in brain tissue on the observed oseltamivir and OC concentrations in the i.v. studies, brain samples were obtained by whole-brain removal and homogenization in one group per test substance, while in the other group, brain tissue was perfused transcardially with physiological sodium chloride solution (0.9%; ca. 30 ml) before tissue collection and homogenization. Brain tissue perfusion was also performed in the oral study before collection and homogenization. All samples were stored at −20°C.

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Female 6-week-old BALB/c mice are anesthetized with isofluorane and intranasally inoculated with 50 μL of 10-fold serial dilutions of VN1203/04 virus in PBS. The mouse lethal dose (MLD50) is calculated after a 16-day observation period. Oseltamivir is administered by oral gavage twice daily for 5 or 8 days to groups of 10 mice at dosages of 0.1, 1, and 10 mg/kg/day. Control (infected but untreated) mice received sterile PBS (placebo) on the same schedule. Four hours after the first dose of Oseltamivir, the mice are inoculated intranasally with 5 MLD50 of VN1203/04 virus in 50 μL of PBS. Survival and weight change are observed for 24 days. Virus titers in the mouse organs are determined on days 3, 6, and 9 after inoculation. Three mice from each experimental and placebo group are killed, and the lungs and brains are removed. The organs are homogenized and suspended in 1 mL of PBS. The cellular debris is cleared by centrifugation at 2000 g for 5 min. The limit of virus detection is 0.75 log10 EID50. For calculation of the mean, samples with a virus titer <0.75 log10 EID50/mL are assigned a value of 0. Virus titers in each organ are calculated by use of the method of Reed and Muench and are expressed as mean log10 EID50/mL±SE.[3]

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Several studies are performed to characterize the pharmacokinetics of Oseltamivir and OC in the plasma, cerebrospinal fluid (CSF), and brain of Sprague-Dawley rats following single-dose bolus administration of Oseltamivir (intravenous [i.v.] and oral) and OC (i.v.). In the i.v. studies, nonfasted adult rats (two groups of 35 animals for each test substance) received a dose of 30 mg/kg body weight of either Oseltamivir or Oseltamivir carboxylate (OC) in aqueous solution with sodium chloride (0.9%; pH 4.0) via slow injection into the tail vein over 20 to 30 s. In both i.v. studies, pharmacokinetic sampling took place at 5 min and at 0.25, 0.5, 1, 2, 4, and 8 h postdose (four or five rats/time point).[4]

[1]. Identification of GS 4104 as an orally bioavailable prodrug of the influenza virus neuraminidase inhibitor GS 4071. Antimicrob Agents Chemother. 1998 Mar;42(3):647-53.

[2]. Oseltamivir carboxylate, the active metabolite of oseltamivir phosphate (Tamiflu), detected in sewage discharge and river water in Japan. Environ Health Perspect. 2010 Jan;118(1):103-7.

[3]. 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.

[4]. Comparison of the activities of zanamivir, oseltamivir, and RWJ-270201 against clinical isolates of influenza virus and neuraminidase inhibitor-resistant variants.Antimicrob Agents Chemother. 2001 Dec;45(12):3403-8.

[5]. Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice. J Infect Dis. 2005 Aug 15;192(4):665-72.

[6]. Nonclinical pharmacokinetics of oseltamivir and oseltamivir carboxylate in the central nervous system. Antimicrob Agents Chemother. 2009 Nov;53(11):4753-61.

Oseltamivir acid is a cyclohexenecarboxylic acid that is cyclohex-1-ene-1-carboxylic acid which is substituted at positions 3, 4, and 5 by pentan-3-yloxy, acetamido, and amino groups, respectively (the 3R,4R,5S enantiomer). An antiviral drug, it is used as the corresponding ethyl ester prodrug, oseltamivir, to slow the spread of influenza. It has a role as an antiviral drug, an EC 3.2.1.18 (exo-alpha-sialidase) inhibitor and a marine xenobiotic metabolite. It is a cyclohexenecarboxylic acid, an acetate ester, an amino acid and a primary amino compound.

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Oseltamivir acid is a Neuraminidase Inhibitor. The mechanism of action of oseltamivir acid is as a Neuraminidase Inhibitor.

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Influenza virus neuraminidase inhibitors (NAIs) were introduced in clinical practice in various parts of the world since 1999 but were only scarcely distributed in France. Prior to the generalization of zanamivir and oseltamivir utilization in our country, we decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs, based upon a fluorometric neuraminidase enzymatic test. Our study was performed on clinical samples collected by practitioners of the GROG network (Groupe Régional d'Observation de la Grippe) in the south of France during the 2002-2003 influenza season. Out of 355 isolates tested in the fluorometric neuraminidase activity assay, 267 isolates could be included in inhibition assay against anti-neuraminidase drugs. Differences in IC50 range were found according to the subtype and the anti-neuraminidase drug. Influenza B and A/H1N1 viruses appeared to be more sensitive to zanamivir than to oseltamivir (mean B IC50 values: 4.19 nM versus 13 nM; mean H1N1 IC50 values: 0.92 nM versus 1.34 nM), while A/H1N2 and A/H3N2 viruses were more sensitive to oseltamivir than to zanamivir (mean H3N2 IC50 values: 0.67 nM versus 2.28 nM; mean H1N2 IC50 values: 0.9 nM versus 3.09 nM). Out of 128 N2 carrying isolates, 10 isolates had zanamivir or oseltamivir IC50 values in upper limits compared to their respective data range. Sequencing of the neuraminidase of these outliers N2 highlighted several mutations, but none of them were associated with resistance to neuraminidase inhibitors.[3]

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Background: Oseltamivir phosphate (OP; Tamiflu) is a prodrug of the anti-influenza neuraminidase inhibitor oseltamivir carboxylate (OC) and has been developed for the treatment and prevention of both A and B strains of influenza. The recent increase in OP resistance in influenza A virus (H1N1; commonly called "swine flu") has raised questions about the widespread use of Tamiflu in seasonal epidemics and the potential ecotoxicologic risk associated with its use in the event of a pandemic.
Objectives: The objectives of this study were to develop an analytical method for quantitative determination of OC in sewage treatment plant (STP) effluent and receiving river water, and to investigate the occurrence of OC in STP effluent and river water in Japan during a seasonal flu outbreak.
Methods: We developed an analytical method based on solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. Using this method, we analyzed samples from three sampling campaigns conducted during the 2008-2009 flu season in Kyoto City, Japan.
Results: The highest concentration of OC detected in STP discharge was 293.3 ng/L from a conventional activated-sludge-based STP; however, we detected only 37.9 ng/L from an advanced STP with ozonation as a tertiary treatment. In the receiving river water samples, we detected 6.6-190.2 ng/L OC, during the peak of the flu season.
Conclusion: OC is present in STP effluent and river water only during the flu season. Ozonation as tertiary treatment in STP will substantially reduce the OC load in STP effluent during an influenza epidemic or pandemic.[2]

C14H25CLN2O4

320.81

320.15

C, 52.41; H, 7.86; Cl, 11.05; N, 8.73; O, 19.95

1415963-60-8

Oseltamivir acid;187227-45-8;Oseltamivir acid-d3;1242184-43-5; 1415963-60-8 (HCl)

66545297

Typically exists as solid at room temperature

2.7

4

5

6

21

391

3

CCC(CC)O[C@H]1[C@H](NC(C)=O)[C@@H](N)CC(C(O)=O)=C1.Cl

OTBMMOBYSNFNOE-LUHWTZLKSA-N

InChI=1S/C14H24N2O4.ClH/c1-4-10(5-2)20-12-7-9(14(18)19)6-11(15)13(12)16-8(3)17/h7,10-13H,4-6,15H2,1-3H3,(H,16,17)(H,18,19)1H/t11-,12+,13+/m0./s1

(3R,4R,5S)-4-Acetamido-5-amino-3-pentan-3-yloxycyclohexene-1-carboxylic acid hydrochloride

Oseltamivir carboxylate hydrochloride, Oseltamivir carboxylate HCI, Oseltamivir carboxylate(HCI); Oseltamivir carboxylate HCl; Oseltamivir acid (hydrochloride); 1415963-60-8; 1415963-60-8 (HCl); (3R,4R,5S)-4-Acetamido-5-amino-3-pentan-3-yloxycyclohexene-1-carboxylic acid hydrochloride; OSELTAMIVIR CARBOXYLATE HYDROCHLORIDE; 1-Cyclohexene-1-carboxylic acid, 4-(acetylamino)-5-amino-3-(1-ethylpropoxy)-, hydrochloride (1:1), (3R,4R,5S)-; Oseltamivir acid hydrochloride; (3R,4R,5S)-4-Acetamido-5-amino-3-(1-ethylpropoxy)cyclohex-1-ene-1-carboxylic acid hydrochloride; Ro 64-0802 hydrochloride, GS4071 HCl.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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