HCV/hepatitis C virus NS5A; GT1a (EC50 = 34 pM); GT1b (EC50 = 4 pM)[1]

Leadipasvir diacetone is regarded the active ingredient, which is transformed into a spray-dried dispersion of ledipasvir, an amorphous free base.

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

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.

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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[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.

J Med Chem.2014 Mar 13;57(5):2033-46.

See also: Ledipasvir acetone (annotation moved to).

C₅₅H₆₆F₂N₈O₈

1005.16

1004.5

1502655-48-2

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

92044391

Typically exists as solid at room temperature

10.455

4

12

12

73

1850

6

FC1(C2C=C(C=CC=2C2=CC=C(C=C21)C1=CN=C([C@@H]2CC3(CN2C([C@H](C(C)C)NC(=O)OC)=O)CC3)N1)C1C=CC2=C(C=1)NC([C@@H]1[C@H]3CC[C@H](C3)N1C([C@H](C(C)C)NC(=O)OC)=O)=N2)F.O=C(C)C.O=C(C)C

LXKDKHCANHWUPC-YGWQTYEPSA-N

InChI=1S/C49H54F2N8O6.2C3H6O/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;2*1-3(2)4/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);2*1-2H3/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;propan-2-one

GS-5885 diacetone; GS 5885; GS5885; Ledipasvir diacetone; 1502655-48-2; Ledipasvir (diacetone); Z6GY125S9S; UNII-Z6GY125S9S; 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;propan-2-one; GS-5885 diacetone; 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; 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)

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.)