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

After oral treatment, oseltamivir phosphate (OP) is a prodrug that is easily absorbed from the gastrointestinal tract. Hepatic esterases are primarily responsible for the prodrug's substantial conversion to oseltamivir carboxylate (OC) [1]. One commonly used anti-influenza sialidase inhibitor is oseltamivir phosphate. The metabolic activity of CMA07 and CMT-U27 cell lines was considerably decreased following 305 μM oseltamivir phosphate treatment (p=0.005 and p<0.0001, respectively), according to a one-way ANOVA test. On the other hand, when oseltamivir phosphate was treated at 0.305 μM (p=0.9781), 3.05 μM (p=0.7436), and 30.5 μM (p=0.9623) in contrast to control cells, no statistically significant changes were seen. In order to assess the impact of oseltamivir phosphate on CMA07 and CMT-U27 programmed cell death, the TUNEL test was employed, taking into account that 305 μM oseltamivir phosphate administration reduces cellular metabolic activity. When compared to lower oseltamivir concentrations or PBS, 24-hour oseltamivir phosphate administration, specifically 305 μM, significantly increased CMA07 (p=0.001) and CMT-U27 (p=0.0002) DNA fragmentation[2].

Ki-67 antigen and caspase-3 protein were used to assess CMT-U27 xenograft tumor cell proliferation and apoptosis, respectively. Almost no differences were found in Ki-67 and caspase 3 (p=0.2) expression between oseltamivir-treated and untreated mice [2]. Oseltamivir phosphate-treated mice showed significantly more inflammatory infiltrates in primary tumors (p=0.01).

Sialidase activity assay[2]
Higher sialylation levels are expected in malignant cells when compared with benign counterparts. This is as likely to be dependent on the activity both sialyltranferases and on sialidase. To evaluate the effect of oseltamivir phosphate on the activity of sialidases, an in vitro assay using a modified sialic acid (4-methyl-umbelliferyl-Nacetylneuraminic acid-4-MuNana), was performed using CMA07 and CMT-U27 canine mammary tumor cells. Sialidase activity was determined by obtaining the metabolic conversion of the sialic acid analog, 4-MuNana, into the fluorescent compound methyl-umbelliferone (blue color), upon treatment with different doses of oseltamivir phosphate. Cells were grown in 12 mm circular glass until confluence and then incubated for 24 h with medium containing different oseltamivir phosphate concentrations (0.305 μM, 3.05 μM, 30.5 μM and 305 μM oseltamivir phosphate dissolved in PBS), and the vehicle of the drug (PBS) was used as control. After 24 hours of treatment, each 12 mm circular glass was placed on a slide and incubated in a 2μM 4-MuNana (2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid) solution. Slides were immediately observed with epi-fluorescent microscopy under UV light (excitation wave length at 360 nm, and emission wave length at 440 nm), as has been previously described. Slides were analyzed and images were taken with a Carl Zeiss fluorescent microscope.

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Antiviral Determination[3]
For antiviral determination, infected cells (0.01 MOI) were cultured in Opti-MEM (2 μg/mL TPCK–trypsin) containing increasing concentrations of ribavirin or oseltamivir phosphate. At around 24 h post infection (p.i.), aliquots were removed, and Gaussia luciferase activity was determined.

Cell morphology analysis[2]
CMA07 and CMT-U27 cells were plated at a density of 1x104 cells per well in 6-well plates, in triplicate. Three different oseltamivir phosphate concentrations were studied: 0.305 μM, 3.05 μM and 30.5 μM, and PBS was used as control. Analysis of cell confluence and morphology was performed using a contrast inverted microscope over a period of 7 days. Photographs were taken at days 0 and 7 under 200x magnification.

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Cell proliferation assay[2]
CMA07 and CMT-U27 cells were cultured in 24-well plates in triplicate for each condition: 0.305 μM, 3.05 μM, 30.5 μM and 305 μM oseltamivir phosphate and PBS was used as control. Cells were counted every day for 7 days in a Neubauer’s chamber in a 1:2 dilution of cells in 0.4% trypan blue and cell count was done using the volume conversion factor for 1 mm3, which is 1x104. This assay was repeated 3 times and growth curves were traced.

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Cell growth assay[2]
Cell growth was determined in CMA07 and CMT-U27 cell lines using a commercially available kit CellTiter 96 AQueous One Solution reagent, and performed according to manufacturer’s instructions. Briefly, cells were plated in 96-well plates in triplicate, at a density of 5x103 cells per well. After cell attachment, oseltamivir phosphate was added at 0.305 μM, 3.05 μM, 30.5 μM and 305 μM final concentrations and PBS was used as control. Cellular metabolism was measured by adding MTS tetrazolium reagent and absorbance was recorded at 490nm. Measurements were performed at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hours. An additional control measurement was performed at time-point 0h, in a culture well without cells. The experiments were performed twice.

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TUNEL assay[2]
CMA07 and CMT-U27 cell lines were cultured in 6-well plates and then treated with different concentrations of oseltamivir phosphate (0.305 μM, 3.05 μM, 30.5 μM and 305 μM, and PBS was used as control). After 24 hours of treatment, culture medium and tripsinized cells were collected and centrifuged for 10 minutes at 2000 rpm. Cells were washed in PBS and fixed in cold methanol for 20 minutes. After fixation, cells were ressuspended in 1 mL of PBS for cytospin procedure. Briefly, 100 μL of cell suspension were centrifuged in a cytospin3 centrifuge using polilysine coated slides. Slides were then used for in situ cell death detection using a commercially available kit (In situ cell death detection kit, fluorescein from Roche) based on labeling DNA double strand breaks, according to manufacturer's instructions. Slides were observed under a fluorescence microscope using a 488nm excitation wavelength and percentage of dead cells was calculated by recording positive TUNEL cells in relation to total cells using the ImageJ software. This assay was performed twice.

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Wound-healing The wound-healing assay was performed using a benign (CMA07) and a highly metastatic (CMT-U27) canine mammary tumor cell line in a time-lapse microscope. Briefly, 20x104 cells were plated onto a 24-well culture plate and after reaching high confluence an artificial "wound" was made with a pipette tip. Culture medium was replaced with the different oseltamivir phosphate concentrations: 0.305 μM, 3.05 μM and 30.5 μM oseltamivir phosphate and PBS as control. Wound image acquisition was done with 5 minutes intervals for 48 hours, using the program Axio Vision Release 4.8.2. and converted in video. Treatment of cells with 305 μM oseltamivir phosphate was not performed due to its previously shown cytotoxicity. This assay was performed twice.

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Fluorescent cytochemistry[2]
Cells were cultured in glass coverslips and the culture medium was supplemented with 0.305 μM, 3.05 μM and 30.5 μM oseltamivir phosphate and PBS as control, for 24 hours. Cells were then washed with PBS and fixed with cold methanol for 20 minutes. Following fixation, cells were re-hydrated with PBS and blocked with 10% BSA for 20 minutes. Plant lectins SNA, MAL I, and MAL II (Biotinylated Maackia amurensis lectin II, B-1265, Vector Laboratories) were diluted 1:300 in 5% BSA in PBS and incubated on slides for 1 hour at room temperature. Slides were then washed three times with PBS and incubated 1 hour with streptavidin-FITC. After two washes with PBS, slides were incubated for 10 minutes with DAPI in PBS and slides were mounted in Vectashield mounting medium for fluorescence analysis. Slides were analysed and images were taken in a Carl Zeiss fluorescent microscope.

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Western Blot analysis[2]
Cells from CMA07 and CMT-U27 cell lines were grown to confluence in 6 well-plates and different concentrations of oseltamivir phosphate were added to the medium (0.305 μM, 3.05 μM and 30.5 μM oseltamivir phosphate). After 24 hours of incubation, cells were washed three times with PBS and lysed using RIPA lysis buffer (50 mM Tris HCl, pH 8; 150 mM NaCl; 1% NP-40; 0,5% sodium desoxicolate; 0,1% SDS) containing complete protease inhibitor cocktail, 1mM PMSF (phenylmethyl sulfonyl fluoride), and 1mM Na3VO4 (sodium orthovanadate). Protein concentration was determined using the biocinchoninic acid method from Pierce BCA Protein Assay Kit, according to the manufacturer’s instructions.

Experimental mice groups and drug treatment[2]
Female NIH(S)II-nu/nu nude mice, aged 4–6 weeks, were orthotopically inoculated with 1 x 106 viable CMT-U27 canine breast cancer cells in the mammary fat pad using a 25 gauge needle. A total of 8 mice were inoculated. When nodules reached a volume of approximately 500mm3, mice (n = 8) were randomized and divided into control group (n = 4) and treatment group (n = 4).The animals received intraperitoneally (IP) dailly either 100 μL of PBS (control group) or 100mg/Kg of Oseltamivir phosphate purchased from the pharmacy, diluted in PBS (treatment group) until time of death. Tumor size was measured using calipers, and tumor volume (mm3) was estimated by width x length x height. To observe metastization, primary tumors of all mice were surgically removed when a mean volume of ~1000–1500mm3 was reached. Mice were anesthetized by IP administration of 100 μL of a mixture containing 50 mg/kg of Ketamin (IMALGENE 1000) and 1 mg/kg of medetomidine hydrochloride (Medetor) and the tumor was excised. We used 2.5 mg/kg of atipamezole (Revertor) per mice to antagonize the effect of anesthesia. Mice were treated with an oral solution of 10 mg/kg of tramadol chloridrate (Tramal) every 8h for 24–48h to prevent pain. Animals were followed up after surgical excision of primary tumors for invasion and/or metastization signs.

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Mouse Infections[3]
Female BALB/c mice (4 to 6 weeks old) were inoculated intranasally with the indicated amount of virus in 30 μL PBS under light isoflurane anesthesia. Body weight was monitored daily. Mice losing 20% of their original body weight were humanely euthanized. At the indicated time, the mice were euthanized, and the lungs were removed for further analysis. Viral load in lung homogenates was determined by both TCID50 and the luciferase assay. For antiviral treatments, mice were treated with either 80 mg/kg/day of ribavirin or 20–50 mg/kg/day of oseltamivir phosphate in PBS, administered by intraperitoneal injection. The treatments were started 2 h before infection and were given twice daily until the end of the experiment.

Absorption
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.

Route of Elimination
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.

Volume of Distribution
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.

Clearance
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). 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. Drugs and Lactation Database (LactMed) 11.1.5 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. Thomson/Micromedex. Drug Information for the Health Care Professional. Volume 1, Greenwood Village, CO. 2006., p. 2305 Hazardous Substances Data Bank (HSDB) 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. Physicians Desk Reference 60th ed, Thomson PDR, Montvale, NJ 2006., p. 2811 Hazardous Substances Data Bank (HSDB) Coadministration with amoxicillin does not alter plasma levels of either compound, indicating that competition for the anionic secretion pathway is weak. 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.

[1]. Transplacental transfer of Oseltamivir phosphate and its metabolite Oseltamivir carboxylate using the ex vivo human placenta perfusion model in Chinese Hans population. J Matern Fetal Neonatal Med. 2016 Aug 8:1-5.

[2]. Anti-influenza neuraminidase inhibitor Oseltamivir phosphate induces canine mammary cancer cell aggressiveness. PLoS One. 2015 Apr 7;10(4):e0121590.

[3]. A Simple and Robust Approach for Evaluation of Antivirals Using a Recombinant Influenza Virus Expressing Gaussia Luciferase. Viruses. 2018 Jun 13;10(6). pii: E325.

Oseltamivir phosphate is a phosphate salt. It contains an oseltamivir.
Oseltamivir Phosphate is the phosphate salt of oseltamivir, a synthetic derivative prodrug of ethyl ester with antiviral activity. By blocking neuraminidases on the surfaces of influenza viruses, oseltamivir interferes with host cell release of complete viral particles.
An acetamido cyclohexene that is a structural homolog of SIALIC ACID and inhibits NEURAMINIDASE.
See also: Oseltamivir Acid (has active moiety); Oseltamivir (has active moiety); Oseltamivir carboxylate (has active moiety).

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Drug Indication
Treatment and prevention of influenza

C16H31N2O8P

410.3997

410.181

C, 46.83; H, 7.61; N, 6.83; O, 31.19; P, 7.55

204255-11-8

Oseltamivir;196618-13-0;Oseltamivir acid;187227-45-8;Oseltamivir-d5 phosphate;Oseltamivir-d3 phosphate

78000

White to off-white solid powder

1.08g/cm3

473.3ºC at 760 mmHg

196-198°C

240ºC

1.448

5

9

8

27

468

3

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

PGZUMBJQJWIWGJ-ONAKXNSWSA-N

InChI=1S/C16H28N2O4.H3O4P/c1-5-12(6-2)22-14-9-11(16(20)21-7-3)8-13(17)15(14)18-10(4)19;1-5(2,3)4/h9,12-15H,5-8,17H2,1-4H3,(H,18,19);(H3,1,2,3,4)/t13-,14+,15+;/m0./s1

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

GS-4071, GS-4104, GS4071, GS4104, GS 4071, Oseltamivir phosphate; 204255-11-8; Tamiflu; Oseltamivir (phosphate); Oseltamir Phosphate; Ro 64-0796/002; Oseltamivir (as phosphate); 4A3O49NGEZ; GS 4104, Tamiflu

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, 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)

H2O : ~100 mg/mL (~243.66 mM)
DMSO : ~100 mg/mL (~243.66 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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 (6.09 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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 (Please use freshly prepared in vivo formulations for optimal results.)