HCV/hepatitis C virus NS5A
The target of Ledipasvir (GS-5885) is the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV). For HCV genotype 1a (GT1a) replicon, the half-maximal effective concentration (EC₅₀) is 31 pM [1]
; For JFH1/3a-NS5A hybrid replicon (constructed from HCV genotype 3a, GT3a), the EC₅₀ is 141 nM; against JFH1/3a-NS5A-A30K variant replicon, the EC₅₀ is 1770 nM; against JFH1/3a-NS5A-Y93H variant replicon, the EC₅₀ is 4300 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]

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Ledipasvir (also known as GS5885) is a HCV NS5A polymerase inhibitor that is used for the treatment of hepatitis C virus infection. The combination product of ledipasvir 90 mg/sofosbuvir 400 mg (trade name Harvoni) was approved by FDA in October 2014. The ledipasvir/sofosbuvir combination is a direct-acting antiviral agent that interferes with HCV replication and can be used to treat patients with genotypes 1a or 1b without PEG-interferon or ribavirin.Ledipasvir hasan extended plasma half-life of 37-45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. It has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.Kinase Assay:GT1a replicon EC50 = 31 pMCell Assay:Ledipasvir is a specific inhibitor of HCV NS5A protein to inhibit HCV replication in the HCV subgenomic replicon system. NS5A replication complex inhibitors are novel antiviral factors for HCV treatment. Typically, these inhibitors have high efficiency and low viral resistance when compared to traditional HCV replication inhibitor targeted on NS3 helicase and NS5B RNA polymerasae. NS5A inhibitors are supposed to bind across the NS5A dimer interface, proximal to N-terminal domain 1. The binding is thought to distort dimer association directly or allosterically, which may disrupt NS5A function in HCV RNA replication. When a JFH1/3a-NS5A hybrid replicon was used to assess susceptibility to NS5A, another inhibitor DCV was shown to be more potent than ledipasvir. Additionally, NS5A-A30K and -Y93H variants exhibited reduced sensitivity to ledpasvir (EC50 value of 1770 nM and 4300 nM respectively).

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1. Against HCV GT1a replicon, Ledipasvir (GS-5885) exhibits potent antiviral activity with an EC₅₀ of 31 pM [1]
2. When evaluated using JFH1/3a-NS5A hybrid replicon (derived from HCV GT3a), Ledipasvir shows an EC₅₀ of 141 nM. For JFH1/3a-NS5A variants, its antiviral activity varies: EC₅₀ is 1770 nM against JFH1/3a-NS5A-A30K variant and 4300 nM against JFH1/3a-NS5A-Y93H variant [2]
3. Phylogenetic analysis of the NS5A region from 96 HCV GT3 treatment-naive patients (across North America, Europe, and Australia) reveals no significant geographic clustering. Naturally occurring NS5A polymorphisms (e.g., 28M/V, 30A/K/S/T/V, 31L/M, E92A, Y93H) are detected in these patients, with frequencies of 1%, 10%, 1%, 1%, and 8.3% respectively [2]

In Vivo: 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]
In clinical trials, it was observed ledpasvir was well tolerated and exhibited median maximal reduction of HCV RNA ranging from 2.3 log10 IU/ml to 3.3 log10 IU/ml. Emax modeling also showed administration of 30 mg ledpasvir after 3 days resulted in >95% maximal response of HCV RNA reduction to genotype 1a.Finally, it was also observed that HCV RNA was more sustained in genotype 1b compared to 1a.

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1. In healthy volunteers, Ledipasvir (GS-5885) has an extended plasma half-life of 37–45 hours [1]
2. In HCV GT1a-infected patients, oral administration of Ledipasvir at doses of 3 mg or higher (once daily) as monotherapy results in a rapid >3 log reduction in viral load [1]
3. When Ledipasvir is combined with direct-acting antivirals (DAAs) with complementary mechanisms, the sustained virologic response at 12 weeks (SVR12) rates can reach up to 100% in HCV-infected patients [1]

Competitive Protein Binding Assay[1]
\nHuman 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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\nMetabolic Stability[1]
\nMetabolic 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.
\nMetabolic 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.

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\nEnzyme Assay: 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]
\n \nFor 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]

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\n1. For evaluating the antiviral activity of Ledipasvir (GS-5885) against HCV GT1a replicon: A cell-based assay system containing HCV GT1a replicon is established. Different concentrations of Ledipasvir are added to the culture system, and after a specific incubation period, the level of HCV RNA in the cells is detected (e.g., via quantitative reverse transcription-polymerase chain reaction, qRT-PCR). The EC₅₀ value is calculated based on the reduction of HCV RNA to determine the antiviral potency [1]
\n;
\n2. For assessing the activity against JFH1/3a-NS5A hybrid replicon: First, a JFH1/3a-NS5A hybrid replicon is constructed using a consensus NS5A sequence from HCV GT3 patients. The replicon is then introduced into host cells to establish a stable cell line. Ledipasvir at various concentrations is added to the cell culture, and after incubation, the viral replication level is measured (e.g., via qRT-PCR for HCV RNA). The EC₅₀ is calculated to evaluate the inhibitory effect on the hybrid replicon and its variants [2]

Cell Assay: 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]
\n \\nFor 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]

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\\n\\n\\nGT1a and GT1b Replicons[1]
\n\\nThe 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.
\n\\n\\nPlasmid 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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\\n\\nReplicon Antiviral Assays[1]
\n\\nTo 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

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\n1. HCV GT1a replicon cell assay for Ledipasvir (GS-5885): Host cells stably harboring HCV GT1a replicon are cultured in appropriate medium. The cells are seeded into culture plates and allowed to adhere. Different concentrations of Ledipasvir are added to the wells, and the cells are incubated under standard conditions (e.g., 37°C, 5% CO₂) for a set time. After incubation, the cells are harvested, and total RNA is extracted. The amount of HCV RNA is quantified using qRT-PCR, and the EC₅₀ is determined by analyzing the dose-response relationship between Ledipasvir concentration and HCV RNA reduction [1]
\n;
\n2. JFH1/3a-NS5A hybrid replicon cell assay: Host cells are transfected with the JFH1/3a-NS5A hybrid replicon to establish a stable replication system. The transfected cells are plated and treated with different concentrations of Ledipasvir. Following incubation, the cell supernatant or cell lysate is collected, and HCV RNA levels are measured via qRT-PCR. For variant replicons (e.g., A30K, Y93H), the same procedure is repeated, and EC₅₀ values are calculated to compare the antiviral activity of Ledipasvir against wild-type and mutant replicons [2]

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; Pharmacokinetic studies are performed in male naı̈ve 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. [1]

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Animal Protocol: 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]

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1. Pharmacokinetic study in healthy non-human primates (Macaca fascicularis): Ledipasvir (GS-5885) is formulated into an oral dosage form (e.g., suspension or tablet) using appropriate excipients. The animals are fasted for a specified period before administration. Ledipasvir is administered orally at a predetermined dose, and blood samples are collected at multiple time points (e.g., 0.5, 1, 2, 4, 8, 12, 24, 48 hours) after dosing. Plasma is separated from the blood samples, and the concentration of Ledipasvir in plasma is determined using a sensitive analytical method (e.g., liquid chromatography-tandem mass spectrometry, LC-MS/MS). Pharmacokinetic parameters such as half-life, maximum plasma concentration (Cₘₐₓ), and time to reach Cₘₐₓ (Tₘₐₓ) are calculated [1]
2. Pharmacokinetic study in Sprague-Dawley rats: Ledipasvir is prepared into an injectable or oral formulation. Rats are randomly divided into groups, and Ledipasvir is administered via the intended route (e.g., oral gavage or intravenous injection). Blood samples are collected at various time intervals post-administration, and plasma drug concentrations are measured using LC-MS/MS. Pharmacokinetic parameters are analyzed to evaluate the absorption, distribution, and elimination of Ledipasvir in rats [1]

Absorption, Distribution, and Excretion
Absorption
After oral administration, ledipasvir reaches peak plasma concentrations in approximately 4 to 4.5 hours, with a maximum concentration (Cmax) of 323 ng/mL.
Excretion Routes
Following a single oral dose of 90 mg [14C]-ledipasvir, the average total recovery of the [14C]-radioactive substance in feces and urine is approximately 87%, with the majority of the radioactive dose recovered in feces (approximately 86%). On average, 70% of the administered dose of unchanged ledipasvir is excreted in feces, and 2.2% is excreted as the oxidative metabolite M19. These data suggest that bile excretion is the primary route of excretion for unchanged ledipasvir, while renal excretion is a secondary route (approximately 1%). Metabolites/Metabolites
In vitro studies did not detect metabolism of ledipasvir by human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Evidence of slow oxidative metabolism via an unknown mechanism has been observed. Following a single 90 mg [14C]-ledipasvir dose, systemic exposure is almost entirely unchanged (>98%). Unmetabolized ledipasvir is predominantly present in feces.
Biological Half-Life
The median terminal half-life of ledipasvir is 47 hours.

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1. Plasma half-life: In healthy volunteers, the plasma half-life of leadipasvir (GS-5885) is prolonged to 37–45 hours, supporting once-daily administration [1]
2. Oral absorption: Oral administration of leadipasvir in patients with HCV GT1a infection, at a dose of 3 mg or higher once daily, produces effective antiviral activity (rapid reduction in viral load >3 log), indicating good oral absorption [1]
;

Effects during pregnancy and lactation
◉ Summary of medication use during lactation
Studies of ledipasvir in breastfeeding women receiving treatment for hepatitis C virus infection have not been conducted. Because it binds to maternal plasma proteins at a rate as high as 99.8%, its concentration in breast milk may be very low. Breastfeeding does not need to be discontinued if the mother is using ledipasvir alone or in combination with sofosbuvir (Harvoni). Some sources suggest that breastfeeding should be avoided when ledipasvir is used in combination with ribavirin.
Hepatitis C virus is not transmitted through breast milk, and breast milk has been shown to inactivate hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that breastfeeding should be considered if an HCV-infected mother experiences nipple fissures or bleeding. It is currently unclear whether this warning applies to mothers receiving treatment for hepatitis C. Infants born to mothers infected with hepatitis C virus (HCV) should be tested for HCV; nucleic acid testing is recommended because maternal antibodies are present in the first 18 months after birth and before the infant develops an immune response. ◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on breastfeeding and breast milk
No relevant published information was found as of the revision date.
Drug and lactation database (LactMed)
Protein binding rate
Leadipasvir binds to human plasma proteins >99.8%.

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1. Safety profile: Clinical evaluations have shown that leadipasvir (GS-5885) is safe. No serious toxicities have been reported in patients with HCV GT1a infection, either alone (orally once daily at a dose ≥3 mg) or in combination with other DAAs [1];

[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.

We have discovered a new class of highly effective NS5A inhibitors with an asymmetric benzimidazole-difluorofluorene-imidazolium ring system at the core and a [2.2.1] azabicyclic ring system at the distal end. By optimizing antiviral activity and pharmacokinetics, we screened compound 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has a plasma half-life of 37-45 hours in healthy volunteers and rapidly reduces viral load by more than 3 logs in patients with genotype 1a HCV infection with a single oral dose of 3 mg or higher once daily. Studies have shown that ledipasvir is safe and effective, with an SVR12 rate of up to 100% when used in combination with direct-acting antiviral drugs with complementary mechanisms of action. [1]

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Ledipasvir is a benzimidazole derivative used in combination with sofosbuvir (trade name: Harvoni) to treat chronic hepatitis C genotype 1 infection. It is both an antiviral drug and a hepatitis C protease inhibitor. It is a carbamate, L-valine derivative, bridging compound, carboxamide, benzimidazole, fluorene compound, organofluorine compound, imidazole compound, N-acylpyrrolidine, and azaspirocyclic compound. Leadipasvir is a direct-acting antiviral agent (DAA) used as part of combination therapy to treat chronic hepatitis C, an infectious liver disease caused by infection with the hepatitis C virus (HCV). Hepatitis C virus (HCV) is a single-stranded RNA virus with nine different genotypes, of which genotype 1 is the most common in the United States, accounting for 72% of all chronic hepatitis C cases. Since 2011, significant progress has been made in the treatment of chronic hepatitis C with the development of direct-acting antiviral agents (DAAs) such as leadipasvir. More specifically, leadipasvir is an inhibitor of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A), which is essential for viral RNA replication and HCV viral particle assembly. Although its exact mechanism of action is not fully understood, it is presumed to prevent the hyperphosphorylation of NS5A, which is essential for viral protein synthesis. Leadipasvir is effective against genotypes 1a, 1b, 4a, and 5a, but less active against genotypes 2a and 3a. Leadipasvir, along with other direct-acting antiviral agents, is an effective treatment option for hepatitis C due to its high resistance barrier. This is a significant advantage compared to hepatitis C drugs that target other viral enzymes, such as proteases, where rapid development of resistance has been a major cause of treatment failure. In a joint guideline published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) recommended leadipasvir in combination with sofosbuvir as a first-line treatment for hepatitis C virus genotypes 1a, 1b, 4, 5, and 6. The goal of leadipasvir treatment is cure or sustained virological response (SVR), which is typically achieved after 12 weeks of daily treatment. Sustained virological response (SVR) and eradication of hepatitis C virus (HCV) infection are 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. Side effects from treatment with direct-acting antiviral agents (such as ledipasvir) are minimal, with headache and fatigue being the most common. Significant advantages of ledipasvir treatment compared to previous interferon- and ribavirin-based regimens are fewer side effects and shorter treatment durations, the latter being limited by infusion site reactions, decreased blood cell counts, and neuropsychiatric side effects. Since 2014, ledipasvir has been marketed in combination with sofosbuvir (brand name Harvoni) as a fixed-dose combination for the treatment of chronic hepatitis C. Harvoni was approved by the U.S. Food and Drug Administration (FDA) in October 2014 for the treatment of HCV genotypes 1, 4, 5, and 6 infection, with or without ribavirin, depending on the degree of liver damage or cirrhosis. When used in combination with sofosbuvir, the combination therapy Harvoni achieved a sustained virological response rate (SVR) of 93% to 99% after 12 weeks of treatment. This formulation has also shown success in treating hepatitis C virus (HCV) infection in patients with concurrent HIV infection. Leadipasvir is a hepatitis C virus NS5A inhibitor. Its mechanism of action is as a P-glycoprotein inhibitor and a breast cancer resistance protein inhibitor. Leadipasvir is an oral inhibitor of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) replication complex and has potential anti-HCV activity. After oral administration, leadipasvir is absorbed by cells and binds to the NS5A protein, blocking its activity. This leads to the disruption of the viral RNA replication complex, blocking HCV RNA production and thus inhibiting viral replication. NS5A is a proline-rich hydrophilic phosphoprotein that binds zinc and plays a crucial role in HCV RNA replication. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family; HCV infection is closely associated with the development of hepatocellular carcinoma (HCC).

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Drug Indications
When used in combination with the antiviral drug sofosbuvir, ledipasvir is indicated for the treatment of chronic hepatitis C virus (HCV) infection in adults aged 3 years and older and children meeting the following criteria: - Genotype 1, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis. - Genotype 1 infection with decompensated cirrhosis, requiring combination with ribavirin. - Genotype 1 or 4 infection in liver transplant recipients without cirrhosis or with compensated cirrhosis, requiring combination with ribavirin. Leadipasvir has also been successful in treating hepatitis C virus (HCV) patients with co-infection with HIV.

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Pharmacodynamics
Leadipasvir is an antiviral agent against HCV and belongs to the direct-acting antiviral (DAA) class. Twice daily at 120 mg (equivalent to 2.67 times the maximum recommended dose), ledipasvir does not prolong the QTc interval to any clinically significant extent.

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Mechanism of Action
Leadipasvir is an inhibitor of the hepatitis C virus (HCV) NS5A protein, which is essential for viral RNA replication and HCV viral particle assembly. While its exact mechanism of action is not fully understood, it is presumed to prevent the hyperphosphorylation of NS5A, which is necessary for viral production.

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Leadipasvir (GS5885) is a potent, once-daily oral NS5A inhibitor used to treat 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 cell proteins. The design goal of this compound is to have high activity, high resistance barrier and good pharmacokinetic properties to support once-daily dosing [1]

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1. Leadipasvir (GS-5885) belongs to a new class of NS5A inhibitors with an asymmetric benzimidazole-difluorofluorene-imidazole core structure and a distal [2.2.1] azabicyclic system. Its discovery is based on the optimization of antiviral activity and pharmacokinetic properties [1]
;
2. Before the era of direct-acting antiviral drugs (DAAs), the standard treatment for hepatitis C virus (HCV) infection included ribavirin (RBV) and interferon (IFN), but its sustained virological response rate (SVR) was low (e.g., the SVR of genotype 1 patients was about 30-50%) and its toxicity was high (flu-like symptoms, depression, anemia). As a DAA, ledipasvir revolutionized the treatment of HCV[1];
3. The above literature mainly focuses on ledipasvir (GS-5885) and does not mention "ledipasvir acetone" (there is currently no data on the acetone form of ledipasvir)[1,2];
4. In untreated HCV GT3 patients, naturally occurring NS5A polymorphisms (e.g., Y93H, A30K) can reduce the antiviral activity of ledipasvir, which may affect the clinical efficacy of ledipasvir in HCV GT3 infected patients[2].

C52H60F2N8O7

947.08

946.455

C, 65.95; H, 6.39; F, 4.01; N, 11.83; O, 11.82

1441674-54-9

Ledipasvir;1256388-51-8;Ledipasvir D-tartrate;1502654-87-6;Ledipasvir-d6;2050041-12-6;Ledipasvir hydrochloride;2128695-48-5;Ledipasvir (diacetone);1502655-48-2

78357793

Off-white to yellow solid powder

9.86

4

11

12

69

1850

6

O=C(OC)N[C@H](C(N([C@H](C1=NC=C(C2=CC(C(F)(F)C3=C4C=CC(C5=CC=C6N=C([C@H]7N(C([C@@H](NC(OC)=O)C(C)C)=O)[C@]8([H])CC[C@@]7([H])C8)NC6=C5)=C3)=C4C=C2)N1)C9)CC%109CC%10)=O)C(C)C.CC(C)=O

IEYHPNJBXNWRRN-ABBTUPPKSA-N

InChI=1S/C49H54F2N8O6.C3H6O/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;1-3(2)4/h8-10,12-14,18-20,22,24-25,29-30,38-41,54-55H,7,11,15-17,21,23H2,1-6H3,(H,56,62)(H,57,63);1-2H3/b27-26+,37-28+;/t29-,30+,38-,39-,40-,41-;/m0./s1

methyl ((S)-1-((S)-6-(5-(9,9-difluoro-7-(2-((1R,3S,4S)-2-((methoxycarbonyl)-L-valyl)-2-azabicyclo[2.2.1]heptan-3-yl)-1H-benzo[d]imidazol-6-yl)-9H-fluoren-2-yl)-1H-imidazol-2-yl)-5-azaspiro[2.4]heptan-5-yl)-3-methyl-1-oxobutan-2-yl)carbamate compound with propan-2-one (1:1)

GS-5885 acetone; Ledipasvir acetone; Ledipasvir (acetone); GS-5885 acetone; 3J78ET35HX; UNII-3J78ET35HX; Ledipasvir acetonate (JAN); Carbamic acid, N-((1S)-1-(((6S)-6-(5-(9,9-difluoro-7-(2-((1R,3S,4S)-2-((2S)-2-((methoxycarbonyl)amino)-3-methyl-1-oxobutyl)-2-azabicyclo(2.2.1)hept-3-yl)-1H-benzimidazol-6-yl)-9H-fluoren-2-yl)-1H-imidazol-2-yl)-5-azaspiro(2.4)hept-5-yl)carbonyl)-2-me; GS5885 acetone; GS 5885; trade name: Harvoni;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~105.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)