GT5a (EC50 = 1.4 pM); GT1a H77 (EC50 = 1.8 pM); GT2b (EC50 = 1.9 pM); GT4a (EC50 = 1.9 pM); GT3a (EC50 = 2.1 pM)
Pibrentasvir is a potent pan-genotypic next-generation HCV NS5A inhibitor that retains activity against common amino acid substitutions of HCV genotypes 1–6, which are known to confer resistance to NS5A inhibitors that are currently approved.[1]

Pibrentasvir is a potent pan-genotypic next-generation HCV NS5A inhibitor that retains activity against common amino acid substitutions of HCV genotypes 1–6, which are known to confer resistance to NS5A inhibitors that are currently approved.[1]

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Pibrentasvir demonstrated potent, pan-genotypic antiviral activity against a panel of stable HCV subgenomic replicons containing NS5A from genotypes 1 to 6. The 50% effective concentration (EC50) values in 0% human plasma ranged from 1.4 to 5.0 pM: 1.8 pM against genotype 1a-H77, 4.3 pM against genotype 1b-Con1, 5.0 pM against genotype 2a-JFH-1, and between 1.4 and 2.8 pM against chimeric replicons containing NS5A from genotypes 2a, 2b, 3a, 4a, 5a, and 6a.
The antiviral activity ofpibrentasvirwas attenuated 35- to 47-fold in the presence of 40% human plasma due to plasma protein binding, with EC50s increasing to 64 pM (genotype 1a) and 200 pM (genotype 1b).
Pibrentasvir showed similar activity (median EC50s ranging from 0.50 to 2.7 pM) against a panel of 64 replicons containing NS5A genes derived from HCV-infected patient samples across genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a/e/p, including samples with baseline polymorphisms at positions associated with resistance to other NS5A inhibitors.
Pibrentasvirmaintained full activity against a wide range of single-position NS5A amino acid substitutions known to confer resistance to other NS5A inhibitors (e.g., daclatasvir, elbasvir, ledipasvir, ombitasvir, velpatasvir) in genotypes 1 to 6, with EC50 fold changes typically ≤7. For example, against genotype 1a single substitutions: M28T (2.1-fold), Q30E (2.4-fold), L31M (1.1-fold), Y93H (6.7-fold), Y93N (7.1-fold). Against genotype 3a Y93H, it showed only a 2.3-fold increase in EC50.
It also retained activity against replicons containing key resistance-associated substitutions for NS3/4A protease inhibitors (e.g., R155K, D168V) and NS5B polymerase inhibitors (e.g., S282T, C316Y), with EC50 fold changes ≤1.7, indicating no cross-resistance.
Pibrentasvir had no measurable antiviral activity against human immunodeficiency virus type 1 (HIV-1 EC50 >900,000 pM) or hepatitis B virus (HBV EC50 >32,000,000 pM).
Combination studies in genotype 1b replicon cells using checkerboard assays and MacSynergy II analysis showed that pibrentasvirproduced minor to moderate synergistic antiviral activity when combined with interferon-alpha (synergy volume 43 ± 4.7 μM²%), ribavirin (synergy volume 29 ± 7.4 μM²%), or the HCV NS3/4A protease inhibitor glecaprevir (synergy volume 73 ± 17 μM²%). No antagonism was observed. [1]

The study mentions that the potent in vitro activity of pibrentasvir translated to robust antiviral activity in a 3-day monotherapy clinical trial. In genotype 1-infected, treatment-naive adults, daily doses of 40 to 400 mg resulted in a mean maximal decrease in HCV plasma RNA from baseline of ≥4.1 log₁₀ IU/ml at the end of the 3-day period. The 120 mg dose, selected for phase 3 studies, achieved a 4.5 log₁₀ IU/ml decrease. During this monotherapy study, NS5A resistance-associated substitutions emerged in only 3 out of 40 (7.5%) patients. [1]

Southern Research Institute conducts assays for antiviral activity against HBV and HIV-1. Pibrentasvir is evaluated in an HIV-1 antiviral cytoprotection assay with the IIIB strain of the virus and CEM-SS cells. In short, the virus and cells are combined and incubated for six days in the presence of pibrentasvir or zidovudine (AZT; positive control). The virus titers are predetermined so that the control wells infected with the virus showed between 85% and 95% of the cells' viability being lost as a result of virus replication. Consequently, the observation of an antiviral effect or cytoprotection occurs when a compound hinders the replication of viruses. Twenty to twenty-five microliters of MTS reagent are added to each well six days after infection, and the microtiter plates are then incubated for four to six hours in order to measure the viability of the cells. Plates are read using a Molecular Devices Vmax or SpectraMax Plus plate reader spectrophotometrically at 490/650 nm.

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Antiviral Activity in Stable HCV Replicon Cells: Stable HCV subgenomic replicon cell lines (e.g., containing NS5A from genotypes 1a, 1b, 2a, or chimeric replicons) were cultured. The cells were incubated with serial dilutions of pibrentasvir for 3 days in medium containing 5% fetal bovine serum, with or without 40% human plasma. HCV replication inhibition was determined by measuring luciferase reporter activity from cell lysates using a luminometer. The EC50 was calculated using nonlinear regression curve fitting.
Cytotoxicity Assay (MTT): Cytotoxicity was assessed in Huh-7 cells (with genotype 1a replicon), HepG2 cells, and MT4 cells. Cells were plated in 96-well plates and incubated with pibrentasvir. After an incubation period, cell viability was measured using an MTT colorimetric assay. The 50% cytotoxic concentration (CC50) was calculated. The CC50 for pibrentasvir was >32,000,000 pM in Huh-7 cells and >10,000,000 pM in HepG2 and MT4 cells, indicating a high therapeutic index (>10⁷-fold).
Antiviral Activity Against HIV-1 and HBV: For HIV-1, an antiviral cytoprotection assay was used. CEM-SS cells were infected with HIV-1 (IIIB strain) in the presence of pibrentasvir and incubated for 6 days. Cell viability was assessed using an MTS reagent, with protection indicating antiviral effect. For HBV, HepG2 2.2.15 cells were treated with pibrentasvir for 6 days. Extracellular HBV DNA in the culture supernatant was quantified by real-time quantitative PCR (TaqMan) after protease treatment. The EC50 was calculated from the reduction in HBV DNA levels.
Resistance Selection Assay: HCV stable replicon cells containing NS5A from specific genotypes were plated and grown in the presence of G418 and pibrentasvir at concentrations 10- or 100-fold above the respective EC50. The medium was refreshed every 3-4 days for approximately 3 weeks. Surviving colonies were picked, expanded, and their NS5A coding region was amplified by RT-PCR and sequenced to identify resistance-associated amino acid substitutions. The colony survival rate was calculated.
Transient Replicon Assay for Mutant Susceptibility: HCV replicons engineered with specific amino acid substitutions in NS5A, NS3, or NS5B were constructed. Plasmids were linearized, transcribed into RNA, and transfected into Huh-7 cells. Inhibition of replicon replication by pibrentasvir was measured using a luciferase assay. Replication efficiency was calculated as a percentage of wild-type replication.
Combination Study (Checkerboard Assay): In genotype 1b-Con1 replicon cells, pibrentasvir was combined with another HCV inhibitor (e.g., IFN-α, ribavirin, glecaprevir) in a checkerboard format using serial 2-fold dilutions. HCV replication was determined by a luciferase reporter assay. The data were analyzed using the MacSynergy II program to calculate synergy/antagonism volumes and determine the interaction type (e.g., minor synergy, moderate synergy). [1]

Absorption, Distribution and Excretion
In healthy subjects, the time to peak plasma concentration (Tmax) is approximately 5 hours. The mean peak plasma concentration (Cmax) in non-cirrhotic HCV-infected individuals is 110 ng/mL. Food intake increases Pibrentasvir absorption by 40-53% compared to a fasting state. The primary route of excretion is the bile-fecal route, with 96.6% of the administered drug excreted in feces and 0% in urine. Metabolism/Metabolites Pibrentasvir is not metabolized. Biological Half-Life The elimination half-life (t1/2) is approximately 13 hours.

Effects During Pregnancy and Lactation
◉ Summary of Lactation Use
No studies have been conducted on Pibrentasvir in breastfeeding women undergoing treatment for hepatitis C. Because it binds to maternal plasma proteins at a rate exceeding 99.9%, its levels in breast milk are likely to be very low.
Hepatitis C is not transmitted through breast milk, and breast milk has been shown to inactivate the hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that breastfeeding should be considered if the mother has cracked or bleeding nipples. It is unclear whether this warning applies to mothers undergoing treatment for hepatitis C.
Infants born to HCV-infected mothers should be tested for HCV infection; nucleic acid testing is recommended because maternal antibodies are present in the first 18 months of life and before the infant develops an immune response.
◉ Impact on Breastfed Infants
No published information was found as of the revision date.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Pibrentasvir binds to human plasma proteins >99.9%. The plasma-to-serum ratio is approximately 0.62.

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This study provides in vitro cytotoxicity data (CC50), as described in the cell assay section, indicating low cytotoxicity. [1]

[1]. In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir. Antimicrob Agents Chemother. 2017 Apr 24;61(5). pii: e02558-16.

Pibrentasvir is a direct-acting antiviral drug and an NS5A inhibitor of hepatitis C virus (HCV), targeting viral RNA replication and viral particle assembly. Pibrentasvir, in combination with [DB13879], is effective in treating patients who have failed other NS5A inhibitor treatments. In cell culture, amino acid substitutions known to be associated with NS5A inhibitor resistance in HCV genotype 1a, 2a, or 3a replicons lead to decreased sensitivity and resistance to Pibrentasvir. These resistance-associated amino acid substitutions include: Q30D/deletion, Y93D/H/N, or H58D+Y93H in genotype 1a replicons; F28S+M31I or P29S+K30G in genotype 2a replicons; and Y93H in genotype 3a replicons. The NS5A amino acid substitutions that reduce sensitivity to Pibrentasvir include M28G or Q30D in the genotype 1a replicon and P32 deletion in the genotype 1b replicon. Pibrentasvir is marketed in combination with [DB13879] as an oral combination under the brand name Mavyret. This fixed-dose combination was approved by the FDA in August 2017 for the treatment of adult patients with chronic hepatitis C virus (HCV) genotypes 1–6 without cirrhosis (liver disease) or with mild cirrhosis, including patients with moderate to severe renal disease and those undergoing dialysis. Mavyret is also indicated for patients with HCV genotype 1 infection who have previously received treatment with an NS5A inhibitor or an NS3/4A protease inhibitor (but not both simultaneously). Hepatitis C virus infection often leads to decreased liver function, eventually progressing to liver failure, which severely impacts patients' quality of life. The ultimate goal of combination therapy is to achieve sustained virological response (SVR) and cure the patient. Clinical trials have shown that this combination therapy achieved an SVR12 rate of ≥93% in genotypes 1a, 2a, 3a, 4, 5, and 6, meaning undetectable hepatitis C virus for 12 weeks or longer after treatment. Pibrentasvir is a hepatitis C virus NS5A inhibitor. Pibrentasvir's mechanism of action is as a P-glycoprotein inhibitor, a breast cancer resistance protein inhibitor, an organic anion transporter 1B1 inhibitor, an organic anion transporter 1B3 inhibitor, a cytochrome P450 3A inhibitor, a cytochrome P450 1A2 inhibitor, and a UGT1A1 inhibitor. Indications: This drug is indicated for the treatment of adult patients with chronic hepatitis C virus (HCV) genotypes 1, 2, 3, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis (Child-Pugh A). MAVYRET is also indicated for the treatment of adult patients with HCV genotype 1 infection who have previously received a regimen containing an HCV NS5A inhibitor or an NS3/4A protease inhibitor (PI), but have not received both concurrently.
FDA Label

Mechanism of Action

NS5A is a phosphoprotein that plays a crucial role in the replication, assembly, and maturation of infectious viral proteins. The basal phosphorylated form of NS5A is maintained by a C-terminal serine cluster, which is key to ensuring its interaction with the viral capsid protein (i.e., the core protein). Pibrentasvir inhibits protein assembly and the production of mature HCV particles by blocking this interaction. NS5A also interacts with viral and cellular proteins to form the HCV replicase complex and supports the replication of HCV RNA.
Pharmacodynamics

Pibrentasvir is a pangenotypic drug.
Based on HCV replicon assays, Pibrentasvir showed EC50 values ranging from 0.08 to 4.6 nM for laboratory and clinical isolates of subtypes 1a, 1b, 2a, 2b, 3a, 4a, 4d, 5a, and 6a, or from 0.5 to 4.3 pM for laboratory and clinical isolates of subtypes 1a, 1b, 2a, 2b, 3a, 4a, 4b, 4d, 5a, 6a, 6e, and 6p. It is effective against common resistance mutations in HCV genotypes 1 through 6 that lead to resistance to other NS5A inhibitors and reduced treatment response, including mutations at amino acid positions 24, 28, 30, 31, 58, 92, or 93 of NS5A. In a QT interval study, Pibrentasvir did not show a prolongation of the QTc interval. Pibrentasvir is a novel next-generation HCV NS5A inhibitor, the chemical name of which is provided in the “Materials and Methods” section. Its mechanism of action is the inhibition of the HCV NS5A protein, which plays multiple key roles in viral RNA replication and viral particle assembly. This study highlights that Pibrentasvir has a high genetic barrier to resistance, as evidenced by low clonal selection frequency in vitro and low resistance rates in short-term monotherapy clinical trials. In clinical studies, Pibrentasvir has been used in combination with gliclazide (an HCV NS3/4A protease inhibitor). This combination therapy has achieved high sustained virological response rates in treatment-naïve non-cirrhotic HCV genotypes 1–6 and in genotype 1 infected patients who have failed prior direct-acting antiviral therapy. [1]

C57H65F5N10O8

1113.2

1112.49

C, 61.50; H, 5.89; F, 8.53; N, 12.58; O, 11.50

1353900-92-1

1821461-48-6 (butanamine hydrate);1353900-92-1 (free);

58031952

White to off-white solid powder

1.4±0.1 g/cm3

1.614

8.71

4

17

17

80

2000

8

FC1C=C2C(=CC=1[C@H]1CCC(C3=C(C=C4C(=C3)NC([C@@H]3CCCN3C([C@H]([C@@H](C)OC)NC(=O)OC)=O)=N4)F)N1C1C=C(C(=C(C=1)F)N1CCC(C3C=CC(=CC=3)F)CC1)F)NC([C@@H]1CCCN1C([C@H]([C@@H](C)OC)NC(=O)OC)=O)=N2

VJYSBPDEJWLKKJ-NLIMODCCSA-N

InChI=1S/C57H65F5N10O8/c1-29(77-3)49(67-56(75)79-5)54(73)70-19-7-9-47(70)52-63-41-25-35(37(59)27-43(41)65-52)45-15-16-46(72(45)34-23-39(61)51(40(62)24-34)69-21-17-32(18-22-69)31-11-13-33(58)14-12-31)36-26-42-44(28-38(36)60)66-53(64-42)48-10-8-20-71(48)55(74)50(30(2)78-4)68-57(76)80-6/h11-14,23-30,32,45-50H,7-10,15-22H2,1-6H3,(H,63,65)(H,64,66)(H,67,75)(H,68,76)/t29-,30-,45-,46-,47+,48+,49+,50+/m1/s1

methyl N-[(2S,3R)-1-[(2S)-2-[6-[(2R,5R)-1-[3,5-difluoro-4-[4-(4-fluorophenyl)piperidin-1-yl]phenyl]-5-[6-fluoro-2-[(2S)-1-[(2S,3R)-3-methoxy-2-(methoxycarbonylamino)butanoyl]pyrrolidin-2-yl]-3H-benzimidazol-5-yl]pyrrolidin-2-yl]-5-fluoro-1H-benzimidazol-2-yl]pyrrolidin-1-yl]-3-methoxy-1-oxobutan-2-yl]carbamate

ABT-530; ABT 530; ABT530; A-1325912; A1325912; A 1325912; A 1325912.0; A1325912.0; A-1325912.0; Pibrentasvir

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)

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.)