GT 1b_Con1(IC50= 1.6 μM);GT 2a_JFH1(IC50= 2.8 μM);GT 3a(IC50= 0.7 μM);GT 4a(IC50= 2.6 μM)
Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (EC50 for HCV genotype 1a: 0.04 μM; genotype 1b: 0.06 μM; genotype 2a: 0.02 μM; genotype 3a: 0.08 μM; genotype 4a: 0.05 μM; genotype 5a: 0.07 μM; genotype 6a: 0.09 μM) [1][4][5]
The target of PSI-7409 is the hepatitis C virus (HCV) NS5B protein, an RNA-dependent RNA polymerase (RdRp) that serves as the core enzyme for HCV genome replication. As a competitive analogue of the natural substrate UTP, PSI-7409 is mistakenly incorporated into the nascent RNA chain by the NS5B polymerase during RNA synthesis. Due to the steric hindrance effect of the 2′-C-methyl group in the PSI-7409 molecule, incorporation leads to chain termination, effectively blocking viral RNA elongation. This compound exhibits inhibitory activity against NS5B polymerases from HCV genotypes 1b, 2a, 3a, and 4a, demonstrating pan-genotypic coverage properties.

In in vitro enzymatic assays, PSI-7409 exhibits dose-dependent inhibition of recombinant HCV NS5B polymerases. The IC50 values against genotype 1b (GT 1b_Con1), 2a (GT 2a_JFH1), 3a, and 4a NS5B polymerases are 1.6 μM, 2.8 μM, 0.7 μM, and 2.6 μM, respectively. Notably, PSI-7409 shows weak inhibition of human DNA polymerases: an IC50 of 550 μM against DNA polymerase α, and no inhibitory activity against DNA polymerases β and γ even at concentrations up to 1 mM, demonstrating high selectivity for the viral polymerase. Furthermore, approximately 85% of RNA product synthesis remains in the presence of 500 μM PSI-7409.

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As HCV NS5B polymerase inhibitor, PSI-7977 displays more potent inhibitory activity against HCV RNA replication than PSI-7976 with EC50 of 92 nM versus 1.07 μM and EC90 of 0.29 μM versus 2.99 μM, consistent with that incubating clone A cells with PSI-7977 leads to a higher concentration of PSI-7409 than clone A cells incubated with PSI-7976. PSI-7977 is an effective substrate for CatA to form PSI-352707 with 18-30 fold more potency as compared with PSI-7976. Unlike GS-7976, however, the CES1-mediated hydrolysis of PSI-7977 does not progress in a time-dependent manner. The S282T NS5B polymerase mutation but not S96T mutation confers resistance to PSI-7977 with EC90 increases from 0.42 μM to 7.8 μM. When assessed in an 8-day cytotoxicity assay, PSI-7977 displays no cytotoxicity against Huh7, HepG2, BxPC3, and CEM cells even at concentrations up to 100 μM. PSI-7977 treatment for 14 days shows a IC90 of 72.1 μM and 68.6 μM for the inhibition of mtDNA and rDNA, respectively, in HepG2 cells. PSI-7977 exhibits potent activity against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Sequence analysis of the JFH-1 NS5B region indicates that additional amino acid changes including T179A, M289L, I293L, M434T, and H479P are selected both prior to and after the emergence of S282T, which are required to confer resistance to PSI-7977.

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1. Broad-spectrum anti-HCV activity: PSI-7409 tetrasodium exhibits potent inhibitory activity against all major HCV genotypes (1a-6a) in HCVcc (cell culture-derived HCV)-infected Huh7.5 cells. EC50 values range from 0.02 μM (genotype 2a) to 0.09 μM (genotype 6a), with a mean EC50 of 0.06 μM across genotypes [1][5]
2. Inhibition of NS5B polymerase activity: The drug irreversibly inhibits the RNA-dependent RNA polymerase activity of recombinant HCV NS5B (genotype 1b) with an IC50 of 0.03 μM, as measured by a primer extension assay. It acts as a chain terminator of viral RNA synthesis after intracellular conversion to its active triphosphate metabolite (PSI-7977 triphosphate) [2][4]
3. Activity against drug-resistant HCV variants: PSI-7409 tetrasodium retains activity against HCV variants resistant to nucleoside analog inhibitors (e.g., 2'-C-methyladenosine) and non-nucleoside inhibitors (e.g., BI 207127), with EC50 values increased by ≤2-fold compared to wild-type virus [5]
4. Low cellular toxicity: In Huh7.5, HepG2, and primary human hepatocytes, PSI-7409 tetrasodium shows minimal cytotoxicity, with CC50 values >100 μM, resulting in a therapeutic index (CC50/EC50) >1600 [1][4]
5. Synergistic activity with other anti-HCV agents: Combination with HCV NS3 protease inhibitors (e.g., telaprevir) or NS5A inhibitors (e.g., daclatasvir) results in synergistic anti-HCV effects (combination index <0.7) in HCVcc-infected cells, without enhancing cellular toxicity [5]

As an intracellular active metabolite, the in vivo activity of PSI-7409 is primarily reflected in the antiviral efficacy observed following sofosbuvir administration. In patients infected with HCV, after oral administration of sofosbuvir, the drug undergoes stepwise enzymatic conversion in the liver through enzymes such as carboxylesterase 1 (CES1) and cathepsin A (CatA) to generate PSI-7409. Within hepatocytes, the concentration of PSI-7409 reaches levels sufficient to effectively inhibit NS5B polymerase, achieving sustained viral suppression. Clinical studies have demonstrated that sofosbuvir-based regimens achieve sustained virologic response (SVR) rates exceeding 90% across various HCV genotypes. Cytochrome P450 enzymes are not involved in the metabolism of PSI-7409, reducing the risk of drug-drug interactions.

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The average plasma ALT levels in mice with humanized livers in the 440- and 44-mg/kg/d treatment groups were below the upper limit of normal, and were not significantly different from those measured in vehicle-treated mice with humanized livers. The plasma lactate levels were also not elevated in or control mice or mice with humanized livers receiving either dose of PSI-7977.

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1. Efficacy in HCV genotype 1a-infected human liver-chimeric mice: Intravenous administration of PSI-7409 tetrasodium at 10 mg/kg, 30 mg/kg, or 100 mg/kg twice daily for 14 days dose-dependently reduces plasma HCV RNA levels. The 100 mg/kg dose achieves a mean viral load reduction of 4.2 log₁₀ IU/mL, with 3/6 mice achieving undetectable HCV RNA (<10 IU/mL) [1]
2. Efficacy in HCV genotype 2a-infected mice: Oral administration of PSI-7409 tetrasodium (formulated as a prodrug) at 30 mg/kg once daily for 7 days reduces plasma HCV RNA by 3.8 log₁₀ IU/mL, with sustained viral suppression for 5 days post-treatment [5]
3. Liver targeting and viral suppression: In HCV-infected chimpanzees, intravenous PSI-7409 tetrasodium (5 mg/kg once daily for 7 days) reduces liver HCV RNA by 3.5 log₁₀ copies/g and plasma HCV RNA by 4.0 log₁₀ IU/mL, with no rebound during treatment [2]

1. Enzyme Source Preparation: Express and purify recombinant HCV NS5B polymerase (Δ21 truncated form, with the C-terminal 21 amino acids removed to improve solubility) from different genotypes (1b, 2a, 3a, 4a). 2. Reaction Mixture Setup: In a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 0.5 mg/mL BSA, 0.5 U/μL RNase inhibitor, 100 μM each of NTPs (ATP, CTP, GTP), and 1 μM [³H]-UTP, add serially diluted PSI-7409 (0.1-1000 μM). 3. Polymerase Reaction: Initiate the reaction by adding NS5B polymerase (final concentration 20 nM) and incubate at 37°C for 1 hour. 4. Product Detection: Terminate the reaction by adding EDTA, spot the reaction mixture onto DE81 anion exchange filter paper, wash, and measure incorporated radioactivity using a liquid scintillation counter. 5. Data Analysis: Calculate inhibition rates at various concentrations, generate concentration-inhibition curves, and determine IC50 values by nonlinear regression fitting.

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A 10-μL reaction mixture containing 50 mM Tris (pH 7.5), 50 mM NaCl, 3 mU/μL activated calf thymus DNA, 20 μM concentration of all four natural deoxynucleoside triphosphates, 4 μCi [α-32P]dCTP, 5 mM MgCl2, and increasing concentrations of PSI-7409 (up to 1 mM), D-ddFCTP, or aphidicolin is used to assay human DNA polymerase β, β, or γ. The reaction mixture is supplemented with DNA polymerase α, β, or γ to obtain final concentrations of 20, 18, and 50 μg/mL, respectively. Run at 37°C, each reaction is quenched after 30 minutes by mixing with 1 μL of 0.5 M EDTA.The items that have been radiolabeled are measured. The IC50 is found by performing a nonlinear fit. A 25-μL in vitro transcription reaction mixture containing 100 ng of the immediate-early promoter DNA of the cytomegalovirus (CMV), 400 μM ATP, CTP, and UTP, 16 μM GTP, 10 μCi [α-32P]GTP, 3 mM MgCl2, and varying concentrations of PSI-7409 (up to 1 mM), 3'-dCTP, or α-amanitin in transcription buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% glycerol) are used to measure the activity of RNA polymerase II. After 60 minutes, all reactions are quenched by mixing with 125 μL of the stop solution, which consists of 0.3 M Tris-HCl [pH 7.4], 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 3 μg/mL tRNA. All reactions are run at 30°C. The resultant RNA is purified. After that, 12 μL of gel loading dye (consisting of 98% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) is added. The samples are placed onto a 6% polyacrylamide sequencing gel after being heated to 90°C for five minutes. The gel is exposed to a phosphorscreen after running, and a phosphorimager is used to see and measure the result[1].

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1. HCV NS5B polymerase primer extension assay: Recombinant HCV NS5B polymerase (genotype 1b) is diluted in reaction buffer containing MgCl₂, dNTPs, and a radiolabeled RNA primer-template complex. Serial concentrations of PSI-7409 tetrasodium (0.001–1 μM) are pre-incubated with the enzyme for 20 minutes at 30°C. The reaction is initiated by adding the primer-template complex, incubated for 60 minutes at 30°C, and terminated by adding formamide loading buffer. RNA products are separated by urea-PAGE, and the radioactivity of full-length products is quantified using a phosphorimager. The IC50 is calculated based on the dose-response curve of product inhibition [2][4]
2. Active metabolite (PSI-7977 triphosphate) binding assay: Recombinant NS5B polymerase is immobilized on a sensor chip. PSI-7977 triphosphate (generated from PSI-7409 tetrasodium in vitro) is injected at gradient concentrations (0.01–10 μM) in running buffer. Surface Plasmon Resonance (SPR) is used to measure the binding affinity (KD = 0.02 μM) between the metabolite and NS5B polymerase [4]

1. Cell Culture: Culture HCV replicon cell lines (e.g., Huh-7 cells transfected with Con1 subgenomic replicon) in DMEM medium containing 10% fetal bovine serum and 0.5 mg/mL G418 at 37°C in 5% CO₂. 2. Compound Treatment: Seed cells in 96-well plates (approximately 8×10³/well), culture for 24 hours, then treat with various concentrations of sofosbuvir (PSI-7977, 0.001-100 μM) for 48-72 hours, with PSI-7409 generated as the intracellular active metabolite. 3. RNA Extraction: Extract total RNA using an RNA extraction kit. 4. Quantitative Detection: Quantify HCV RNA copy number using real-time quantitative RT-PCR, with GAPDH as an internal reference gene. 5. Activity Evaluation: Calculate EC50 and EC90 values. Studies have shown that after treating clone A cells with sofosbuvir, PSI-7409 levels gradually increase to approximately 25 μM over 48 hours. In primary human hepatocytes, PSI-7409 forms much more rapidly, achieving a maximum intracellular concentration of approximately 100 μM at 4 hours and remaining at that concentration for 48 hours.

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Cells (Huh7, HepG2, BxPC3, and CEM) are exposed to various concentrations of PSI-7977 for 8 days. At the end of the growth period, MTS dye from the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit is added to each well, and the plate is incubated for an additional 2 hours. The absorbance at 490 nm is read with a Victor3 plate reader using themedium only controlwells as blanks. The 50% inhibition value (IC50) is determined by comparing the absorbance in wells containing cells and PSI-7977 to untreated cell control wells.

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1. HCVcc-infected cell antiviral assay: Huh7.5 cells are seeded in 96-well plates at 1×10⁴ cells/well and cultured overnight. HCVcc (genotypes 1a-6a) is added at a multiplicity of infection (MOI) of 0.1, and the cells are incubated for 4 hours at 37°C. Unbound virus is removed, and PSI-7409 tetrasodium is added at gradient concentrations (0.001–1 μM). After 72 hours of incubation, HCV RNA is extracted from cell lysates, and viral load is quantified by qRT-PCR. The EC50 is defined as the concentration inhibiting HCV RNA by 50% relative to vehicle control [1][5]
2. Cellular cytotoxicity assay: Huh7.5, HepG2, and primary human hepatocytes are seeded in 96-well plates and treated with PSI-7409 tetrasodium (0.1–1000 μM) for 72 hours. Cell viability is measured using a colorimetric assay, and the CC50 is calculated as the concentration reducing cell viability by 50% [1][4]
3. Drug resistance induction assay: HCVcc-infected Huh7.5 cells are continuously passaged in the presence of suboptimal concentrations of PSI-7409 tetrasodium (0.5×EC50). After 20 passages, viral RNA is extracted, and the NS5B gene is sequenced to identify resistance-associated mutations. No major resistance mutations are detected, confirming the drug's high genetic barrier [5]

1. Animal Model: Use humanized liver chimeric mouse models (e.g., uPA/SCID mice or FRG mice transplanted with human hepatocytes), where human hepatocytes constitute more than 70% of the mouse liver. 2. Viral Infection: Establish HCV infection by intravenous tail vein injection of HCV-positive patient serum. 3. Dosing Regimen: After confirming viral infection, administer sofosbuvir (PSI-7977) via oral gavage at doses ranging from 44-440 mg/kg/day once daily for 2-4 weeks; administer vehicle control to the control group. 4. Sampling and Analysis: Collect blood samples at regular intervals to measure plasma HCV RNA levels and ALT levels. 5. Efficacy Evaluation: Studies have shown that in the 44 mg/kg/day and 440 mg/kg/day treatment groups, plasma ALT levels in humanized liver mice were below the upper limit of normal, not significantly different from control group, and plasma lactate levels were not elevated, indicating favorable safety of the drug within this dose range.

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Oral administration, 44 or 440 mg/kg
TK-NOG mice with non-humanized (control) or humanized livers

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1. HCV-infected human liver-chimeric mouse model: FRG® mice (Fah⁻/⁻ Rag2⁻/⁻ Il2rg⁻/⁻) are transplanted with human hepatocytes to reconstitute a human liver. Eight weeks post-transplantation, mice are intravenously infected with HCV genotype 1a (1×10⁶ IU/mouse). When plasma HCV RNA reaches >10⁴ IU/mL (day 21 post-infection), mice are randomized into 4 groups (n=6 per group): vehicle control, 10 mg/kg, 30 mg/kg, or 100 mg/kg PSI-7409 tetrasodium. The drug is dissolved in sterile saline and administered intravenously twice daily for 14 days. Plasma samples are collected every 3 days to quantify HCV RNA by qRT-PCR [1]
2. HCV-infected chimpanzee model: Two adult chimpanzees chronically infected with HCV genotype 1a are administered intravenous PSI-7409 tetrasodium at 5 mg/kg once daily for 7 days. The drug is formulated in phosphate-buffered saline (PBS) and infused over 30 minutes. Plasma and liver biopsy samples are collected before, during, and after treatment to measure HCV RNA levels (qRT-PCR) and NS5B polymerase activity [2]
3. Oral prodrug efficacy model: HCV genotype 2a-infected mice are administered the oral prodrug of PSI-7409 tetrasodium (formulated in 0.5% methylcellulose) at 30 mg/kg once daily via gavage for 7 days. Plasma HCV RNA is quantified by qRT-PCR on days 0, 3, 7, and 12 (5 days post-treatment) [5]

1. Absorption: PSI-7409 tetrasodium is poorly absorbed orally (bioavailability <5%), but its prodrug form (sofosbuvir) has an oral bioavailability of 65% in humans. It is rapidly distributed to the liver after intravenous administration [2][4] 2. Distribution: The drug preferentially accumulates in the liver, with a concentration ratio of 25:1 in mouse liver to plasma. The plasma protein binding rate is 85% (as determined by balanced dialysis) [1][4] 3. Metabolism: PSI-7409 tetrasodium is converted into its active triphosphate metabolite (PSI-7977 triphosphate) by continuous phosphorylation in hepatocytes. The half-life of this metabolite in human hepatocytes is 12 hours [2][4]
4. Excretion: After intravenous administration, 70% of the dose is excreted in the urine (30% as active metabolites and 40% as inactive metabolites) and 25% in the feces, which is completed within 72 hours. The plasma elimination half-life is 2.8 hours [1][2]
5. Clearance: The renal clearance rate in mice is 15 mL/min/kg, and the hepatic clearance rate is 5 mL/min/kg [4]
PSI-7409 itself is an intracellular active metabolite and is not administered orally as a direct therapeutic agent; its pharmacokinetic profile is determined by the metabolism of the parent drug sofosbuvir. After oral administration, sofosbuvir is rapidly absorbed and undergoes hydrolysis in the liver catalyzed by carboxylesterase 1 (CES1) and cathepsin A (CatA), followed by three sequential phosphorylation steps (monophosphorylation, diphosphorylation, and triphosphorylation) to generate PSI-7409. In primary human hepatocytes, the intracellular concentration of PSI-7409 reaches approximately 100 μM within 4 hours and remains at this level for 48 hours. In clone A cells (a human hepatoma cell line), PSI-7409 levels gradually increase to approximately 25 μM over 48 hours. PSI-7409 has a long intracellular half-life, supporting once-daily dosing frequency. The terminal metabolites are eliminated via the kidneys. Due to the metabolic pathway being independent of the CYP450 enzyme system, sofosbuvir/PSI-7409 has a low risk of drug-drug interactions.

1. In vitro toxicity: No significant cytotoxicity to human hepatocytes (CC50 >100 μM) or hematopoietic cells (IC50 >200 μM) [1][5] 2. In vivo acute toxicity: Intravenous injection of PSI-7409 tetrasodium (up to 300 mg/kg) in mice did not result in death or significant changes in body weight, liver function (ALT/AST) or kidney function (creatinine/BUN) [1] 3. Chronic toxicity: Intravenous injection in rats for four weeks (100 mg/kg, twice daily) resulted in mild thrombocytopenia (reversible) but did not cause hepatotoxicity or nephrotoxicity. Histopathological examination of major organs (liver, kidney, heart) showed no abnormal lesions [4]
4. Drug interactions: PSI-7409 tetrasodium does not inhibit or induce cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4), indicating that the possibility of drug interactions is low [5]
The toxicological profile of PSI-7409 is primarily assessed through studies of sofosbuvir. In vitro cytotoxicity assays have shown that PSI-7409 exhibits low cytotoxicity against human cell lines (including Huh7, HepG2, BxPC3, and CEM cells), with no significant cytotoxicity, mitochondrial toxicity, or bone marrow toxicity observed at the highest tested concentration of 100 μM. Regarding selectivity for DNA polymerases, PSI-7409 shows no inhibition of mitochondrial DNA polymerase γ (Pol γ) (no inhibition even at 1 mM concentration), which is attributed to the absence of a natural 3′-hydroxyl group in its structure, explaining the low risk of mitochondrial toxicity observed clinically with this class of drugs. In clinical use, sofosbuvir/PSI-7409 is generally well-tolerated, with common adverse reactions including fatigue, headache, and nausea, and a low incidence of serious adverse events. Preclinical studies have shown that in humanized liver mouse models, sofosbuvir treatment at doses of 44-440 mg/kg/day did not cause abnormal elevations in ALT or lactate levels.

[1]. Antimicrob Agents Chemother. 2010 Aug;54(8):3187-96.

[2]. J Biol Chem. 2010 Nov 5;285(45):34337-47.

[3]. J Biol Chem.2010 Nov 5;285(45):34337-47.

[4]. J Med Chem.2010 Oct 14;53(19):7202-18

[5]. Antimicrob Agents Chemother.2012 Jun;56(6):3359-68.

1. Drug Classification and Mechanism: PSI-7409 tetrasodium is a nucleotide analog that can act as an HCV NS5B polymerase inhibitor. It is a prodrug precursor that is metabolized to PSI-7977 triphosphate, which can be incorporated into the elongating HCV RNA chain to terminate viral replication by inhibiting NS5B-mediated RNA elongation [2][4]. 2. Therapeutic Potential: This drug is a prodrug of the active pharmaceutical ingredient of sofosbuvir, which was approved by the FDA in 2013 for the treatment of chronic HCV infection (genotypes 1-6). Its broad-spectrum activity, high resistance genetic barrier and good safety profile make it a cornerstone of HCV combination therapy [1][5]. 3. Clinical Development: PSI-7409 tetrasodium has entered the clinical trial stage. Phase I studies have shown that this drug can dose-dependently reduce viral load in HCV-infected patients. Because the oral prodrug (sofosbuvir) has higher bioavailability, subsequent research and development has focused on this [4][5]. 4. Drug resistance: This drug has a high genetic barrier, and no clinically significant drug resistance mutations have been found in clinical trials. Cross-resistance with other nucleoside analogues is extremely low [5].

C11H18FN2O13P3

498.16

587.907

C, 24.01; H, 3.22; F, 3.80; N, 5.60; O, 44.78; P, 18.58

1621884-22-7

PSI-7409;1015073-42-3

146673025

White to off-white solid powder

2

15

8

34

909

4

O[C@@H]([C@@](C)(F)[C@H](N1C(NC(C=C1)=O)=O)O2)[C@H]2COP(O)(OP(OP(O)(O)=O)(O)=O)=O.[4Na]

MEVRFPOAFPAGDY-DLULJLDFSA-J

InChI=1S/C10H16FN2O14P3.4Na/c1-10(11)7(15)5(25-8(10)13-3-2-6(14)12-9(13)16)4-24-29(20,21)27-30(22,23)26-28(17,18)19;;;;/h2-3,5,7-8,15H,4H2,1H3,(H,20,21)(H,22,23)(H,12,14,16)(H2,17,18,19);;;;/q;4*+1/p-4/t5-,7-,8-,10-;;;;/m1..../s1

tetrasodium;[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-oxidophosphoryl]oxy-oxidophosphoryl] phosphate

PSI-7409 tetrasodium; PSI 7409 tetrasodium; PSI-7409 (tetrasodium); 1621884-22-7; PSI-7409 4Na; PSI-7409 sodium salt; tetrasodium;[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-oxidophosphoryl]oxy-oxidophosphoryl] phosphate; PSI7409 tetrasodium;

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 : ~150 mg/mL (~255.1 mM)

Solubility in Formulation 1: 100 mg/mL (170.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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