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]

Oseltamivir, a potent and selective inhibitor of influenza A and B virus neuraminidases, is a prodrug that is systemically converted into the active metabolite oseltamivir carboxylate. In light of reported neuropsychiatric events in influenza patients, including some taking oseltamivir, and as part of a full assessment to determine whether oseltamivir could contribute to, or exacerbate, such events, we undertook a series of nonclinical studies. In particular, we investigated (i) the distribution of oseltamivir and oseltamivir carboxylate in the central nervous system of rats after single intravenous doses of oseltamivir and oseltamivir carboxylate and oral doses of oseltamivir, (ii) the active transport of oseltamivir and oseltamivir carboxylate in vitro by transporters located in the blood-brain barrier, and (iii) the extent of local conversion of oseltamivir to oseltamivir carboxylate in brain fractions. In all experiments, results showed that the extent of partitioning of oseltamivir and especially oseltamivir carboxylate to the central nervous system was low. Brain-to-plasma exposure ratios were approximately 0.2 for oseltamivir and 0.01 for oseltamivir carboxylate. Apart from oseltamivir being a good substrate for the P-glycoprotein transporter, no other active transport processes were observed. The conversion of the prodrug to the active metabolite was slow and limited in human and rat brain S9 fractions. Overall, these studies indicate that the potential for oseltamivir and oseltamivir carboxylate to reach the central nervous system in high quantities is low and, together with other analyses and studies, that their involvement in neuropsychiatric events in influenza patients is unlikely.[6]

[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.
Oseltamivir acid is a Neuraminidase Inhibitor. The mechanism of action of oseltamivir acid is as a Neuraminidase Inhibitor.
See also: Oseltamivir (is active moiety of); Oseltamivir Phosphate (active moiety of); Oseltamivir carboxylate (annotation moved to).

C14H24N2O4

284.356

284.173

C, 59.14; H, 8.51; N, 9.85; O, 22.51

187227-45-8

Oseltamivir;196618-13-0;Oseltamivir acid hydrochloride;1415963-60-8;Oseltamivir acid-d3;1242184-43-5;Oseltamivir phosphate;204255-11-8

449381

White to off-white solid powder

1.2±0.1 g/cm3

508.7±50.0 °C at 760 mmHg

183-185°C

261.5±30.1 °C

0.0±2.8 mmHg at 25°C

1.525

0.45

3

5

6

20

391

3

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

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

NENPYTRHICXVCS-YNEHKIRRSA-N

Oseltamivir carboxylate

GS-4071; Ro 64-0802; GS 4071; Ro-64-0802; GS4071; Ro64-0802; Oseltamivir acid; 187227-45-8; Oseltamivir carboxylate; GS4071; Oseltamivir free acid; (-)-;Oseltamivir acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : ≥ 230 mg/mL (~808.86 mM)
H2O : ~125 mg/mL (~439.60 mM)

Solubility in Formulation 1: ≥ 5.75 mg/mL (20.22 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 57.5 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: ≥ 5.75 mg/mL (20.22 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 57.5 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: ≥ 5.75 mg/mL (20.22 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 57.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (351.68 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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