HCV/hepatitis C virus NS5A; GT1a (EC50 = 34 pM); GT1b (EC50 = 4 pM)[1]
The target of Ledipasvir (GS5885) is the NS5A protein of hepatitis C virus (HCV). For HCV genotype 1a, the EC50 was reported to be 0.03 nM; for genotype 1b, the EC50 was 0.09 nM [1]
. Against HCV genotype 3a with wild-type NS5A, the EC50 of Ledipasvir (GS5885) was 0.02 nM, but it showed reduced activity against certain NS5A polymorphisms (e.g., Y93H variant with EC50 > 100 nM) [2]

In Vitro: Ledipasvir (GS5885) exhibited potent antiviral activity against HCV in vitro. In HCV replicon assays, it inhibited the replication of genotype 1a and 1b replicons with EC50 values of 0.03 nM and 0.09 nM, respectively. It also showed activity against genotype 3a wild-type virus with an EC50 of 0.02 nM, but its activity was significantly reduced against genotype 3a variants with NS5A polymorphisms such as Y93H (EC50 > 100 nM), L31M (EC50 1.8 nM), and P58S (EC50 0.13 nM) [1]
[2]
The compound demonstrated a high barrier to resistance in vitro, with resistant variants emerging only after prolonged culture at high concentrations. Additionally, it showed additive or synergistic effects when combined with other HCV inhibitors such as sofosbuvir [1]

---

Ledipasvir's protein-adjusted EC50 values are 210 pM for GT1a and 27 pM for GT1b, and its intrinsic EC50 of 39 is 310 fM for GT1a and 40 fM for GT1b. Its GT1a and 1b EC50 values are 31 and 4 pM, respectively. Ledipasvir exhibits high levels of protein binding in both human serum and the cell-culture medium used in the replicon assay, which contains 10% BSA[1]. Ledipasvir's EC50 value against the JFH/3a-NS5A replicon is 141 nM [2].

In a chimeric mouse model with human hepatocytes infected with HCV genotype 1a, oral administration of Ledipasvir (GS5885) at doses of 0.1, 1, and 10 mg/kg once daily for 14 days resulted in a dose-dependent reduction in HCV RNA levels. At the highest dose (10 mg/kg), HCV RNA was undetectable in some mice. The antiviral effect was sustained during treatment, and no rebound was observed until treatment cessation [1]
Ledipasvir is unique due to its low clearance, good bioavailability, long half-lives in rats, dogs, and monkeys, as well as its low projected clearance in humans, in addition to its high replicon potency. Ledipasvir's pharmacokinetics are assessed in dogs and rats. Ledipasvir exhibits low systemic clearance (CL), moderate volumes of distribution (Vss) that are more than total body water volume, and good half-lives (dog 2.63 ± 0.18 hr, rat 1.83 ± 0.22 hr) in plasma[1].

Competitive Protein Binding Assay[1]
Human plasma and cell-culture medium containing 10% fetal bovine serum (CCM) were spiked with the test compound at a final concentration of 2 μM. Spiked plasma (1 mL) and CCM (1 mL) were placed into opposite sides of the assembled dialysis cells, which are separated by a semipermeable membrane. The dialysis cells were rotated slowly in a 37 °C water bath for the time necessary to reach equilibrium. Postdialysis plasma and CCM weights were measured, and the test compound concentrations in plasma and CCM were determined with LC/MS/MS.

---

Metabolic Stability[1]
Metabolic stability in vitro was determined using pooled hepatic microsomal fractions (final protein concentration of 0.5 mg/mL) at a final test compound concentration of 3 μM. The reaction was initiated by the addition of an NADPH-regenerating system. Aliquot of 25 μL of the reaction mixture were transferred at various time points to plates containing a quenching solution. The test compound concentration in the reaction mixture was determined with LC/MS/MS. Hepatic intrinsic clearance was calculated as described previously by Obach, and the predicted clearance was calculated using the well-stirred liver model without protein restriction.
Metabolic stability was also determined in cryopreserved hepatocytes using tritiated test compounds. The incubation mixture contained 1 × 106 hepatocytes/mL and 1 μM tritiated test compound (2.5 μCi). The incubation was carried out with gentle shaking at 37 °C under a humid atmosphere of 95% air/5% CO2 (v/v). Aliquots of 50 μL were removed after 0, 1, 3, and 6 h and added to 100 μL of quenching solution. The samples were analyzed on a flow scintillation radio detector coupled to an HPLC system. The metabolites were quantified on the basis of the peak areas from the radio detector, with the cell-free control samples used as a reference. Metabolic stabilities in hepatocytes were determined by measuring the rate of disappearance of the test compound as the percent of total peak areas of the formed radiolabeled metabolites and the test compound.

---

To evaluate the binding affinity of Ledipasvir (GS5885) to NS5A, a fluorescence polarization assay was used. Purified NS5A domain I protein was incubated with a fluorescently labeled peptide ligand, and the change in fluorescence polarization upon addition of the compound was measured. The assay allowed determination of the compound's ability to compete with the ligand for binding to NS5A [1]
For assessing antiviral activity, HCV subgenomic replicons (genotypes 1a and 1b) were transfected into Huh-7 cells. Cells were treated with serial dilutions of Ledipasvir (GS5885), and after 72 hours, the level of HCV RNA was measured using quantitative PCR. The EC50 was calculated as the concentration required to reduce HCV RNA levels by 50% compared to untreated controls [1] [2]

GT1a and GT1b Replicons[1]
The stable genotype 1a (GT1a) subgenomic replicon cell line 1a-57C-RlucP (H77 strain) was used to determine compound GT1a antiviral activity and was established as described previously. The compound GT1b antiviral activity was determined in the stable GT1b subgenomic replicon cell line 1b-Rluc-2 (Con-1 strain). To establish 1b-Rluc-2, replicon plasmid pCon1/SG-hRlucNeo (G+I+T) was generated from plasmid I389luc-ubineo/NS3-3′/ET, which encodes a subgenomic replicon of the Con-1 strain and was obtained from ReBLikon. The hRluc-Neo gene was PCR amplified from pF9 CMV hRluc-Neo Flexi by PCR using Accuprime Super Mix I and the primers AscI hRLuc Fwd and NotI hRluc Rev. These two primers have the following sequence and carry restriction sites for subsequent cloning: AscI hRLuc Fwd: 5′-ACT GAC GGC GCG CCA TGG CTT CCA AGG TGT ACG-3′ (AscI site underlined) and NotI hRluc Rev: 5′-GTC AGT GCG GCC GCT CAG AAG AAC TCG TCA AGA-3′ (NotI site underlined). The hRluc-Neo amplification product was subcloned into pCR2.1-TOPO. The resulting plasmid was digested with AscI and NotI, and the excised fragment (hRluc-Neo) was ligated using T4 DNA ligase into I389luc-ubi-neo/NS3-3′/ET digested with the same enzymes. The resulting vector, pCon1/SG-hRlucNeo (G+I+T), was sequenced to confirm the correct orientation and sequence of the hRluc-Neo fusion gene.
Plasmid pCon1/SG-hRlucNeo (G+I+T) was linearized with SpeI and purified using a PCR purification kit. Replicon RNA was in vitro synthesized with T7MEGAScript reagents following the manufacturer’s suggested protocol. RNA was purified by column purification using an RNeasy Kit according to the manufacturer’s instructions. RNA concentrations were determined by measurement of absorbance at 260 nm, and integrity was verified by 0.8% agarose gel electrophoresis and ethidium bromide staining. Ten micrograms of in vitro transcribed pCon1/SG-hRlucNeo (G+I+T) RNA was electroporated into 4 × 106 Huh7-Lunet cells as described previously. Briefly, electroporated cells were plated onto 100 mm cell culture dishes. Twenty-four hours after plating, the media was replaced with propagation media supplemented with 1.0 mg/mL of G418 (selection lasted for approximately 3 weeks). G418-resistant clones were isolated and expanded. HCV replication was quantified using a commercial Renilla luciferase assay per the manufacturer’s instructions. Clones with the highest luciferase signal-to-background ratios were selected for validation in high-throughput antiviral susceptibility assays. The final clonal cell line selected for GT1b antiviral studies was designated 1b-Rluc-2.

---

Replicon Antiviral Assays[1]
To determine compound GT1 antiviral activities, either 1a-57C-RlucP or 1b-Rluc-2 replicon cells were plated at 2000 cells per well in 384-well plates ( cell-culture treated). Compounds were 3-fold serially diluted in DMSO and added to the cells using an automated instrument at a final concentration of 0.44% DMSO in a total volume of 90 μL. For each drug concentration, quadruple wells were set up in the 384-well plate. DMSO was used as a negative (solvent; no inhibition) control, and a combination of three HCV inhibitors, including a protease inhibitor, an NS5A inhibitor, and a nucleoside inhibitor, was used at concentrations >100× EC50 as a positive control (100% inhibition). Plates were incubated for 3 days at 37 °C in an atmosphere of 5% CO2 and 85% humidity. Culture medium was aspirated with a Biotek ELX405 plate washer. Twenty microliters of Dual-Glo luciferase buffer was added to each well of the plate with a Biotek μFlow Workstation. The plate was incubated for 10 min at room temperature. Twenty microliters of a solution containing a 1:100 mixture of Dual-Glo Stop & Glo substrate and Dual-Glo Stop & Glo buffer was added to each well with a Biotek μFlow Workstation. The plate was incubated at room temperature for 10 min before the luminescence signal was measured with an Envision plate reader

---

HCV-infected Huh-7 cells or cells containing HCV replicons were seeded in 96-well plates and treated with various concentrations of Ledipasvir (GS5885). After 72–96 hours of incubation, cell viability was assessed using a colorimetric assay to determine the cytotoxicity (CC50). Concurrently, HCV RNA levels were measured by quantitative PCR to determine the antiviral EC50. The selectivity index (SI) was calculated as the ratio of CC50 to EC50 [1] [2]
For evaluating resistance, HCV replicon cells were cultured in the presence of increasing concentrations of Ledipasvir (GS5885) over several weeks. Emerging resistant variants were sequenced to identify mutations in the NS5A gene, and the EC50 of the compound against these variants was determined [1]

Pharmacokinetic studies are performed in male na ve Sprague-Dawley(SD) rats, non-naive beagle dogs, and cynomolgus monkeys (three animals per dosing route). Intravenous (IV) administration is dosed via infusion over 30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH adjusted to 3.0 with HCl). Oral dosing is administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% of 50 mM citrate buffer, pH 3. Blood samples are collected over a 24 h period postdose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of the test compound in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile.

---

PK studies in Rats, Dogs and Monkeys; Ledipasvir is remarkable not only on the basis of its high replicon potency but also on the basis of its low clearance, good bioavailability, and long half-lives in rat, dog, and monkey and low predicted clearance in human. The pharmacokinetics of Ledipasvir is measured in rats and dogs. Ledipasvir shows good half-lives (rat 1.83 ± 0.22 hr, dog 2.63 ± 0.18 hr) in plasma, low systemic clearance (CL), and moderate volumes of distribution (Vss) that are greater than total body water volume

---

Chimeric mice with human hepatocytes were infected with HCV genotype 1a. Ledipasvir (GS5885) was formulated in a vehicle (consisting of a mixture of surfactants and water) and administered orally via gavage at doses of 0.1, 1, and 10 mg/kg once daily for 14 days. Control mice received the vehicle alone. HCV RNA levels in serum and liver were measured at various time points during and after treatment using quantitative PCR [1]

Absorption, Distribution and Excretion
When given orally, ledipasvir reaches its maximum plasma concentration in about 4 to 4.5 hours with a maximum concentration (Cmax) of 323 ng/mL.
Following a single 90 mg oral dose of [14C]-ledipasvir, mean total recovery of the [14C]-radioactivity in feces and urine was approximately 87%, with most of the radioactive dose recovered from feces (approximately 86%). Unchanged ledipasvir excreted in feces accounted for a mean of 70% of the administered dose and the oxidative metabolite M19 accounted for 2.2% of the dose. These data indicate that biliary excretion of unchanged ledipasvir is a major route of elimination, with renal excretion being a minor pathway (approximately 1%).

---

Metabolism / Metabolites
In vitro, no detectable metabolism of ledipasvir was observed by human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Evidence of slow oxidative metabolism via an unknown mechanism has been observed. Following a single dose of 90 mg [14C]-ledipasvir, systemic exposure was almost exclusively to the parent drug (>98%). Unchanged ledipasvir is the major species present in feces.

---

Biological Half-Life
The median terminal half-life of ledipasvir is 47 hours.

---

ADME/Pharmacokinetics: In preclinical studies, Ledipasvir (GS5885) showed good oral bioavailability in mice, rats, and dogs. In mice, the oral bioavailability was approximately 40%. The compound had a long half-life in animals (e.g., ~24 hours in dogs), supporting once-daily dosing. It was extensively distributed to the liver, with liver-to-plasma concentration ratios >100 in preclinical species. Metabolism studies indicated that it was primarily excreted unchanged in feces, with minimal metabolism [1]

oxicity/Toxicokinetics: In in vitro cytotoxicity assays, Ledipasvir (GS5885) showed low cytotoxicity, with CC50 values >10 μM in Huh-7 cells, resulting in high selectivity indices (SI > 100,000). In preclinical toxicology studies in rats and dogs, no significant adverse effects were observed at doses up to 100 mg/kg/day for 4 weeks. The compound did not show evidence of genotoxicity or hepatotoxicity in these models [1]
Protein Binding
Ledipasvir is >99.8% bound to human plasma proteins.

[1]. Discovery of ledipasvir (GS-5885): a potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J Med Chem. 2014 Mar 13;57(5):2033-46.

[2]. Natural prevalence of NS5A polymorphisms in subjects infected with hepatitis C virus genotype 3 and their effects on the antiviral activity of NS5A inhibitors. J Clin Virol. 2013 May;57(1):13-8.

[3]. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Ledipasvir (GS5885) is a potent, once-daily oral NS5A inhibitor developed for the treatment of chronic hepatitis C virus infection. Its mechanism of action involves binding to the NS5A protein, which is essential for viral replication and assembly, thereby inhibiting HCV replication. It is highly selective for HCV NS5A and does not inhibit host cellular proteins. The compound was designed to have high potency, a high barrier to resistance, and favorable pharmacokinetic properties to support once-daily dosing [1]
Pharmacodynamics
Ledipasvir acts against HCV and is categorized as a direct-acting antiviral agent (DAA). At a dose of 120 mg twice daily (2.67 times the maximum recommended dosage), ledipasvir does not prolong QTc interval to any clinically relevant extent.

C49H54F2N8O6

889.0

888.413

C, 66.20; H, 6.12; F, 4.27; N, 12.60; O, 10.80

1256388-51-8

Ledipasvir (acetone);1441674-54-9;Ledipasvir D-tartrate;1502654-87-6;Ledipasvir-d6;2050041-12-6;Ledipasvir hydrochloride;2128695-48-5;Ledipasvir (diacetone);1502655-48-2; 1256388-51-8 (free); 1499193-68-8 (D-tartrate); 1441674-54-9 (acetone)

67505836

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.677

6.77

4

10

12

65

1820

6

CC(C)[C@@H](C(=O)N1CC2(CC2)C[C@H]1C3=NC=C(N3)C4=CC5=C(C=C4)C6=C(C5(F)F)C=C(C=C6)C7=CC8=C(C=C7)N=C(N8)[C@@H]9[C@H]1CC[C@H](C1)N9C(=O)[C@H](C(C)C)NC(=O)OC)NC(=O)OC

VRTWBAAJJOHBQU-KMWAZVGDSA-N

InChI=1S/C49H54F2N8O6/c1-24(2)39(56-46(62)64-5)44(60)58-23-48(15-16-48)21-38(58)42-52-22-37(55-42)28-9-13-32-31-12-8-26(18-33(31)49(50,51)34(32)19-28)27-10-14-35-36(20-27)54-43(53-35)41-29-7-11-30(17-29)59(41)45(61)40(25(3)4)57-47(63)65-6/h8-10,12-14,18-20,22,24-25,29-30,38-41H,7,11,15-17,21,23H2,1-6H3,(H,52,55)(H,53,54)(H,56,62)(H,57,63)/t29-,30+,38-,39-,40-,41-/m0/s1

Methyl N-[(2S)-1-[(6S)-6-[5-[9,9-Difluoro-7-[2-[(1S,2S,4R)-3-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]-3-azabicyclo[2.2.1]heptan-2-yl]-3H-benzimidazol-5-yl]fluoren-2-yl]-1H-imidazol-2-yl]-5-azaspiro[2.4]heptan-5-yl]-3-methyl-1-oxobutan-2-yl]carbamate

Ledipasvir; GS-5885, GS5885; GS 5885; Ledipasvir acetonate; CHEBI:85089; WHO 9796; trade name: Harvoni;

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: ≥ 2.5 mg/mL (2.81 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (2.81 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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