HCV NS5A (EC50 = 9 pM-50 pM)

BMS-790052 is among the strongest HCV replication inhibitors found to date. For HCV genotype 1a and 1b replicons, the mean EC50 values of BMS-790052 are 50 and 9 pM, respectively. BMS-790052 is inactive against a panel of 10 RNA and DNA viruses, with an EC50 greater than 10 μM, and exhibits a therapeutic index (CC50/EC50) of at least 10⁵. This validates the HCV specificity of BMS-790052.[1] BMS-790052, with EC50 values ranging from 1 to 15 pM, inhibits both transient and stable HCV genome replication in Huh7 cells expressing the HCV genotype 1b replicons. It has been demonstrated that BMS-790052 (100 pM or 1 nM) modifies the subcellular localization and biochemical fractionation of NS5A.[2] With an EC50 of 7–13 pM, BMS-790052 suppresses hybrid replicons carrying HCV genotype–4 NS5A genes. In the hybrid replicons, residue 30 of NS5A plays a crucial role in BMS-790052-mediated resistance.[3]

Humanized liver chimeric mice, with an estimated 40% chimeric liver, receive intravenous injections of 100 µL of human serum samples positive for HCV. Every one to four weeks following the vaccination, their blood is drawn from an external jugular vein. With a lower measurement range of 3.2 log IU/mL serum, the COBAS TaqMan HCV test measures the HCV RNA levels in 100-fold diluted serum. Once serum HCV RNA levels plateau, mice are given 40 mg/kg of Asunaprevir plus 30 mg/kg of Daclatasvir, 15 mg/kg of Ledipasvir plus 50 mg/kg of GS-558093, or 50 mg/kg of GS-558093 plus 400 mg/kg of Telaprevir orally once a day for four weeks.

The peptide (Ac-Asp-Glu-Asp [EDANS]-Glu-Glu-Abu-[COO] Ala-Ser-Lys [DABCYL]-NH2) contains a fluorescence donor {EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid} near one end of the peptide and an acceptor {DABCYL, 4-[(4-dimethylamino)phenyl]azo)benzoic acid} near the other end. Intermolecular resonance energy transfer between the donor and the acceptor quenches the fluorescence of the peptide, but as the NS3 protease cleaves the peptide, the products are released from resonance energy transfer quenching. The fluorescence of the donor increases over time as more substrate is cleaved by the NS3 protease. The assay reagent consists of 20 μM FRET peptide, 150 mM NaCl, and 5× luciferase cell culture lysis reagent diluted to 1× with dH₂O. In a 96-well plate, HCV-Huh-7 cells are added and left to attach for the entire night (1×10⁴ cells per well). The plate is incubated for 72 hours after BMS-790052 is added to the wells the following day. After that, the plate is cleaned with PBS and prepared for the FRET test by adding 30 μL of the previously mentioned FRET peptide assay reagent to each well. Signals are acquired by reading the plate in the kinetic mode with the Cytofluor 4000 instrument, which is programmed to operate in the automatic mode at 340 nm (excitation)/490 nm (emission) for 20 cycles or less. Once FRET is completed, each well is filled with 40 μL of luciferase substrate, and the amount of luciferase is evaluated.

BMS-790052 is added to 96-well plates that have HCV replicon cells seeded in 200 µL media about 12 hours earlier.After being incubated for 72 hours, the cell plates are examined for cytotoxicity and replication activity. Following the measurement of cytotoxicity using CellTiter-Blue, the media and dye are removed, the plates are inverted, and the liquid that remains is blotted with paper towels. Renilla luciferase is used to measure the HCV genotype 1a cell lines' replication activity. After adding 1× Renilla luciferase lysis buffer (30 µL) to each well, the plates are incubated for 15 minutes with light shaking. The signals are then detected using a Top Count luminometer that is configured for light emission quantification after adding 40 µL of renilla luciferase substrate. The DMSO-only wells calculate 100% activity for each cell line; the average value for compound-containing wells is divided by the average value for DMSO-containing wells to determine the percentage activity for each inhibitor concentration.

At the termination of experiments, all mice were euthanized by CO2 inhalation. To evaluate in vivo efficacy of antiviral agents on HCV, NOD/SCID mice bearing HCV-replicating Huh7 xenografts were used21. Briefly, HCV RNA-transfected cells mixed with Matrigel were injected into the large lobes of the livers of anesthetized immunodeficient NOD/SCID male mice (5 weeks of age, 18–20 g body weight). Four weeks after implantation, compounds dissolved in saline were orally administered to mice using a feeding needle. Serum HCV titer was monitored by RT-qPCR.[Sci Rep. 2018 Aug 20;8(1):12469]

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30 mg/kg; oral

Mice

Absorption, Distribution and Excretion
Studies demonstrated that peak plasma concentrations typically occurred within 2 hours after administration of multiple oral doses ranging from 1 - 100 mg once daily. Steady state is reached after approximately 4 days of once-daily daclatasvir administration. The absolute bioavailability of the tablet formulation is 67%.
Approximately 88% of total dose of daclatasvir is eliminated into bile and feces in which 53% remains as unchanged form, while 6.6% of the total dose is eliminated primarily unchanged in the urine.
The approximate volume of distribution of daclatasvir is 47 L in patients who was orally administered 60 mg tablet followed by 100 µg [13C,15N]-daclatasvir intravenously.
In subjects who received daclatasvir 60 mg tablet orally followed by 100 µg radiolabeled daclatasvir intravenously, the total clearance was 4.2 L/h.

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Metabolism / Metabolites
Daclastavir is a substrate of CYP3A enzymes where its metabolism is predominantly mediated by CYP3A4 isoform. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. High proportion of the drug in the plasma (greater than 97%) is in the unchanged form.

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Biological Half-Life
Following multiple dose administration of daclatasvir in HCV-infected subjects, with doses ranging from 1 mg to 100 mg once daily, the terminal elimination half-life of daclatasvir ranged from approximately 12 to 15 hours.

Hepatotoxicity
In large randomized controlled trials, daclatasvir was not associated with serum enzyme elevations during therapy. A difficulty in assessing side effects of daclatasvir and other anti-HCV agents, however, was that they are never used as monotherapy, but were also combined with agents active against other HCV targets, such as the viral protease (NS3) or polymerase (NS5B). Daclatasvir was also commonly used in combination with the more traditional agents used for hepatitis C, such as peginterferon and ribavirin, both of which have prominent adverse effects. In combination with asunaprevir (an HCV protease inhibitor), daclatasvir was associated with serum ALT elevations in 3% to 11% of patients and with several instances of acute hypersensitivity and hepatitis, some of which were severe. The cause of the hypersensitivity reaction, however, appeared to be asunaprevir. In combination with sofosbuvir, daclatasvir was not associated with serum enzyme elevations or with clinically apparent liver injury.
Daclatasvir has, however, been implicated in rare instances of acute decompensation of HCV related cirrhosis. The role of daclatasvir and the other HCV antivirals in this syndrome, however, was unclear. The liver injury usually arose within 2 to 6 weeks of starting therapy, but occasionally later and even after discontinuation of therapy. The injury was marked by worsening jaundice and signs of hepatic failure. In some instances, lactic acidosis was present early. In most but not all instances, the serum enzymes increased minimally if at all, despite the worsening hepatic failure. Several instances resulted in death or need for emergency liver transplantation. For this reason, it was recommended that patients with cirrhosis undergoing antiviral therapy with potent direct acting agents should be monitored carefully, particularly during the first few weeks of treatment.
Finally, reactivation of hepatitis B has occurred in rare patients being treated for chronic hepatitis C some of whom had received daclatasvir. The relationship of HBV reactivation to the antiviral treatment of HCV infection is not clear, but it may be due to clearance of HCV replication which allows HBV DNA levels to increase.
Likelihood score: C (probable rare cause of clinically apparent liver injury in patients with cirrhosis or preexisting hepatitis B virus coinfection).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Daclatasvir has been removed from the US market. It has not been studied in nursing mothers being treated for hepatitis C infection. Because it is 99% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. If daclatasvir used alone or in combination with sofosbuvir is required by the mother, it is not a reason to discontinue breastfeeding. Some sources recommend against breastfeeding when daclatasvir 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.

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Protein Binding
Daclatasvir is highly protein bound (99%).

[1]. Nature . 2010 May 6;465(7294):96-100.

[2]. Virology . 2011 May 25;414(1):10-8.

[3]. Antimicrob Agents Chemother . 2012 Mar;56(3):1588-90.

[4]. Antimicrob Agents Chemother . 2005 Apr;49(4):1346-53.

Pharmacodynamics
Daclatasvir is a direct-acting antiviral agent that targets the NS5A and causes a decrease in serum HCV RNA levels. It disrupts HCV replication by specifically inhibiting the critical functions of an NS5A protein in the replication complex. It is shown to cause downregulation of the hyperphosphorylation of NS5A. It does not appear to prolong the QT interval even when given at 3 times the maximum recommended dose.

C40H50N8O6

738.890

738.385

C, 65.02; H, 6.82; N, 15.17; O, 12.99

1009119-64-5

Daclatasvir dihydrochloride;1009119-65-6;Daclatasvir-d6;1801709-41-0;Daclatasvir-d16

25154714

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1071.2±65.0 °C at 760 mmHg

601.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.595

5.44

4

8

13

54

1190

4

O=C([C@@H](NC(OC)=O)C(C)C)N1CCC[C@H]1C2=NC=C(N2)C3=CC=C(C=C3)C4=CC=C(C5=CN=C([C@@H]6CCCN6C([C@@H](NC(OC)=O)C(C)C)=O)N5)C=C4

FKRSSPOQAMALKA-CUPIEXAXSA-N

InChI=1S/C40H50N8O6/c1-23(2)33(45-39(51)53-5)37(49)47-19-7-9-31(47)35-41-21-29(43-35)27-15-11-25(12-16-27)26-13-17-28(18-14-26)30-22-42-36(44-30)32-10-8-20-48(32)38(50)34(24(3)4)46-40(52)54-6/h11-18,21-24,31-34H,7-10,19-20H2,1-6H3,(H,41,43)(H,42,44)(H,45,51)(H,46,52)/t31-,32-,33-,34-/m0/s1

methyl N-[(2S)-1-[(2S)-2-[5-[4-[4-[2-[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl]phenyl]phenyl]-1H-imidazol-2-yl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate dihydrochloride

BMS-790052; Daclatasvir; BMS790052; BMS 790052; EBP883; EBP 883; EBP-883; BMS 790052; Daclatasvir [USAN];Daklinza (trade name)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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