PSI-7976 is a prodrug that must be metabolized to the active triphosphate form (PSI-7409) to inhibit the HCV NS5B RNA-dependent RNA polymerase (RdRp). The active triphosphate metabolite PSI-7409 has been previously shown to be a potent inhibitor of HCV NS5B RdRp with a Ki value of 0.42 μM. [1]

PSI-7976 inhibited HCV RNA replication in clone A HCV replicon cells with an EC50 of 1.07 ± 0.04 μM and an EC90 of 2.99 ± 0.82 μM. In comparison, PSI-7977 (the other diastereoisomer) had an EC50 of 0.092 ± 0.005 μM and EC90 of 0.29 ± 0.08 μM, and the isomeric mixture PSI-7851 had an EC50 of 0.149 ± 0.001 μM and EC90 of 0.51 ± 0.16 μM. [1]
In cellular metabolism studies using clone A cells (which express CatA but not CES1), incubation with 50 μM PSI-7976 resulted in lower intracellular concentrations of the active triphosphate PSI-7409 compared to PSI-7977. In primary human hepatocytes (which express both CatA and CES1), similar amounts of PSI-7409 were formed when cells were incubated with PSI-7976, PSI-7977, or PSI-7851. [1]

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Combining PSI-7976 and PSI-7977, two diastereoisomers that are known to be a more potent inhibitor of HCV RNA replication in HCV replicon tests, results in PSI-7851. PSI-7976 is hydrolyzed more favorably by carboxylesterase 1 (CES1) than PSI-7977. Additionally, PSI-7976 is a more effective substrate for CES1 than cathepsin A (CatA) according to kinetic data[1].

PSI-7976 hydrolysis by cathepsin A (CatA): Purified recombinant human CatA was activated by incubation with cathepsin L (1 μg/ml) in 25 mM MES buffer pH 6.0 with 5 mM DTT for 30 min at 37°C, followed by CatL inactivation with 10 μM E-64. The CatA assay was performed in a 100-μl reaction mixture containing activated CatA (0.1 μg), 100 μM PSI-7976 in 25 mM MES pH 6.0, 100 mM NaCl, 1 mM DTT, and 0.1% Nonidet P-40. After incubation at 37°C for 1 h, the reaction mixture was filtered through a YM-10 Microcon filter to remove protein, and the flow-through was analyzed by HPLC using a PARTISIL 10 SAX column with a linear gradient of buffer B (1 M KH2PO4 pH 3.5) from 0 to 100% over 90 min. Time-dependent formation of the common intermediate PSI-352707 was monitored. When CatA was incubated with PSI-7976 for 150 min, approximately 18-fold less PSI-352707 was formed compared to PSI-7977 as substrate. [1]
Steady-state kinetic parameters for PSI-7976 with CatA were determined using 14C-labeled compound. Reactions were performed at 37°C in a 100-μl volume containing varied concentrations of PSI-7976 in buffer containing 50 mM Hepes pH 7.0, 100 mM NaCl, and 0.1% Nonidet P-40, with final activated CatA protein concentration of 0.1 μg/ml. Aliquots (10 μl) were spotted on DE81 paper at each time point, washed three times with 1 mM ammonium formate for 5 min followed by an ethanol wash, dried, and counted in a liquid scintillation counter. The kinetic parameters were: Km = 880 ± 430 μM, kcat = 0.30 ± 0.07 min⁻¹, kcat/Km = 0.0003 μM⁻¹•min⁻¹. [1]
PSI-7976 hydrolysis by carboxylesterase 1 (CES1): CES1 assays were performed in 50 mM Tris/HCl buffer pH 7.5 containing 100 μM PSI-7976 and 0.4 μg of CES1. After incubation at 37°C for 1 h, samples were filtered and analyzed by HPLC as described above. PSI-7976 showed a time-dependent increase in product formation with CES1. Kinetic parameters for PSI-7976 with CES1 were: Km = 51 ± 1 μM, kcat = 0.27 ± 0.02 min⁻¹, kcat/Km = 0.0053 μM⁻¹•min⁻¹. In contrast, PSI-7977 exhibited complex kinetics with CES1 and kinetic parameters could not be determined. [1]

PSI-7976 HCV replicon assay: Clone A HCV replicon cells were seeded at 1,500 cells/well in 96-well plates with 2-fold serial drug dilutions (total volume 100 μl/well). Plates were incubated at 37°C in 5% CO2 for 4 days. Supernatant was discarded, total RNA was extracted using an RNeasy 96 kit, and amplified as described. ΔCt values for HCV RNA were determined, and EC90 was calculated. A "no drug" control was used to determine maximum HCV RNA. PSI-7976 had EC50 of 1.07 ± 0.04 μM and EC90 of 2.99 ± 0.82 μM. [1]
Cellular metabolism study: Clone A cells (approximately 5×10⁶ cells/T75 flask) or primary human hepatocytes (approximately 5×10⁶ cells/T75 flask) were seeded and incubated overnight to allow attachment. Cells were then incubated with 50 μM PSI-7976 in fresh medium for up to 24 h at 37°C in 5% CO2. At selected times, cells were trypsinized, counted, centrifuged at 1,200 rpm for 5 min, and cell pellets suspended in 1 ml of cold 60% methanol and incubated overnight at -20°C. Samples were centrifuged at 14,000 rpm for 5 min, supernatants dried using a SpeedVac concentrator, and stored at -20°C. Residues were suspended in 100 μl of water and analyzed by ion exchange HPLC with a SAX column using a linear gradient of buffer B (1 M KH2PO4 pH 3.5) from 0 to 100% over 90 min, with UV detection at 254 nm. Intracellular PSI-7409 concentration was calculated using a standard curve and converted to micromolar based on 3 μl volume per 1×10⁶ cells for normal human liver parenchymal cells. In clone A cells (which lack CES1 expression), PSI-7977 produced higher PSI-7409 concentrations than PSI-7976, while PSI-7851 gave intermediate concentrations. In primary human hepatocytes (expressing both CatA and CES1), similar PSI-7409 concentrations were observed with PSI-7976, PSI-7977, and PSI-7851. [1]

PSI-7976 is metabolized intracellularly to the active triphosphate PSI-7409 through a multi-step pathway. In primary human hepatocytes, which express both CatA and CES1, similar amounts of PSI-7409 were formed from PSI-7976, PSI-7977, and the isomeric mixture PSI-7851. In contrast, in clone A cells (which lack CES1 expression), PSI-7976 resulted in lower intracellular concentrations of PSI-7409 compared to PSI-7977. [1]
PSI-7976 is a substrate for both CatA and CES1. The kinetic parameters for PSI-7976 with CatA were: Km = 880 ± 430 μM, kcat = 0.30 ± 0.07 min⁻¹, kcat/Km = 0.0003 μM⁻¹•min⁻¹. With CES1, the parameters were: Km = 51 ± 1 μM, kcat = 0.27 ± 0.02 min⁻¹, kcat/Km = 0.0053 μM⁻¹•min⁻¹. [1]

Toxicity Summary
Identification and Use: Sofosbuvir is a white to off-white crystalline solid. Sofosbuvir is an antiviral drug (pan-genotypic polymerase inhibitor) that acts directly on the hepatitis C virus. It is used in combination with other antiviral drugs to treat chronic hepatitis C virus (HCV) genotypes 1, 2, 3, or 4 infection in adults, including patients with hepatocellular carcinoma awaiting liver transplantation and patients co-infected with human immunodeficiency virus (HIV). Sofosbuvir must be used as part of a multidrug regimen and should not be used alone to treat chronic HCV infection. Human Exposure and Toxicity: The highest recorded sofosbuvir dose was a single administration of 1200 mg over-therapeutic dose to 59 healthy subjects. No adverse events were observed at this dose level, and the frequency and severity of adverse events were similar to those reported in the placebo group and the sofosbuvir 400 mg treatment group. Sofosbuvir did not induce chromosomal aberrations in human peripheral blood lymphocytes. Animal studies: Single-dose toxicity studies of GS-9851/PSI-7851 (a mixture of diastereomers) were conducted in rats. No death, clinical signs, weight changes, gross pathological changes, or changes in liver and kidney organ weight were observed at the highest dose of 1800 mg/kg. Repeated-dose oral toxicity studies lasting up to 13 weeks were conducted in mice, up to 26 weeks in rats, and up to 39 weeks in dogs, evaluating sofosbuvir or GS-9851 (a 1:1 diastereomer mixture of sofosbuvir and its stereoisomers). Major target organs include the cardiovascular, hepatobiliary, gastrointestinal, and hematopoietic (erythroid) systems. In a 7-day GS-9851 toxicity study, rats were given a daily dose of 2000 mg/kg and dogs a daily dose of 1500 mg/kg. Results showed (but were not limited to) increased gastric mucus secretion, glycogen depletion, elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin; dogs also showed associated liver histopathological changes. Cardiac adverse reactions were observed in both rats (e.g., multifocal myocardial fibrosis) and dogs (e.g., QT/QTc interval prolongation). In long-term studies of GS-9851 or sofosbuvir, the aforementioned hepatic and cardiac adverse reactions were not observed. In chronic toxicity studies in rats (26 weeks) and dogs (39 weeks), observed adverse reactions included (but were not limited to) gastrointestinal clinical symptoms (e.g., loose stools and vomiting) and a decrease in mean erythrocyte index (e.g., a decrease of approximately 10%), primarily observed in the high-dose group of dogs. When evaluated in rats, sofosbuvir had no effect on embryo-fetal viability or fertility. No teratogenic effects were observed in sofosbuvir developmental toxicity studies in rats and rabbits. In prenatal and postnatal developmental studies in rats, sofosbuvir had no adverse effects on offspring behavior, reproduction, or development. At the highest tested dose, exposure to the major circulating metabolite GS-331007 was at least 8 times the recommended human clinical exposure. Rats exposed to GS-331007 (AUC) daily from prenatal (in utero) to day 20 of lactation had offspring with normal fertility. Daily exposure to GS-331007 was approximately 12 times the recommended human clinical exposure. A two-year carcinogenicity study of sofosbuvir was conducted in mice and rats. Doses were up to 200 mg/kg/day in male mice and up to 600 mg/kg/day in female mice; doses were up to 750 mg/kg/day in both male and female rats. At the highest test doses in mice and rats, no increase in drug-related tumor incidence was observed, resulting in AUC exposures of the major circulating metabolite GS-331007 that were approximately 7-fold (mice) and 30-fold (rats), and 13-fold and 17-fold (rats), respectively, to the human exposure at the recommended clinical dose. Sofosbuvir did not show genotoxicity in a range of in vitro and in vivo studies, including bacterial mutagenicity assays and in vivo mouse micronucleus assays. Interactions Rifampin (a potent intestinal P-gp inducer) may lead to decreased plasma concentrations of both sofosbuvir and GS-331007, potentially reducing the therapeutic effect of sofosbuvir. Rifampin and sofosbuvir should not be used concurrently. Rifabutin is expected to decrease plasma concentrations of both sofosbuvir and GS-331007, which may reduce the efficacy of sofosbuvir. Concurrent use of rifabutin and sofosbuvir is not recommended. When used concomitantly with sofosbuvir, certain antiepileptic drugs (such as carbamazepine, oxcarbazepine, phenobarbital, and phenytoin sodium) are expected to decrease plasma concentrations of both sofosbuvir and GS-331007, which may reduce the efficacy of sofosbuvir. Concomitant use of these antiepileptic drugs and sofosbuvir is not recommended. Sofosbuvir is a substrate of breast cancer resistance protein (BCRP); GS-331007 is not a BCRP substrate. BCRP inhibitors may increase plasma concentrations of sofosbuvir but will not increase plasma concentrations of GS-331007. Sofosbuvir and GS-331007 are not BCRP inhibitors; the likelihood of pharmacokinetic interactions with BCRP substrate drugs is very low. For more complete data on interactions with sofosbuvir (13 items in total), please visit the HSDB record page.

[1]. Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977. J Biol Chem. 2010 Nov 5;285(45):34337-47.

PSI-7976 is the Rp diastereoisomer of PSI-7851, while PSI-7977 is the Sp diastereoisomer. The two isomers differ in their stereospecificity at the first hydrolysis step: CatA preferentially hydrolyzes PSI-7977, while CES1 preferentially hydrolyzes PSI-7976. Once the carboxyl ester moiety is hydrolyzed, the remaining steps leading to the formation of the active triphosphate PSI-7409 are identical for both isomers. [1]
In primary human hepatocytes, where both CatA and CES1 are expressed, both isomers produce similar levels of the active triphosphate, suggesting that both will be equally active in liver cells where HCV infection occurs. However, in the commonly used HCV replicon screening system (using Huh7-derived clone A cells where CES1 expression is significantly lower than in primary hepatocytes), the anti-HCV activity of phosphoramidate compounds may not directly correlate with their in vivo activity. [1]

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Therapeutic Uses
Sovaldi is a hepatitis C virus (HCV) nucleotide analog NS5B polymerase inhibitor indicated for the treatment of chronic hepatitis C (CHC) infection as part of a combination antiviral therapy regimen. /US product label includes/
The following should be considered when initiating treatment with Sovaldi: Sovaldi monotherapy is not recommended for chronic hepatitis C (CHC). Treatment regimen and duration depend on viral genotype and patient population. Treatment response varies depending on host and viral baseline factors.

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Drug Warnings The FDA warns that severe bradycardia may occur when the antiarrhythmic drug amiodarone is used in combination with the hepatitis C drug Harvoni (ledipasvir/sofosbuvir) or Sovaldi (sofosbuvir) with another direct-acting antiviral drug used to treat hepatitis C infection. The U.S. Food and Drug Administration (FDA) has added information regarding severe bradycardia (a condition known as symptomatic bradycardia) to the drug labels for Harvoni and Sovaldi. The FDA advises healthcare professionals not to use Harvoni or Sovaldi in combination with another direct-acting antiviral drug (such as the investigational drugs daclatasvir or oricioso (simeprevir)) and amiodarone. Following a review of submitted post-marketing adverse event reports, the FDA found that patients may experience severe and life-threatening symptomatic bradycardia when Harvoni or Sovaldi is used in combination with another direct-acting antiviral drug and amiodarone. The reports indicate that one patient died from cardiac arrest, and three patients required pacemakers to regulate their heart rhythm. Other patients recovered after discontinuing hepatitis C medication or amiodarone (or both). The causes of these events are currently undetermined. The FDA will continue to monitor the risk of severe symptomatic bradycardia with Harvoni and Sovaldi and further investigate the causes of cardiac-related events resulting from the combination of amiodarone and these hepatitis C medications.
It is not recommended to use sofosbuvir in combination with potent inducers of the intestinal P-glycoprotein (P-gp) transport system (such as rifampin and St. John's wort), as this may significantly reduce sofosbuvir plasma concentrations and potentially decrease its efficacy.
Anemia has been reported in patients receiving sofosbuvir in combination with ribavirin or in combination with pegylated interferon-alpha and ribavirin. In clinical trials, 21% of patients receiving sofosbuvir, pegylated interferon-alpha, and ribavirin for 12 weeks reported anemia, compared to 12% of patients receiving pegylated interferon-alpha and ribavirin for 24 weeks without sofosbuvir. In addition, among patients treated with sofosbuvir, pegylated interferon-alpha, and ribavirin for 12 weeks, 23% reported hemoglobin concentrations below 10 g/dL, compared to 14% among patients treated with pegylated interferon-alpha and ribavirin for 24 weeks (without sofosbuvir). Adverse reactions reported in over 20% of patients treated with sofosbuvir in combination with ribavirin and pegylated interferon-alpha included fatigue, headache, nausea, insomnia, and anemia. For more complete data on sofosbuvir warnings (13 in total), please visit the HSDB record page.

C22H29FN3O9P

529.45

529.163

C, 49.91; H, 5.52; F, 3.59; N, 7.94; O, 27.20; P, 5.85

1190308-01-0

Sofosbuvir;1190307-88-0;Sofosbuvir impurity C;1496552-28-3;Sofosbuvir impurity A;1496552-16-9;Sofosbuvir-d6;1868135-06-1;Sofosbuvir-13C,d3

45375809

White to off-white solid powder

2.047

3

11

11

36

913

6

C[C@@H](C(=O)OC(C)C)NP(=O)(OC[C@@H]1[C@H]([C@@]([C@@H](O1)N2C=CC(=O)NC2=O)(C)F)O)OC3=CC=CC=C3

TTZHDVOVKQGIBA-IAAJYNJHSA-N

InChI=1S/C22H29FN3O9P/c1-13(2)33-19(29)14(3)25-36(31,35-15-8-6-5-7-9-15)32-12-16-18(28)22(4,23)20(34-16)26-11-10-17(27)24-21(26)30/h5-11,13-14,16,18,20,28H,12H2,1-4H3,(H,25,31)(H,24,27,30)/t14-,16+,18+,20+,22+,36?/m0/s1

propan-2-yl (2S)-2-[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate

psi-7976; psi7976; psi 7976

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.)