NA/neuraminidase（IC50 = 0.9-4.3 nM); IKK-α;STAT3;ERK1;ERK2
Influenza virus neuraminidase (NA). Peramivir (BCX-1812, RWJ-270201, S-021812) demonstrated an IC50 of 1.1 nM against influenza B virus (B/Brisbane/60/2008, BR/08) in a fluorescence-based NA inhibition assay [2].
- The anti-cytokine storm effect is mediated partly through intervention of NF-κB activity in LPS-induced macrophage models [1].

For macrophages, peramivir (0.3125–40 μM, 4 h) is nontoxic.In LPS-induced hPBMCs, peramivir (2–10 μM, 6–12 h) inhibits the release of cytokines[1].

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Antiviral activity in cell culture[5] 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.

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Viral neuraminidase inhibition studies[5] 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).

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In LPS-induced mouse peritoneal macrophages, Peramivir (BCX-1812, RWJ-270201, S-021812) at 10 μM inhibited TNF-α release by 67.2%, showing the strongest effect among three neuraminidase inhibitors (oseltamivir, zanamivir, and peramivir). The IC50 for TNF-α inhibition was 4.3 μM [1].
- In LPS-induced human peripheral blood mononuclear cells (hPBMCs) from healthy donors, Peramivir (BCX-1812, RWJ-270201, S-021812) (2.5, 5, and 10 μM) significantly and dose-dependently counteracted the elevated TNF-α levels at both 6 and 12 hours post-LPS stimulation [1].
- In MDCK cells infected with influenza A/NWS/33 (H1N1) virus, Peramivir (BCX-1812, RWJ-270201, S-021812) alone at concentrations of 32 and 100 μM reduced virus yield by ≥5 log10 below the detection limit [5].
- Peramivir (BCX-1812, RWJ-270201, S-021812) potently inhibited the NA activity of influenza A/Vietnam/1203/04 (H5N1) in vitro with an IC50 of 0.171 nM [4].

Peramivir (20–60 mg/kg, intraperitoneal injection, single dose) reduces acute lung injury, prevents LPS-induced cytokine storm, and increases the survival time of mice modelled by cytokine storm syndrome[1].
In immunocompromised murine models of influenza B virus infection, peramivir (75 mg/kg, intramuscular injection, once daily for 7 days) saved BALB scid mice from a lethal challenge with BR/08[2].

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In a mouse model of LPS-induced cytokine storm syndrome (CSS), pre-treatment with Peramivir (BCX-1812, RWJ-270201, S-021812) (60 mg/kg, i.p.) significantly reduced serum levels of TNF-α, IFN-α, IFN-γ, MCP-1, GM-CSF, IL-1β, IL-6, and IL-12. In bronchoalveolar lavage fluid (BALF), it significantly reduced TNF-α and IL-6. It also alleviated acute lung injury and prolonged survival time in mice challenged with a lethal dose of LPS [1].
- In BALB/c mice infected with a lethal dose of influenza B virus (B/Brisbane/60/2008, BR/08), treatment with Peramivir (BCX-1812, RWJ-270201, S-021812) (75 mg/kg/day, i.m.) in a single dose (1×), double dose (2×), or four-dose (4×) regimen conferred complete protection (100% survival). In immunocompromised BALB scid mice, the same treatment regimens resulted in 40% (1×), 60% (2×), and 60% (4×) survival, respectively [2].
- In a lethal mouse model of influenza A/NWS/33 (H1N1) virus infection, Peramivir (BCX-1812, RWJ-270201, S-021812) administered intramuscularly (i.m.) at 0.4 and 1 mg/kg/day (twice daily for 5 days) provided 100% protection [5]. When combined with suboptimal doses of oseltamivir (0.4 mg/kg/day, p.o.), peramivir at 0.1 and 0.2 mg/kg/day (i.m.) significantly increased survival rates to 80% and 100%, respectively, compared to either drug alone [5].
- In mice infected with a lethal dose of highly pathogenic avian influenza A/Vietnam/1203/04 (H5N1), post-exposure treatment with Peramivir (BCX-1812, RWJ-270201, S-021812) (30 mg/kg, i.m., single or multiple doses) resulted in 70-80% survival, comparable to oseltamivir (70% survival) and significantly better than the vehicle control (36% survival) [4].
- In ferrets infected with a lethal dose of influenza A/Vietnam/1203/04 (H5N1), intramuscular administration of Peramivir (BCX-1812, RWJ-270201, S-021812) (30 mg/kg, multiple doses) significantly improved survival rates to 70-75% compared to untreated/vehicle-treated controls (11-30%) in two out of three trials [4].

Viral neuraminidase inhibition assay[5]
The effects of compounds on viral neuraminidase activity were determined using a commercially available kit 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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The susceptibility of influenza B viruses to neuraminidase inhibitors (NAIs) was assessed in a fluorescence-based assay. A fluorogenic substrate was used. The concentration of drug required to inhibit a standardized amount of NA activity by 50% (IC50) was calculated. For the BR/08 virus, the IC50 for Peramivir (BCX-1812, RWJ-270201, S-021812) was determined to be 1.1 nM [2].
- A commercial chemiluminescent neuraminidase inhibition assay kit was used to evaluate the effects of Peramivir (BCX-1812, RWJ-270201, S-021812) and oseltamivir carboxylate, alone and in combination. The virus (influenza A/NWS/33) was used as the source of NA. Compounds in half-log dilution increments were incubated with the virus. After a pre-incubation, a chemiluminescent substrate was added. Neuraminidase activity was measured using a luminometer. The percentage of chemiluminescent counts at each compound concentration was normalized to 100% under untreated conditions [5].

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.

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For anti-inflammatory screening, peritoneal macrophages were stimulated with LPS (100 ng/ml) for 4 hours in the presence of a test compound at 10 μM. The cell supernatant was collected, and the concentration of TNF-α was measured using an ELISA kit. The remaining cells were examined for cytotoxicity using a CCK-8 assay [1].
- For the virus yield reduction assay, confluent MDCK cells in 96-well plates were infected with influenza A/NWS/33 (H1N1) virus (approximately 50 CCID50). Peramivir (BCX-1812, RWJ-270201, S-021812) and oseltamivir carboxylate were added at concentrations ranging from 0.32 to 100 μM. After three days of culture, the plates were frozen. The virus titers in the supernatants were then determined by an endpoint dilution method on fresh MDCK cells. Virus titers were expressed as log10 CCID50 per 0.1 ml [5].
- The viability of macrophages treated with Peramivir (BCX-1812, RWJ-270201, S-021812) was assessed using a Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in 96-well plates. After treatment, the CCK-8 solution was added to each well, and the plates were incubated for 1 hour at 37°C. Absorbance was measured at 450 nm. No apparent cytotoxicity was observed at concentrations up to 40 μM [1].
- For NF-κB luciferase activity assay, RAW264.7 cells stably transfected with an NF-κB-responsive luciferase construct were seeded in 96-well plates. Cells were pretreated with Peramivir (BCX-1812, RWJ-270201, S-021812) for 1 hour, followed by stimulation with LPS (100 ng/ml) for 6 hours. Cells were then collected, and luciferase activity was measured using a luciferase assay system [1].
- Western blotting was performed to assess the effect of Peramivir (BCX-1812, RWJ-270201, S-021812) on LPS-induced activation of NF-κB and MAPK pathways. Protein samples were separated by SDS-PAGE, transferred to NC membranes, and probed with specific primary antibodies against phosphorylated and total forms of p65, IκBα, p38, Erk1/2, and others, followed by HRP-linked secondary antibodies. The signal was detected by chemiluminescence [1].

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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Pharmacokinetic (PK) analysis[4]
PK data was obtained in ferrets and mice, as follows . Male ferrets (three per group) were injected via i.m. route with a dose of 1, 3 or 9 mg/kg peramivir. Prior to dosing (time 0) and at time intervals between 0.083 and 72 hrs following dosing, blood samples of approximately 0.5 ml were collected and analyzed via liquid chromatography with tandem mass spectrophotometry (LS/MS/MS). Mice PK data were generated based upon modeling from a 10 mg/kg i.m. dose. PK parameters were calculated and modeling performed using WinNonlin® 5.0.1. PK data were obtained following a single i.m. injection of ferrets (3 per group) with doses of 1, 3 or 9 mg/kg peramivir. Animals were monitored for peramivir concentration in plasma over a 72 hour period.

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Mice[4]
Ten-week-old mice (10-11 per group) were infected intranasally with a dose of 6 × 102 TCID50 of influenza A H5N1 (A/Vietnam/1203/04) grown in MDCK cells. Subsequently, mice were treated with peramivir (30 mg/kg) in a single dose at 1 h post-inoculation (+1 h) or multiple doses (+1 h, daily on day +1 through day +4) via i.m. injection. As control, mice were treated with drug diluent (“vehicle” at +1 h, daily on day +1 through day +4. For comparison, mice were treated orally (per os, p.o.) with oseltamivir (10 mg/kg/day) at +1 h and daily on day +1 through day +4. Animals were monitored daily for a period of 15 days for death, disease development, and body temperature. Body mass (weight) was recorded on days indicated in figure legend. Severe disease was defined as the loss of ≥20% of their initial body mass.

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Ferrets[4]
Six- to eight-week-old ferrets were infected intranasally with 1.5 × 103 TCID50 (trial 1), 1.5 × 104 TCID50 (trial 2), or 1.7 × 104 TCID50 of influenza A H5N1 (A/Vietnam/1203/04) using virus stock prepared in MDCK cells (trial 1) or in eggs (trial 2 and 3). Ferrets were then treated with multiple doses of peramivir (30 mg/kg) or, as control, were treated with vehicle at +1 h, and daily on day +1 through day +4 (trial 1 and 3) or were untreated (trial 2). Following infection and drug treatment, animals were monitored daily for a period of 16-18 days following infection for death and disease development. Daily telemetric monitoring of body temperature was performed. For trial 3, five animals per group were randomly pre-selected to be euthanized on day 4 and on day 6 post-infection for organ harvest and infectious virus titration.

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Animal Model: Cytokine storm syndrome model mice [1]
Dosage: 20 mg/kg, 60 mg/kg
Administration: Intraperitoneal injection (i.p.)
Result: reduced levels of eight cytokines, including GM-CSF, IL-1β, IL-6, IL-12, chemokines (MCP-1), TNF-a, IFN-a, and IFN-γ.demonstrated a decrease in bleeding sites or congestion, a mild thickening of the alveoli, and an infiltration of inflammatory cells.demonstrated a strong protective effect on the lung tissues.

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Cytokine Storm Syndrome (CSS) Model in Mice: CSS was induced by a single intraperitoneal (i.p.) injection of LPS (15 mg/kg). Peramivir (BCX-1812, RWJ-270201, S-021812) (60 mg/kg) was administered i.p. 1 hour before LPS injection. Mice were sacrificed 4 hours post-LPS for serum collection or 8 hours post-LPS for bronchoalveolar lavage fluid (BALF) and lung tissue collection. For survival studies, a lethal dose of LPS (30 mg/kg) was given, and mice were treated with low-dose (20 mg/kg) or high-dose (60 mg/kg) peramivir i.p. 1 hour before LPS challenge. Survival was monitored every 2 hours for 40 hours [1].
- Influenza B Virus Infection Model in Mice: Female 6-week-old BALB/c, NOD scid, and BALB scid mice were inoculated intranasally (i.n.) with influenza B virus (MA/12, PH/13, or BR/08). Peramivir (BCX-1812, RWJ-270201, S-021812) was administered by intramuscular (i.m.) injection at a dose of 75 mg/kg/day. Treatment regimens included a single dose at +24 hpi (1×), double dose at +24 and +72 hpi (2×), or four doses at +24, +72, +120, and +168 hpi (4×). Control animals received sterile water. Mice were monitored daily for weight loss, clinical signs, and survival up to 30 days post-infection (dpi) [2].
- Influenza A/NWS/33 (H1N1) Mouse Model: Female BALB/c mice were infected intranasally with the virus (approximately 3 MLD50). Peramivir (BCX-1812, RWJ-270201, S-021812) was administered intramuscularly (i.m.) twice a day at 12-hour intervals for 5 days, starting 2 hours before virus challenge. The drug was prepared in sterile saline. Oseltamivir was administered orally (p.o.) by gavage. Ten mice per group were used, and they were observed for death for 21 days. Body weights were recorded every other day [5].
- H5N1 Influenza A Virus Model in Mice and Ferrets: Mice (10-week-old) were infected intranasally with a lethal dose (6×10^2 TCID50) of A/Vietnam/1203/04 (H5N1). Peramivir (BCX-1812, RWJ-270201, S-021812) was given intramuscularly at 30 mg/kg as a single dose at 1 hpi, or as multiple doses (daily on day +1 through +4). For comparison, oseltamivir was given orally at 10 mg/kg/day. Ferrets (6-8 week-old) were infected intranasally with a lethal dose (1.5×10^3 to 1.7×10^4 TCID50) of the same virus and treated with multiple doses of peramivir (30 mg/kg, i.m.) at +1 hpi and daily on day +1 through +4. Control animals received vehicle or were untreated. Animals were monitored for survival and disease development for 15-18 days [4].

0.025 mg/kg/d, 0.05 mg/kg/d, and 0.1 mg/kg/d

Mice

Researchers also evaluated the post-exposure therapeutic effect of peramivir in non-inbred animals (ferrets), as pharmacokinetic analysis of peramivir in ferrets showed that the drug rapidly entered the bloodstream after intramuscular injection. Pharmacokinetic analysis also showed that the injectable form of peramivir rapidly entered the bloodstream of these mice after intramuscular injection. https://pmc.ncbi.nlm.nih.gov/articles/PMC2680697/

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In mice, after a single intramuscular (i.m.) injection of 10 mg/kg, the plasma concentration of Peramivir (BCX-1812, RWJ-270201, S-021812) peaked immediately, and the elimination half-life is more than 24 hours, supporting once-daily dosing [2].
- In ferrets, following a single i.m. injection of 1, 3, or 9 mg/kg, Peramivir (BCX-1812, RWJ-270201, S-021812) was rapidly absorbed into the circulation. The maximum plasma concentrations (Cmax) achieved were up to 20,000-fold above the IC50 for H5N1 isolates [4].
- In humans, oral peramivir has low bioavailability (≤3%). The elimination half-life of peramivir following a single IV administration of 600 mg to healthy subjects is approximately 20 hours. It is almost entirely eliminated by renal excretion, with 90% of the drug excreted unchanged. Clearance is linearly related to creatinine clearance (CrCl) when CrCl values are below 115 mL/min. Dose adjustment is required for patients with renal impairment (e.g., 200 mg for CrCl 30-49 mL/min, 100 mg for CrCl 10-29 mL/min) [3].

Hepatotoxicity
Despite the widespread use of peramivir, there is little evidence that a single intravenous infusion of peramivir at the recommended dose causes liver injury, whether it is elevated serum enzymes or clinically apparent liver disease. Some influenza patients may experience mild elevations in serum enzymes during the acute phase, but this appears to be unrelated to treatment and is not exacerbated by peramivir. Probability score: E (unlikely to be the cause of clinically apparent liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Due to poor oral absorption of peramivir, it is unlikely to enter the infant's bloodstream at clinically significant concentrations. However, since there is currently no information on the use of peramivir during lactation, alternative medications may be preferred, especially for breastfed newborns or premature infants. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No relevant published information was found as of the revision date.

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In cell viability assays (CCK-8) on macrophages, Peramivir (BCX-1812, RWJ-270201, S-021812) showed no apparent cytotoxicity at concentrations up to 40 μM [1].
- In clinical studies, Peramivir (BCX-1812, RWJ-270201, S-021812) demonstrated good tolerability and safety. The most common adverse events were mild to moderate and included gastrointestinal disorders (diarrhea, nausea, vomiting) and decreased neutrophil counts, which were self-limiting. No significant drug interactions were observed with oseltamivir or rimantadine [3].
- In the ferret model, no significant toxicity was observed with multiple doses of peramivir (30 mg/kg, i.m.) [4].

[1]. Peramivir, an anti-influenza virus drug, exhibits potential anti-cytokine storm effects [J]. Frontiers in Immunology, 2022, 13: 856327.

[2]. Pathogenicity and peramivir efficacy in immunocompromised murine models of influenza B virus infection [J]. Scientific reports, 2017, 7(1): 7345.

[3]. Peramivir: a novel intravenous neuraminidase inhibitor for treatment of acute influenza infections [J]. Frontiers in microbiology, 2016, 7: 450.

[4]. Virology.2008 Apr 25;374(1):198-209.

[5]. Antiviral Res.2010 Oct;88(1):38-44.

Peramivir belongs to the guanidine class of drugs, and its trihydrate is used to treat acute uncomplicated influenza in patients aged 18 years and older with symptoms lasting no more than two days. It is an antiviral drug and an EC 3.2.1.18 (exo-α-sialidase) inhibitor. Peramivir belongs to the cyclopentanol, acetamide, guanidine, and 3-hydroxymonocarboxylic acid class of compounds. It contains peramivir hydrate. Peramivir is an antiviral drug developed by Biocryst Pharmaceuticals for the treatment of influenza A/B. The development of peramivir was supported by the U.S. Department of Health and Human Services as part of the government's pandemic response. As an influenza virus neuraminidase inhibitor, peramivir's mechanism of action is to prevent the release of new virus from infected cells. Due to low oral bioavailability, Johnson & Johnson had previously discontinued production of the oral formulation of this drug. In September 2017, the U.S. Food and Drug Administration (FDA) approved an injectable form of peramivir for the treatment of acute uncomplicated influenza in children aged 2 years and older with symptoms lasting no more than two days. Peramivir is an influenza virus neuraminidase inhibitor used to treat acute influenza A and B. No elevation of serum enzymes or clinically significant liver damage has been observed during peramivir treatment. Peramivir is a cyclopentane derivative active against both influenza A and B viruses. As a neuraminidase inhibitor, peramivir prevents the normal processing of viral particles, thus preventing their release from infected cells. Drug Indications Peramivir is indicated for the treatment of acute uncomplicated influenza in patients 6 months or older with symptoms presenting for no more than two days. FDA Label Peramivir is indicated for the treatment of uncomplicated influenza in adults 2 years of age and older and children. Influenza Treatment Mechanism of Action Peramivir is an influenza virus neuraminidase inhibitor that prevents new viral particles from leaving infected cells.

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Peramivir (BCX-1812, RWJ-270201, S-021812) is a novel cyclopentane neuraminidase inhibitor. It was approved by the U.S. Food and Drug Administration (FDA) in December 2014 for the treatment of acute uncomplicated influenza in patients 18 years and older. It is administered as a single intravenous (IV) dose of 600 mg [3].
- During the 2009 H1N1 influenza pandemic, the FDA issued an Emergency Use Authorization (EUA) for IV peramivir for hospitalized adult and pediatric patients who were not responsive to approved antivirals or for whom alternative routes were not feasible [3].
- The unique structure of Peramivir (BCX-1812, RWJ-270201, S-021812) (a cyclopentane ring with a guanidino group and a hydrophobic side chain) allows it to form eight strong hydrogen bonds with conserved arginine residues in the NA active site, contributing to its tight binding and slow dissociation rate (t1/2 > 24 h), compared to oseltamivir and zanamivir (t1/2 = 1.25 h) [3].

C15H28N4O4

328.41

328.211

C, 54.86; H, 8.59; N, 17.06; O, 19.49

330600-85-6

Peramivir trihydrate;1041434-82-5

154234

White to off-white solid powder

1.4±0.1 g/cm3

1.614

-1.37

5

5

7

23

460

5

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

XRQDFNLINLXZLB-CKIKVBCHSA-N

InChI=1S/C15H28N4O4/c1-4-8(5-2)12(18-7(3)20)11-10(19-15(16)17)6-9(13(11)21)14(22)23/h8-13,21H,4-6H2,1-3H3,(H,18,20)(H,22,23)(H4,16,17,19)/t9-,10+,11+,12-,13+/m0/s1

(1S,2S,3R,4R)-3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino-2-hydroxycyclopentanecarboxylic acid

RWJ 270201; RWJ270201; BCX-1812; Rapiacta; 229614-55-5; Peramivir anhydrous; RAPIVAB; Brand name: Rapivab; Rapiacta and Peramiflu; BCX1812; BCX1812; BCX 1812; RWJ270201;

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.)