Peramivir Trihydrate (also known as BCX-1812, RWJ-270201, S-021812; trade name Rapivab) is a trihydrate form of the antiviral agent peramivir which is a transition-state analogue and a specific influenza viral neuraminidase inhibitor approved for the treatment of influenza. It inhibits with neuraminidase an IC50 of median 0.09 nM. Peramivir protects mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets. Peramivir combined with Oseltamivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Peramivir (30 mg/kg) is effective in promoting the survival of mice infected with systemically replicating H5N1 virus.

Physicochemical Properties

Molecular Formula	C15H28N4O4.3H2O
Molecular Weight	382.45
Exact Mass	382.242
Elemental Analysis	C, 47.11; H, 8.96; N, 14.65; O, 29.28
CAS #	1041434-82-5
Related CAS #	Peramivir;330600-85-6
PubChem CID	11954371
Appearance	White to off-white solid powder
LogP	1.698
Hydrogen Bond Donor Count	8
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	7
Heavy Atom Count	26
Complexity	460
Defined Atom Stereocenter Count	5
SMILES	CCC(CC)[C@@H]([C@H]1[C@@H](C[C@@H]([C@H]1O)C(=O)O)N=C(N)N)NC(=O)C.O.O.O
InChi Key	RFUCJKFZFXNIGB-ZBBHRWOZSA-N
InChi Code	InChI=1S/C15H28N4O4.3H2O/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);3*1H2/t9-,10+,11+,12-,13+;;;/m0.../s1
Chemical Name	(1S,2S,3R,4R)-3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino-2-hydroxycyclopentanecarboxylic acid trihydrate
Synonyms	BCX-1812,RWJ-270201, S-021812; BCX1812; BCX 1812; RWJ 270201; RWJ270201; Peramivir trihydrate; 1041434-82-5; Peramivir (trihydrate); Peramivir hydrate; Peramivir [USAN]; Rapiacta; QW7Y7ZR15U; Peramivir hydrate (JAN); Peramivir. Brand name: Rapivab; Rapiacta and Peramiflu
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	NA/neuraminidase（IC50 = 0.9-4.3 nM)[2]
ln Vitro	Antiviral activity in cell culture[2] 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. Viral neuraminidase inhibition studies[2] 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).
ln Vivo	Peramivir protects mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets. Peramivir combined with Oseltamivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Peramivir (30 mg/kg) is effective in promoting the survival of mice infected with systemically replicating H5N1 virus. Peramivir is 30% protective against H1N1 virus in mice at 0.5 mg/kg/d, but ineffective at lower doses when used as monotherapy. Peramivir combined with favipiravir increases the numbers of survivors by 10-50% when the 0.025 mg/kg/d, 0.05 mg/kg/d, and 0.1 mg/kg/d doses of Peramivir are combined with 20 mg/kg/d Favipiravir and when all doses of Peramivir are combined with 40 mg/kg/d Favipiravir. Peramivir combined with favipiravir results in an increase in lifespan, improved body weight and significant reductions in lung hemorrhage score and lung weight in H1N1 infected mice. Peramivir (1 mg/kg/d) combined with Rimantadine (5 mg/kg/d and 10 mg/kg/d) produces weight losses of 1.69 and 0.69, respectively, whereas the combination of 3.0 mg/kg/d Peramivir with 10 and 30 mg/kg/d Rimantadine does not show any weight loss in sub-lethal influenza A (H3N2) virus mouse model.
Enzyme Assay	Viral neuraminidase inhibition assay[2] 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.
Cell Assay	Cell culture antiviral studies[2] 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.
Animal Protocol	Animal experiment design[2] 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. Pharmacokinetic (PK) analysis[1] 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. Mice[1] 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. Ferrets[1] 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. 0.025 mg/kg/d, 0.05 mg/kg/d, and 0.1 mg/kg/d Mice
ADME/Pharmacokinetics	The post-exposure therapeutic efficacy of peramivir was also evaluated in outbred animals (ferrets), as pharmacokinetic analysis of peramivir in ferrets showed rapid uptake into the circulation following i.m. inoculation. Pharmacokinetic analysis indicated that the parenteral formulation of peramivir was rapidly introduced into the circulation of these mice following intramuscular inoculation. https://pmc.ncbi.nlm.nih.gov/articles/PMC2680697/
Toxicity/Toxicokinetics	Hepatotoxicity Despite widespread use, there is little evidence that peramivir, when given as recommended as a single intravenous infusion, is associated with liver injury, either in the form of serum enzyme elevations or clinically apparent liver disease. A proportion of patients with influenza may have minor serum enzyme elevations during the acute illness, but these appear to be independent of therapy and are not exacerbated by peramivir. Likelihood score: E (unlikely cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Because peramivir is poorly absorbed orally, it is not likely to reach the bloodstream of the infant in clinically important amounts. However, because no information is available on the use of peramivir breastfeeding, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. ◉ 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.
References	[1]. Virology.2008 Apr 25;374(1):198-209; [2]. Antiviral Res.2010 Oct;88(1):38-44; [3]. Antiviral Res.2008 Nov;80(2):150-7.
Additional Infomation	Peramivir is an antiviral agent developed by Biocryst Pharmaceuticals to treat influenza A/B. The development of peramivir has been supported by the US Department of Health and Human Services as part of the government's effort to prepare for a flu pandemic. Being an influenza virus neuraminidase inhibitor, peramivir works by preventing new viruses from emerging from infected cells. Due to the poor oral bioavailability, the oral formulation of the drug was previously abandoned by Johnson and Johnson Company. The injectable intravenous formulation of peramivir was approved by the FDA in September 2017 for the treatment of acute uncomplicated influenza to pediatric patients 2 years and older who have been symptomatic for no more than two days. Peramivir is a Neuraminidase Inhibitor. The mechanism of action of peramivir is as a Neuraminidase Inhibitor. Peramivir is an inhibitor of the influenza neuraminidase enzyme and is used as therapy of acute symptomatic influenza A and B. Peramivir has not been associated with serum enzyme elevations during therapy or with clinically apparent liver injury. Peramivir is a cyclopentane derivative with activity against influenza A and B viruses. Peramivir is a neuraminidase inhibitor which prevents normal processing of virus particles such that virus particles are not released from infected cells. Drug Indication Peramivir is indicated for the treatment of acute uncomplicated influenza in patients six months and older who have been symptomatic for no more than two days. FDA Label Alpivab is indicated for the treatment of uncomplicated influenza in adults and children from the age of 2 years. Treatment of influenza Mechanism of Action Peramivir is an inhibitor of influenza neuraminidase, preventing new virus particles from leaving infected cells.

Solubility Data

Solubility (In Vitro)	5%TFA : ~3 mg/mL H2O : ~5 mg/mL (~13.07 mM) DMSO : ~0.2 mg/mL ( ~0.52 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 4.55 mg/mL (11.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6147 mL	13.0736 mL	26.1472 mL
	5 mM	0.5229 mL	2.6147 mL	5.2294 mL
	10 mM	0.2615 mL	1.3074 mL	2.6147 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.