Neuraminidase; influenza A/H3N2, A/H1N2, A/H1N1, and B viruses
Sialidase (neuraminidase) inhibitor; inhibits viral sialidase activity of influenza virus. In canine mammary cancer cells (CMA07 and CMT-U27), it also inhibits endogenous mammalian sialidase activity, leading to increased sialylation.

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].

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Treatment with oseltamivir phosphate (0.305 μM, 3.05 μM, 30.5 μM, and 305 μM) for 24 hours impaired sialidase activity in CMA07 and CMT-U27 canine mammary cancer cells, as shown by decreased fluorescence from 4-MuNana substrate conversion.
At 305 μM, oseltamivir phosphate significantly decreased metabolic activity in both CMA07 and CMT-U27 cells (MTS assay) and increased DNA fragmentation (TUNEL assay), indicating cytotoxicity and apoptosis promotion.
At lower concentrations (3.05 μM and 30.5 μM), oseltamivir phosphate significantly increased migration (wound-healing assay) and invasion (Matrigel assay) of CMT-U27 cells in a dose-dependent manner.
Oseltamivir phosphate increased α2,6-linked sialylation and SLe(x) expression in both cell lines, as shown by Western blot, 2D electrophoresis, and fluorescent lectin staining.

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).

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In nude mice xenografted with CMT-U27 cells, daily intraperitoneal administration of oseltamivir phosphate (100 mg/kg) increased α2,6 sialylation and SLe(x) expression in primary tumors.
Treated mice showed a trend toward increased lung metastases compared to controls, though not statistically significant (p=0.07).
Treated mice also exhibited more inflammatory infiltrate in primary tumors and earlier signs of disease progression.

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.

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Sialidase activity was assessed using a fluorescent substrate, 4-methyl-umbelliferyl-N-acetylneuraminic acid (4-MuNana). Cells were treated with oseltamivir phosphate for 24 hours, then incubated with 2 μM 4-MuNana. Fluorescence from methyl-umbelliferone was measured under UV light to indicate sialidase activity. Decreased fluorescence indicated inhibition of sialidase activity by oseltamivir phosphate.

Cell morphology analysis[2]
\nCMA07 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.\n

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\nCell proliferation assay[2]
\nCMA07 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.\n

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\nCell growth assay[2]
\nCell 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.\n

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\nTUNEL assay[2]
\nCMA07 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.\n

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\nWound-healing\nThe 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.\n

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\nFluorescent cytochemistry[2]
\nCells 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.\n

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\nWestern Blot analysis[2]
\nCells 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.

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Cell proliferation was assessed by daily counting for 7 days using trypan blue exclusion.
Metabolic activity was measured using MTS assay over 48 hours.
Apoptosis was evaluated using TUNEL assay after 24-hour treatment.
Cell migration was assessed by wound-healing assay with time-lapse imaging over 48 hours.
Cell invasion was measured using Matrigel-coated chambers over 6 hours.
Glycosylation changes were analyzed by Western blot, 2D electrophoresis, and fluorescent lectin staining (SNA, MAL I, MAL II) for sialylated structures.

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.

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Female nude mice were orthotopically inoculated with CMT-U27 cells. When tumors reached ~500 mm³, mice were divided into control (PBS) and treatment groups (100 mg/kg oseltamivir phosphate daily via intraperitoneal injection). Tumors were surgically removed at ~1000–1500 mm³, and mice were monitored for metastasis. Lungs were examined histologically for metastases.

Absorption
Oseltamivir phosphate is rapidly absorbed from the gastrointestinal tract after oral administration and is primarily converted to the active metabolite, oseltamivir carboxylate, 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. 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, while AUC (0-12h) of oseltamivir and oseltamivir carboxylate are 112 ng·h/mL and 2719 ng·h/mL, respectively.
Elimination Route
After absorption, over 90% of oseltamivir is eliminated through conversion to oseltamivir carboxylate, and subsequently completely excreted via the kidneys. Clinical studies have found that less than 20% of the oral radiolabeled dose is excreted in feces.
Volume of Distribution
The steady-state average volume of distribution of oseltamivir carboxylate in the human body is approximately 23 to 26 liters, roughly equivalent to the extracellular fluid. Because neuraminidase activity is located extracellularly, oseltamivir carboxylate can be distributed to all sites of influenza virus transmission.
Clearance
The renal clearance of this drug (18.8 L/h) exceeds the 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 was 23 to 26 liters after intravenous administration in 24 subjects.
Oral oseltamivir phosphate is readily absorbed and subsequently converted in large quantities by hepatic esterases to its active form—oseltamivir carboxylate. At least 75% of the oral dose enters the systemic circulation as oseltamivir carboxylate. Of the oral dose, less than 5% enters the systemic circulation as oseltamivir phosphate. Clearance: Renal: Oseltamivir carboxylate is primarily cleared by renal excretion (>99%). Renal clearance (18.8 L/h) exceeds glomerular filtration rate (7.5 L/h), indicating tubular secretion. Fecal: Fecal excretion is <20% after 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. Metabolism/Metabolites: Oseltamivir is primarily converted to its active metabolite, oseltamivir carboxylate, in the liver by esterases. Oseltamivir carboxylate is not further metabolized. Neither oseltamivir nor oseltamivir 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 primarily converted to oseltamivir carboxylate in the liver by esterases. Neither oseltamivir nor its carboxylate is a substrate or inhibitor of cytochrome P450 isoenzymes.
Biotransformation: Hepatic metabolism; oseltamivir ethyl ester prodrug is extensively hydrolyzed to its active stellate form—oseltamivir carboxylate.
Biological half-life: After oral administration, plasma oseltamivir concentrations decrease within 1 to 3 hours in most subjects, while plasma oseltamivir carboxylate concentrations decrease within 6 to 10 hours in most subjects.
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 the symptoms were transient. 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 case reports 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).
Effects during Pregnancy and Lactation
◉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, and no adverse effects are expected on breastfed infants. Infants older than 2 weeks can take oseltamivir directly at a much higher dose than 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.
Drug and Lactation Database (LactMed)
11.1.5 Interactions
Concomitant use with probenecid results in approximately a two-fold increase in the concentration of the active metabolite due to reduced secretion of active anion vessels in the kidneys.
Thomson/Micromedex. Drug Information for Healthcare Professionals. Volume 1, Greenwood Village, Colorado, 2006, p. 11. 2305
Hazardous Substances Database (HSDB)
In vitro studies have shown that oseltamivir and its carboxylates are not good substrates for P450 mixed-function oxidases or glucuronyl transferases. Cimetidine is a non-specific cytochrome P450 isoenzyme inhibitor that competitively inhibits the secretion of basic or cationic drugs in the renal tubules, but it has no effect on the plasma concentrations of oseltamivir and its carboxylates.
Physician's Desk Reference, 60th ed., Thomson PDR, Montvale, NJ, 2006, p. 2305. 2811
Hazardous Substances Database (HSDB)
Co-administration with amoxicillin does not alter the plasma concentrations of either compound, indicating weak competition for anion secretion pathways.
Protein Binding
The binding rate of active oseltamivir carboxylate metabolites 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. In in vitro antiviral assays in MDCK cells, the tested concentrations of oseltamivir phosphate (up to the highest concentration used for IC50 assay) showed no significant signs of cytotoxicity, as inferred from the absence of reported impaired cell viability. In in vivo mouse studies, daily doses of 20 or 50 mg/kg oseltamivir phosphate did not induce significant adverse reactions or directly attributable to toxicity. This has been reported in the literature. This study focuses on its protective effect against influenza virus infection.

[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 containing the oseltamivir molecule. Oseltamivir phosphate is the phosphate salt of oseltamivir, a synthetic ethyl ester derivative prodrug with antiviral activity. Oseltamivir interferes with the release of intact viral particles from host cells by blocking neuraminidase on the surface of the influenza virus. Oseltamivir is an acetamidocyclohexene, a structural homologue of sialic acid, that inhibits neuraminidase. See also: Oseltamivir acid (containing the active moiety); Oseltamivir (containing the active moiety); Oseltamivir carboxylate (containing the active moiety).

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Drug Indications Treatment and prevention of influenza. Oseltamivir phosphate is an anti-influenza prodrug converted to active oseltamivir carboxylate by carboxylesterase. This study suggests that it may inhibit mammalian sialylase in cancer cells, thereby altering sialylation and potentially increasing tumor invasiveness. This study used canine mammary tumor cells as a model for human breast cancer research.

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.)