HCV/hepatitis C virus NS5A; GT1a (EC50 = 34 pM); GT1b (EC50 = 4 pM)[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].

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.

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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.

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.

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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

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%).

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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.

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

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.

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.

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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.

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