HCV/hepatitis C virus NS5A

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).

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.

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

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]

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.

A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an 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. 39 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.[1]

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Ledipasvir is a benzimidazole derivative that is used in combination with sofosbuvir (under the trade name Harvoni) for the treatment of chronic hepatitis C genotype 1 infection. It has a role as an antiviral drug and a hepatitis C protease inhibitor. It is a carbamate ester, a L-valine derivative, a bridged compound, a carboxamide, a benzimidazole, a member of fluorenes, an organofluorine compound, a member of imidazoles, a N-acylpyrrolidine and an azaspiro compound.

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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.

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Ledipasvir is a Hepatitis C Virus NS5A Inhibitor. The mechanism of action of ledipasvir is as a P-Glycoprotein Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Ledipasvir is an orally available inhibitor of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) replication complex, with potential activity against HCV. Upon oral administration and after intracellular uptake, ledipasvir binds to and blocks the activity of the NS5A protein. This results in the disruption of the viral RNA replication complex, blockage of HCV RNA production, and inhibition of viral replication. NS5A, a zinc-binding and proline-rich hydrophilic phosphoprotein, 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 associated with the development of hepatocellular carcinoma (HCC).

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Drug Indication
When used in combination with the antiviral medication [sofosbuvir], ledipasvir is indicated for the treatment of treatment of chronic hepatitis C virus (HCV) in adults and pediatric patients 3 years of age and older with the following conditions: - Genotype 1, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis. - Genotype 1 infection with decompensated cirrhosis, in combination with [ribavirin]. - Genotype 1 or 4 infection who are liver transplant recipients without cirrhosis or with compensated cirrhosis, in combination with [ribavirin]. Its use has also proven successful in the treatment of HCV in patients co-infected with HIV.

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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.

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Mechanism of Action
Ledipasvir is an inhibitor of the Hepatitis C Virus (HCV) NS5A protein 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 production.

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.)