HCV ( EC50 = 92±5 nM )

The average plasma ALT levels in the 440- and 44-mg/kg/d treatment groups of mice with humanized livers were below the upper limit of normal and did not differ statistically from those in the vehicle-treated humanized liver mice. Additionally, neither the control mice nor the mice with humanized livers that received either dose of PSI-7977 had elevated plasma lactate levels.

In T75 flasks, clone A cells are deposited at a density of approximately 5×10⁶ cells/flask using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mL Penicillin, and 100 μg/mL streptomycin. In a similar manner, T75 flasks are seeded with approximately 5×10⁶ human primary hepatocytes per flask using cell plating medium. Cells are incubated with 50 μM PSI-7851, PSI-7976, or Sofosbuvir (PSI-7977) in fresh medium for clone A cells or in cell maintenance medium for primary hepatocytes for up to 24 hours at 37°C in a 5% CO₂ atmosphere after being left to attach overnight. When using radiolabeled PSI-7851 in the study, the same protocols are followed, with the exception that 1×10⁶ cells are seeded into each well of a 6-well plate, and the cells are then incubated with 5 μM [³H]PSI-7851. The medium is taken out at predetermined intervals, and the cell layer is cleaned using cold phosphate-buffered saline (PBS). Following trypsinization, cells are tallied and centrifuged for five minutes at 1,200 rpm. The cell pellets are left overnight at −20°C after being suspended in 1 mL of cold 60% methanol. After centrifuging the samples for five minutes at 14,000 rpm, the supernatants are gathered, dried with a SpeedVac concentrator, and kept at -20°C until high performance liquid chromatography (HPLC) analysis. After suspending residues in 100 μL of water, 50 μL aliquots are injected into an HPLC.

Absorption, Distribution and Excretion
When given orally, sofosbuvir reaches its maximum plasma concentration in about 0.5 to 2 hours with a maximal concentration (Cmax) of 567 ng/mL.
Sofosbuvir is eliminated by three routes: urine ( 80%), feces (14%), and respiration (2.5%); however, elimination through the kidneys is the major route.
The volume of distribution for sofosbuvir has yet to be determined.
The clearance of sofosbuvir has yet to be determined.
Sofosbuvir is approximately 61-65% bound to human plasma proteins and the binding is independent of drug concentration over the range of 1 ug/mL to 20 ug/mL. Protein binding of GS-331007 was minimal in human plasma. After a single 400 mg dose of (14)C-sofosbuvir in healthy subjects, the blood to plasma ratio of (14)C-radioactivity was approximately 0.7.
The pharmacokinetic properties of sofosbuvir and the predominant circulating metabolite GS-331007 have been evaluated in healthy adult subjects and in subjects with chronic hepatitis C. Following oral administration of SOVALDI, sofosbuvir was absorbed with a peak plasma concentration observed at approximately 0.5-2 hour post-dose, regardless of dose level. Peak plasma concentration of GS-331007 was observed between 2 to 4 hours post-dose. Based on population pharmacokinetic analysis in subjects with genotype 1 to 6 HCV infection who were coadministered ribavirin (with or without pegylated interferon), geometric mean steady state AUC0-24 was 969 ng*hr/mL for sofosbuvir (N=838), and 6790 ng*hr/mL for GS-331007 (N=1695), respectively. Relative to healthy subjects administered sofosbuvir alone (N = 272), the sofosbuvir AUC0-24 was 60% higher; and GS-331007 AUC0-24 was 39% lower, respectively, in HCV-infected subjects. Sofosbuvir and GS-331007 AUCs are near dose proportional over the dose range of 200 mg to 1200 mg.
Following a single 400 mg oral dose of (14)C-sofosbuvir, mean total recovery of the dose was greater than 92%, consisting of approximately 80%, 14%, and 2.5% recovered in urine, feces, and expired air, respectively. The majority of the sofosbuvir dose recovered in urine was GS-331007 (78%) while 3.5% was recovered as sofosbuvir. These data indicate that renal clearance is the major elimination pathway for GS-331007.
Studies in pregnant rats showed that sofosbuvir crossed the placenta. Fetal blood and brain sofosbuvir derived radioactivity was higher than in dams, but fetal liver and kidney had lower levels than corresponding organs in dams. Sofosbuvir-derived radioactivity was also quantifiable in milk from day 2 postpartum rats, but nursing pups did not appear to be extensively exposed to drug-derived radioactivity. Milk to plasma ratios were 0.1 at 1 hour and 0.8 at 24 hours.
For more Absorption, Distribution and Excretion (Complete) data for Sofosbuvir (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
In vitro studies in human liver microsomes showed that sofosbuvir was an efficient substrate for Cathepsin A (Cat A) and carboxyl esterase 1 (CES1). Sofosbuvir was cleaved by CatA and CES1 and subsequent activation steps included amino acid removal by histidine triad nucleotide-binding protein 1 (HINT1) and phosphorylation by uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase and nucleoside diphosphate (NDP) kinase. In vitro data indicated that Cat A preferentially hydrolysed sofosbuvir (the S-diastereomer) while CES1 did not exhibit stereoselectivity.
In vitro studies in human liver microsomes showed that sofosbuvir was an efficient substrate for Cathepsin A (Cat A) and carboxyl esterase 1 (CES1). There were no indications of metabolism via urdine diphosphate glucuronosyltransferases (UGTs) or flavin-containing monooxygenase (FMO). Sofosbuvir was cleaved by CatA and CES1 and subsequent activation steps included amino acid removal by histidine triad nucleotide-binding protein 1 (HINT1) and phosphorylation by uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase and nucleoside diphosphate (NDP) kinase. In vitro data indicated that Cat A preferentially hydrolysed sofosbuvir (the S-diastereomer) while CES1 did not exhibit stereoselectivity. This would be consistent with studies using GS-9851 showing a less efficient metabolism to the triphosphate in the hepatically-derived cell line containing the Clone A replicon and shown to exhibit low CES 1 activity, but high Cat A activity compared with primary human hepatocytes. Following incubation of hepatocytes from rat, dog, monkey and human GS-9851 was converted to the triphosphate GS-461203 in all species, most efficiently in human. Sofosbuvir was also readily converted to the triphosphate in dog liver after oral doses and was the dominant metabolite at all time points assessed with a long half-life of approx. 18 hours. The active metabolite GS-461203 could not be detected in monkey. Further while GS-461203 was detected in rat liver, it could not be measured in liver from mouse.
Sofosbuvir is extensively metabolized in the liver to form the pharmacologically active nucleoside analog triphosphate GS-461203. The metabolic activation pathway involves sequential hydrolysis of the carboxyl ester moiety catalyzed by human cathepsin A (CatA) or carboxylesterase 1 (CES1) and phosphoramidate cleavage by histidine triad nucleotide-binding protein 1 (HINT1) followed by phosphorylation by the pyrimidine nucleotide biosynthesis pathway. Dephosphorylation results in the formation of nucleoside metabolite GS-331007 that cannot be efficiently 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 material in all species and all matrices. In plasma, urine and feces of all species administered sofosbuvir the primary metabolite detected was GS-331007 accounting for >80% of total exposure. In rat liver and plasma GS-566500 was also detected. The metabolite profile was overall comparable between non-pregnant, pregnant and postpartum rats and in milk of postpartum rats with GS-331007 and 2 sulfate conjugates of GS-331007 being the major metabolites.
In dog following a single oral dose of 20 mg/kg of sofosbuvir three metabolites in plasma were identified, GS-331007, GS-566500 and M4 (proposed glucuronidation product of GS-606965), accounting for 93.4%, 1.6% and 0.5%, respectively of total plasma AUC. Parent compound amounted to 4.5%. In dog (and mouse) the majority of a radioactive dose was recovered in urine within 8 to 12 hours.
For more Metabolism/Metabolites (Complete) data for Sofosbuvir (7 total), please visit the HSDB record page.

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Biological Half-Life
Sofosbuvir has a terminal half life of 0.4 hours.
The median terminal half-lives of sofosbuvir and GS-331007 were 0.4 and 27 hours, respectively.

Toxicity Summary
IDENTIFICATION AND USE: Sofosbuvir is a white to off-white crystalline solid. Sofosbuvir is a direct-acting antiviral agent (pan-genotypic polymerase inhibitor) against the hepatitis C virus. It is used in conjunction with other antiviral agents for the treatment of chronic hepatitis C virus (HCV) genotype 1, 2, 3, or 4 infections in adults, including those with hepatocellular carcinoma awaiting liver transplantation and those with human immunodeficiency virus (HIV) co-infection. Sofosbuvir must be used as part of a multiple-drug regimen and should not be used alone for the treatment of chronic HCV infection. HUMAN EXPOSURE AND TOXICITY: The highest documented dose of sofosbuvir was a single supratherapeutic dose of sofosbuvir 1200 mg administered to 59 healthy subjects. There were no untoward effects observed at this dose level, and adverse events were similar in frequency and severity to those reported in the placebo and sofosbuvir 400 mg treatment groups. Sofosbuvir did not induce chromosome aberration using human peripheral blood lymphocytes. ANIMAL STUDIES: Single dose toxicity study was performed with GS-9851/PSI-7851 (the diastereomeric mixture) in rats. No mortality, clinical signs, body weight changes, macroscopic pathology, or organ weight changes for liver and kidney up to a highest dose of 1,800 mg/kg. Sofosbuvir or GS-9851, a 1:1 diastereomeric mixture of sofosbuvir and its stereoisomer, were evaluated in repeat-dose oral toxicity studies up to 13 weeks in mice, 26 weeks in rats, and 39 weeks in dogs. The primary target organs identified were the cardiovascular, hepatobiliary, gastrointestinal (GI) and hematopoietic (erythroid) systems. In the 7-day toxicity studies with GS-9851 doses of 2000 mg/kg/day in the rat and 1500 mg/kg/day in the dog resulted (but were not limited to) in increased mucus secretions in the stomach, glycogen depletion, and increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin, with associated histopathologic liver findings in dogs; and heart adverse effects in rats (e.g., multifocal cardiac myofiber degeneration) and dogs (e.g., increased QT/QTc intervals). Findings in the liver and heart were not observed in long-term studies with GS-9851 or sofosbuvir. In chronic toxicity studies in rats (26 weeks) and dogs (39 weeks), effects included (but were not limited to) GI-related clinical signs (e.g., soft feces and emesis) and a decrease (e.g., approximately 10%) in mean red cell indices that were observed mainly in the high-dose group of dogs. Sofosbuvir had no effects on embryo-fetal viability or on fertility when evaluated in rats. No teratogenic effects were observed in rat and rabbit developmental toxicity studies with sofosbuvir. It had no adverse effects on behavior, reproduction, or development of the offspring in the rat pre- and post-natal development study. At the highest dose tested, exposure to the predominant circulating metabolite GS-331007 was at least 8-fold the exposure in humans at the recommended clinical dose. Fertility was normal in the offspring of rats exposed daily from before birth (in utero) through lactation day 20 at daily GS-331007 exposures (AUC) of approximately 12-fold higher than human exposures at the recommended clinical dose. Two-year carcinogenicity studies in mice and rats were conducted with sofosbuvir. Mice were administered doses of up to 200 mg/kg/day in males and 600 mg/kg/day in females, while rats were administered doses of up to 750 mg/kg/day in males and females. No increase in the incidence of drug-related neoplasms were observed at the highest doses tested in mice and rats, resulting in AUC exposure to the predominant circulating metabolite GS-331007 of approximately 7- and 30-fold (in mice) and 13- and 17-fold (in rats), in males and females respectively, the exposure in humans at the recommended clinical dose. Sofosbuvir was not genotoxic in a battery of in vitro or in vivo assays, including bacterial mutagenicity, and in vivo mouse micronucleus assays.

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Interactions
Concomitant use of rifampin, a potent inducer of P-gp in the intestine, and sofosbuvir may cause decreased plasma concentrations of sofosbuvir and GS-331007 and may lead to decreased therapeutic effect of sofosbuvir. Rifampin and sofosbuvir should not be used concomitantly.
Rifabutin is expected to cause decreased plasma concentrations of sofosbuvir and GS-331007, which may lead to decreased therapeutic effect of sofosbuvir. Concomitant use of rifabutin and sofosbuvir is not recommended.
When used concomitantly with sofosbuvir, certain anticonvulsants (i.e., carbamazepine, oxcarbazepine, phenobarbital, phenytoin) are expected to decrease plasma concentrations of sofosbuvir and GS-331007, which may lead to decreased therapeutic effect of sofosbuvir. Concomitant use of these anticonvulsants and sofosbuvir is not recommended.
Sofosbuvir is a substrate of breast cancer resistance protein (BCRP); GS-331007 is not a BCRP substrate. Inhibitors of BCRP may cause increased plasma concentrations of sofosbuvir without increasing plasma concentrations of GS-331007. Sofosbuvir and GS-331007 are not BCRP inhibitors; pharmacokinetic interactions are unlikely with drugs that are BCRP substrates.
For more Interactions (Complete) data for Sofosbuvir (13 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.

[2]. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother. 2012 Jun;56(6):3359-68.

[3]. Discovery of a β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J Med Chem. 2010 Oct 14;53(19):7202-18.

[4]. Discovery and evolution of aloperine derivatives as a new family of HCV inhibitors with novel mechanism. Eur J Med Chem. 2018 Jan 1;143:1053-1065.

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 a component of a combination antiviral treatment regimen. /Included in US product label/
The following points should be considered when initiating treatment with Sovaldi: Monotherapy of Sovaldi is not recommended for treatment of chronic hepatitis C (CHC). Treatment regimen and duration are dependent on both viral genotype and patient population. Treatment response varies based on baseline host and viral factors.

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Drug Warnings
FDA is warning that serious slowing of the heart rate can occur when the antiarrhythmic drug amiodarone is taken together with either the hepatitis C drug Harvoni (ledipasvir/sofosbuvir) or with Sovaldi (sofosbuvir) taken in combination with another direct acting antiviral for the treatment of hepatitis C infection. FDA is adding information about serious slowing of the heart rate, known as symptomatic bradycardia, to the Harvoni and Sovaldi labels. FDA is recommending that health care professionals should not prescribe either Harvoni or Sovaldi combined with another direct acting antiviral, such as the investigational drug daclatasvir or Olysio (simeprevir), with amiodarone. FDA review of submitted postmarketing adverse event reports found that patients can develop a serious and life-threatening symptomatic bradycardia when either Harvoni or Sovaldi combined with another direct-acting antiviral is taken together with amiodarone. The reports included the death of one patient due to cardiac arrest and three patients requiring placement of a pacemaker to regulate their heart rhythms. The other patients recovered after discontinuing either the hepatitis C drugs or amiodarone, or both. The cause of these events could not be determined. FDA will continue to monitor Harvoni and Sovaldi for risks of serious symptomatic bradycardia and further investigate the reason why the use of amiodarone with these hepatitis C drugs led to the heart-related events.
Concomitant use of sofosbuvir with drugs that are potent inducers of the P-glycoprotein (P-gp) transport system in the intestine (e.g., rifampin, St. John's wort) is not recommended since this may result in substantially decreased sofosbuvir plasma concentrations and could lead to reduced therapeutic effect of sofosbuvir.
Anemia has been reported in patients receiving sofosbuvir in conjunction with ribavirin or in conjunction with peginterferon alfa andribavirin. In clinical trials, anemia was reported in 21% of patients who received 12 weeks of treatment with sofosbuvir, peginterferon alfa, and ribavirin compared with 12% of patients who received 24 weeks of treatment with peginterferon alfa and ribavirin without sofosbuvir. In addition, hemoglobin concentrations less than 10 g/dL were reported in 23% of patients who received 12 weeks of treatment with sofosbuvir, peginterferon alfa, and ribavirin compared with 14% of patients who received 24 weeks of treatment with peginterferon alfa and ribavirin without sofosbuvir.
Adverse effects reported in more than 20% of patients receiving sofosbuvir in conjunction with ribavirin and peginterferon alfa include fatigue, headache, nausea, insomnia, and anemia.
For more Drug Warnings (Complete) data for Sofosbuvir (13 total), please visit the HSDB record page.

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Pharmacodynamics
Sofosbuvir acts against HCV and is categorized as a direct-acting antiviral agent (DAA). At a dose 3 times the recommended dose, sofosbuvir does not prolong QTc to any clinically relevant extent.

C22H29FN3O9P

529.45

529.162

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

1190307-88-0

45375808

White solid powder

1.4±0.1 g/cm3

1.573

1.62

3

11

11

36

913

6

O=C1N([C@H]2[C@]([C@H](O)[C@@H](CO[P@](OC3=CC=CC=C3)(N[C@@H](C)C(OC(C)C)=O)=O)O2)(C)F)C=CC(N1)=O

TTZHDVOVKQGIBA-IQWMDFIBSA-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

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4.55 mg/mL (8.59 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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