Absorption, Distribution and Excretion
Sofosbuvir binds to human plasma proteins at a rate of approximately 61-65%, and this binding is independent of drug concentration within the concentration range of 1 μg/mL to 20 μg/mL. GS-331007 exhibits extremely low protein binding in human plasma. Following a single 400 mg (14)C-sofosbuvir administration in healthy subjects, the ratio of (14)C radioactivity in blood to plasma was approximately 0.7. The pharmacokinetic properties of sofosbuvir and its major circulating metabolite, GS-331007, have been evaluated in healthy adult subjects and patients with chronic hepatitis C. Following oral administration of SOVALDI, sofosbuvir is absorbed regardless of dose level, reaching peak plasma concentrations approximately 0.5-2 hours after administration. Peak plasma concentrations of GS-331007 occur 2-4 hours after administration. Based on a population pharmacokinetic analysis of HCV genotypes 1 to 6 patients treated with ribavirin (with or without pegylated interferon), the geometric mean steady-state AUC0-24 for sofosbuvir (N=838) and GS-331007 (N=1695) were 969 nghr/mL and 6790 nghr/mL, respectively. Compared with healthy subjects receiving sofosbuvir alone (N=272), the AUC0-24 of sofosbuvir was increased by 60% in HCV-infected patients, while the AUC0-24 of GS-331007 was decreased by 39%. Within the dose range of 200 mg to 1200 mg, the AUC values of sofosbuvir and GS-331007 were close to dose-proportionate.
After a single oral dose of 400 mg (14)C-sofosbuvir, the mean total recovery was greater than 92%, with approximately 80%, 14%, and 2.5% recovered from urine, feces, and exhaled air, respectively. Sofosbuvir recovered in urine was primarily in the form of GS-331007 (78%), while sofosbuvir itself accounted for only 3.5%. These data suggest that renal clearance is the primary elimination pathway for GS-331007.
Studies in pregnant rats showed that sofosbuvir can cross the placenta. Radioactivity of sofosbuvir in fetal blood and brain tissue was higher than in maternal rats, but lower in fetal liver and kidneys than in the corresponding maternal organs. Sofosbuvir-derived radioactivity was also quantitatively detected in the milk of pups on day 2 postpartum, but lactating pups did not appear to be significantly exposed to drug-derived radioactive substances. The milk-to-plasma ratio was 0.1 at 1 hour and 0.8 at 24 hours. For more complete data on the absorption, distribution, and excretion of sofosbuvir (6 items), please visit the HSDB record page. Metabolism/Metabolites In vitro human liver microsomal studies have shown that sofosbuvir is an effective substrate for cathepsin A (Cat A) and carboxylesterase 1 (CES1). No evidence of metabolism by uridine diphosphate glucuronyl transferase (UGT) or flavin-containing monooxygenase (FMO) was found. Subsequent activation steps after cleavage by Cat A and CES1 include amino acid removal by histidine triplet nucleotide-binding protein 1 (HINT1) and phosphorylation by uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase and nucleoside diphosphate (NDP) kinase. In vitro data indicate that Cat A preferentially hydrolyzes sofosbuvir (S-diastereform), while CES1 does not exhibit stereoselectivity. This is consistent with findings from studies using GS-9851, which demonstrated that GS-9851 is metabolized to triphosphate less efficiently in liver-derived cell lines containing the A clone replicon, exhibiting lower CES1 activity and higher Cat A activity compared to primary human hepatocytes. After incubation with rat, dog, monkey, and human hepatocytes, GS-9851 was converted to triphosphate GS-461203 in all species, with the highest conversion efficiency in human hepatocytes. Following oral administration of sofosbuvir, it was rapidly converted to triphosphate in canine liver and remained the major metabolite at all assessment time points, with a half-life of approximately 18 hours. The active metabolite GS-461203 was not detected in monkeys. Furthermore, while GS-461203 was detected in rat liver, it was not detected in mouse liver. Sofosbuvir is extensively metabolized in the liver to produce the pharmacologically active nucleoside analog triphosphate GS-461203. Its metabolic activation pathway includes: first, partial hydrolysis of the carboxylate ester catalyzed by human cathepsin A (CatA) or carboxylesterase 1 (CES1); second, phosphorylation catalyzed by histidine trinucleotide-binding protein 1 (HINT1); and finally, phosphorylation via the pyrimidine nucleotide biosynthesis pathway. Dephosphorylation yields the nucleoside metabolite GS-331007, which cannot be effectively rephosphorylated and lacks anti-HCV activity in vitro. GS-331007 and GS-566500 were detected in all species, with GS-331007 being the major drug-related substance in all species and all matrices. In all species administered sofosbuvir, the major metabolite detected was GS-331007 in plasma, urine, and feces, accounting for over 80% of total exposure. GS-566500 was also detected in rat liver and plasma. The metabolite profiles in the milk of non-pregnant, pregnant, and postpartum rats were generally similar, with GS-331007 and its two sulfate conjugates being the major metabolites. Following a single oral administration of 20 mg/kg sofosbuvir in dogs, three metabolites were identified in plasma: GS-331007, GS-566500, and M4 (presumably a glucuronidated product of GS-606965), accounting for 93.4%, 1.6%, and 0.5% of the total plasma AUC, respectively. The parent compound accounted for 4.5%. In dogs (and rats), most of the radioactive dose was recovered from urine within 8 to 12 hours. For more complete metabolite/metabolite data on sofosbuvir (7 metabolites in total), please visit the HSDB record page. The median terminal half-lives for sofosbuvir and GS-331007 were 0.4 hours and 27 hours, respectively.

Effects During Pregnancy and Lactation
◉ Summary of Medication Use During Lactation
While one infant was breastfed for 3 weeks during treatment for hepatitis C virus infection without developmental abnormalities, research on sofosbuvir in breastfeeding mothers undergoing treatment is insufficient. Breastfeeding does not need to be discontinued if the mother is using sofosbuvir alone or in combination with ledipasvir (Harvoni). Some sources suggest that breastfeeding should be avoided when sofosbuvir is used in combination with ribavirin.
Hepatitis C virus is not transmitted through breast milk, and breast milk has been shown to inactivate hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that breastfeeding should be considered if an HCV-infected mother experiences nipple fissures or bleeding. It is currently unclear whether this warning applies to mothers undergoing treatment for hepatitis C. Infants born to mothers infected with hepatitis C virus (HCV) should be tested for HCV; nucleic acid testing is recommended because maternal antibodies are present in the infant during the first 18 months of life and before the infant develops an immune response. ◉ Effects on breastfed infants: A mother started taking sofosbuvir 400 mg and ledipasvir 90 mg once daily for 12 weeks at 31 weeks of gestation to treat her chronic hepatitis C infection. The infant was breastfed for 3 weeks postpartum (feeding extent not specified). The infant was followed up for 1 year, and the results showed normal growth and development. ◉ Effects on lactation and breast milk: As of the revision date, no relevant published information was found.

Mechanism of Action
Sofosbuvir is an antiviral drug (pan-genotypic polymerase inhibitor) that acts directly on hepatitis C virus (HCV). HCV RNA replication is mediated by the membrane-associated polyprotein replication complex (MAP). HCV polymerase (NS5B protein) is an RNA-dependent RNA polymerase (RdRp). It is an essential initiation and catalytic subunit of this replication complex and is crucial for the viral replication cycle. There are no human homologs of HCV NS5B RdRp. Sofosbuvir is a monophosphorylated pyrimidine nucleotide prodrug that is metabolized intracellularly to form the pharmacologically active uridine analog triphosphate (GS-461203). GS-461203 competes with the native nucleotide for incorporation into the nascent RNA chain during viral genome replication by HCV NS5B. GS-461203 differs from endogenous pyrimidine nucleotides in that its 2' site is modified with the addition of methyl and fluorine functional groups. After GS-461203 is incorporated into nascent RNA, it significantly reduces the efficiency of RNA-dependent RNA polymerase (RdRp) in further elongating RNA, leading to premature termination of RNA synthesis. The cessation of viral replication results in a rapid decrease in HCV viral load and ultimately the clearance of HCV from the body.

C22H29FN3O9P

529.452530622482

529.162

1496552-28-3

Sofosbuvir;1190307-88-0;PSI-7976;1190308-01-0;Sofosbuvir-d6;1868135-06-1;Sofosbuvir-13C,d3

90055712

White to off-white crystalline solid

1.41±0.1 g/cm3 (20 ºC 760 Torr)

1

3

11

11

36

913

6

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

TTZHDVOVKQGIBA-UHFFFAOYSA-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)

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

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.)