EC50: 34 pM (GT1a), 4 pM (GT1b)[1] IC50: 1.62 Μm (SARS-CoV 3CLpro)[3]

Ledipasvir hydrochloride has intrinsic EC50 values of 39 is 310 fM for GT1a and 40 fM for GT1b. Protein-adjusted EC50 values are 210 pM for GT1a and 27 pM for GT1b. GT1a and 1b EC50 values are 31 and 4 pM, respectively. Both human serum and the cell-culture media used in the replicon experiment, which contains 10% BSA, have significant levels of protein binding for ledipasvir hydrochloride[1]. When used against the JFH/3a-NS5A replicon, ledipasvir hydrochloride has an EC50 value of 141 nM [2].

Ledipasvir hydrochloride 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 hydrochloride's pharmacokinetics are assessed in rats and dogs. Ledipasvir exhibits low systemic clearance (CL), moderate volumes of distribution (Vss) that are more than the entire body water volume, and good half-lives (rat 1.83 hr, dog 2.63 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

Mice: Recombinant adenovirus Ad-WT-HCVpro-SEAP, with 109 IFU per mouse, is injected via the tail vein into five groups of six-week-old SCID mice (six animals per group). Two oral doses of Telaprevir (VX-950) at a dose of 10, 25, 75, 150, or 300 mg/kg are administered to each group of mice. First dose of Telaprevir is administered two hours prior to adenovirus injection; second dose is administered ten hours following injection. A second set of ten mice is given the vehicle on its own. Serum samples are taken 24 hours after injection, and the SEAP activity in each group administered with Telaprevir is contrasted with the vehicle group's. Rat and Canine Rats and dogs are used to assess the oral and intravenous pharmacokinetics of telaprevir (VX-950). One intravenous bolus dose of 0.95 mg/kg Telaprevir is given intravenously to three male Sprague-Dawley rats weighing 250–300 g. Heparinized tubes are used to collect serial blood samples prior to dosage administration and at intervals of 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours following the dose. Telaprevir in 10% ethanol, 40% polyethylene glycol 400, and 50% D5W is given intravenously as a bolus dose to three male beagle dogs (8–12 kg). Heparinized tubes are used to collect serial blood samples prior to dosage administration as well as at 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours later. Telaprevir is formulated in polyvinylpyrrolidone (PVP) K-30 plus 2% sodium lauryl sulfate and dosed as an oral gavage for oral studies in rats and dogs. Oral dosages of 40 mg/kg VX-950 are given to three male Sprague-Dawley rats (250–300 g) and 9.6 mg/kg VX-950 are given to four male Beagle dogs (10.9–12.0 kg). Blood samples are obtained before dosage administration and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours following dose administration in both oral studies. Plasma samples are obtained by centrifugation and kept at -70°C until analysis in both intravenous and oral studies. Samples from the oral studies are analyzed using an achiral LC/MS/MS method, while samples from the intravenous studies are analyzed using a chiral liquid chromatography followed by tandem mass spectrometry (LC/MS/MS) method.

Absorption, Distribution and Excretion
Absorption
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.

Route of Elimination
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.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Ledipasvir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is 99.8% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. If ledipasvir alone or in combination with sofosbuvir (Harvoni) is required by the mother, it is not a reason to discontinue breastfeeding. Some sources recommend against breastfeeding when ledipasvir is used with ribavirin.

Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus (HCV). However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C.

Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended. ◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
Drugs and Lactation Database (LactMed)

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 is a direct acting antiviral (DAA) medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Treatment options for chronic Hepatitis C have advanced significantly since 2011, with the development of Direct Acting Antivirals (DAAs) such as ledipasvir. More specifically, ledipasvir is an inhibitor of the Hepatitis C Virus (HCV) Non-Structural Protein 5A (NS5A), which is required for viral RNA replication and assembly of HCV virions. Although its exact mechanism of action is unknown, it is postulated to prevent hyperphosphorylation of NS5A which is required for viral protein production. It is effective against genotypes 1a, 1b, 4a, and 5a and with a lesser activity against genotypes 2a and 3a of HCV. Ledipasvir and other direct acting antivirals are very potent options for the treatment of Hepatitis C, as they exhibit a high barrier to the development of resistance. This is an important advantage relative to HCV drugs that target other viral enzymes such as the protease, for which rapid development of resistance has proven to be an important cause of therapeutic failure. In a joint recommendation published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) recommend ledipasvir as a first line therapy option in combination with [sofosbuvir] for the treatment of HCV genotypes 1a, 1b, 4, 5, and 6. Treatment with ledipasvir is used with the intent to cure, or achieve a sustained virologic response (SVR), after 12 weeks of daily therapy. SVR and eradication of HCV infection is associated with significant long-term health benefits including reduced liver-related damage, improved quality of life, reduced incidence of Hepatocellular Carcinoma, and reduced all-cause mortality. Treatment with direct acting antivirals such as ledipasvir is associated with very minimal side effects, with the most common being headache and fatigue. Lack of significant side effects and short duration of therapy is a considerable advantage over older interferon- and ribavirin-based regimens, which were limited by infusion site reactions, reduced blood count, and neuropsychiatric effects. Since 2014, ledipasvir has been available as a fixed dose combination product with [sofosbuvir] (tradename Harvoni) used for the treatment of chronic Hepatitis C. Approved in October 2014 by the FDA, Harvoni is indicated for the treatment of HCV genotypes 1, 4, 5, and 6 with or without [ribavirin] depending on the level of liver damage or cirrhosis. When combined together, ledipasvir and sofosbuvir as the combination product Harvoni has been shown to achieve a SVR between 93 and 99% after 12 weeks of treatment. Its use has also proven successful in the treatment of HCV in patients co-infected with HIV.

C49H55CLF2N8O6

925.46

924.3901

2128695-48-5

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

164887328

Typically exists as solid at room temperature

DDPFJRIMZUVTQU-NDANSHMASA-N

InChI=1S/C49H54F2N8O6.ClH/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);1H/t29-,30+,38-,39-,40-,41-;/m0./s1

methyl N-[(2S)-1-[(6S)-6-[5-[9,9-difluoro-7-[2-[(1R,3S,4S)-2-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]-2-azabicyclo[2.2.1]heptan-3-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;hydrochloride

Ledipasvir hydrochloride; Ledipasvir (hydrochloride);

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.)