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| Targets |
Dengue Virus RNA-dependent RNA Polymerase: R1479 is a nucleoside analogue that inhibits DENV replication. The active form, R1479 triphosphate, inhibits the viral polymerase. No direct IC50/Ki values for enzyme inhibition are provided. [3]
Hepatitis C Virus NS5B Polymerase: R1479 was originally developed as an inhibitor of HCV replication. [3] |
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| ln Vitro |
After incubating proliferating replicon cells for 72 hours, R-1479 (R1479) suppresses HCV RNA replication, exhibiting a dose-dependent decrease in Renilla luciferase activity with an average IC50 value of 1.28 μM. At concentrations up to 2 mM, R-1479 has no influence on the viability or proliferation of Huh-7 or HCV replicons [1]. In the HCV replicon system, R-1479 has an IC50 of 1.28 μM, making it the most powerful and non-cytotoxic derivative. An inhibitor of NS5B (HCV-encoded RNA polymerase)-mediated RNA synthesis was demonstrated by the preparation of the triphosphate salt of R-1479 (IC50=320 nM). In replicon experiments, R-1479 exhibited strong activity and no detectable cytotoxic or cytostatic effects [2].
DENV Replication Inhibition in Huh-7 Cells: R1479 inhibited replication of DENV reference strains and clinical isolates in Huh-7 cells with mean EC50 values ranging from 1.9 to 11 μM. For comparison, the known DENV polymerase inhibitor NITD008 had mean IC50 values of 0.1-0.6 μM. [3] DENV Replication Inhibition in Primary Human Cells: R1479 was active against DENV-1, DENV-2, and DENV-4 in primary human macrophages (mean EC50 range: 1.3-3.2 μM) and dendritic cells (mean EC50 range: 5.2-6.0 μM). DENV-3 was not tested in these cell types. [3] These data suggest that R1479 has anti-DENV activity in vitro at concentrations pharmacologically attainable in humans receiving ≥1500 mg balapiravir twice daily. [3] |
| ln Vivo |
This document reports a randomized, double-blind, placebo-controlled trial of balapiravir (the prodrug of R1479) in 64 adult male dengue patients. Key in vivo findings include:
Pharmacokinetics: Patients receiving 1500 mg or 3000 mg balapiravir twice daily showed dose-dependent plasma concentrations of R1479. On day 1, Cmax values: 1500 mg group (median 4.5 μM, range 2.7-19.8 μM); 3000 mg group (median 23.8 μM, range 8.7-90.6 μM). Cmin values: 1500 mg group (median 3.5 μM, range 2.7-4.9 μM); 3000 mg group (median 5.8 μM, range 2.1-15.7 μM). [3] Virological Efficacy: Despite achieving plasma concentrations exceeding in vitro EC50 values in most patients, balapiravir treatment did not significantly affect any predefined virological endpoints: area under the viremia curve, time to first viremia level <1000 copies/mL, or time to first negative NS1 test result. Viremia declined similarly in all treatment groups within 24 hours of enrollment regardless of treatment assignment. [3] Clinical Efficacy: Balapiravir treatment did not affect fever clearance time, hematocrit levels, platelet count nadir, liver transaminases, coagulation markers, or quality of life scores. [3] Immunological Effects: Treatment did not alter plasma cytokine concentrations (including IFN-γ and IL-10) or whole blood transcriptional profiles. [3] Viral Genetics: Mutation rates in virus genome sequences were not significantly different between balapiravir and placebo arms. [3] |
| Enzyme Assay |
Native HCV Replicase Assay: Membrane-associated native HCV replicase complexes were isolated from HCV replicon cells. R1479-TP inhibited RNA synthesis by wild-type replicase with IC50 = 0.79 ± 0.12 μM. For replicase containing the S282T mutation, IC50 = 0.71 ± 0.15 μM, demonstrating no cross-resistance. [2]
Recombinant HCV Polymerase (NS5B) Assay: R1479-TP inhibited NS5B-mediated RNA synthesis in a template-dependent manner. IC50 values varied by template: poly(I) RNA (directs poly(C) synthesis) IC50 = 0.02 ± 0.002 μM; HCV cIRES RNA IC50 = 0.29 ± 0.13 μM (UTP label) and 0.21 ± 0.12 μM (ATP label); HCV 3'-UTR RNA IC50 = 0.67 ± 0.21 μM (UTP label) and 1.43 ± 0.37 μM (ATP label); poly(A) RNA (directs poly(U) synthesis) IC50 = 173 ± 25 μM. [2] Kinetic Analysis: R1479-TP was identified as a competitive inhibitor of CMP incorporation. Increasing CTP concentrations reduced the potency of R1479-TP (shifted IC50 curves), while ATP concentrations had no effect. The Ki for R1479-TP was 40 ± 25 nM, based on Km(app) for CTP of 81.4 ± 10.5 nM. [2] Nucleotide Incorporation Assay (Gel-based): Using a 19-nucleotide RNA template and labeled GG primer, R1479-TP was incorporated into nascent RNA by HCV polymerase. After incorporation, further elongation with UTP was blocked, similar to the obligatory chain terminator 3'-dCTP. [2] Comparison with Other Inhibitors: R1479-TP inhibited NS5B with similar potency to 3'-dCTP. Ribavirin triphosphate was much weaker (IC50 = 255 ± 59 μM). [2] |
| Cell Assay |
Huh-7 Cell Assay: Human hepatoma Huh-7 cells were infected with DENV reference strains and clinical isolates. Cells were treated with R1479, and viral replication was measured. EC50 values were calculated as the concentration inhibiting 50% of viral replication. [3]
Primary Human Cell Assays: Primary human macrophages and dendritic cells were infected with DENV-1, DENV-2, and DENV-4, treated with R1479, and viral replication measured to determine EC50 values. [3] |
| ADME/Pharmacokinetics |
Prodrug Activation: Balapiravir is a prodrug of R1479. R1479 requires intracellular phosphorylation to the active triphosphate form (R1479 triphosphate) to inhibit the DENV polymerase. [3]
Human Pharmacokinetics (Day 1): For 1500 mg balapiravir twice daily: Tmax median 4 h (range 2-8 h); Cmax median 4.5 μM (range 2.7-19.8 μM); Cmin median 3.5 μM (range 2.7-4.9 μM); AUClast median 10.6 h×μM (range 2.6-30.7 h×μM). For 3000 mg balapiravir twice daily: Tmax median 4 h (range 2-12 h); Cmax median 23.8 μM (range 8.7-90.6 μM); Cmin median 5.8 μM (range 2.1-15.7 μM); AUClast median 67.0 h×μM (range 27.1-608.1 h×μM). Similar dose-dependent pharmacokinetics were observed on day 5. [3] Target Concentration Achievement: In the first 12 hours of treatment, 95% of patients receiving 3000 mg balapiravir had plasma Cmax values of R1479 >6 μM, a concentration inhibitory to DENV in vitro. [3] Pharmacokinetic-Pharmacodynamic Relationship: Based on HCV studies, mean Cmin concentrations exceeding 6.7-39 μM or Cmax exceeding 25-145 μM may be needed for observable anti-DENV effects. The doses tested may have been insufficient to achieve these thresholds. [3] |
| Toxicity/Toxicokinetics |
Clinical Safety Profile: Balapiravir was well tolerated at both 1500 mg and 3000 mg twice daily for 5 days. The range of clinical and laboratory adverse events was typical for dengue and similar between balapiravir and placebo arms. [3]
Serious Adverse Events: Four serious adverse events were reported (2 in placebo, 1 in 1500 mg, 1 in 3000 mg arm), all typical of dengue (prolonged thrombocytopenia, transient visual acuity loss, narrowed pulse pressure). None were considered dose-related to balapiravir. [3] Development Discontinuation: Clinical development of balapiravir for HCV was stopped when safety signals emerged in patients receiving extended courses (2-3 months) in combination with pegylated interferon and ribavirin. [3] |
| References |
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| Additional Infomation |
4'-Azidocytidine is an N-glycoside compound.
Background: R1479 (4'-azidocytidine) is a nucleoside analogue originally developed as an inhibitor of hepatitis C virus replication. It is the active moiety released from the prodrug balapiravir. [3] Mechanism of Action: R1479 is intracellularly phosphorylated to R1479 triphosphate, which inhibits the RNA-dependent RNA polymerase of flaviviruses including DENV and HCV. [3] Clinical Trial Context: This was the first-ever randomized controlled trial of an antiviral drug in dengue. The study was stopped early after 64 patients due to the end of dengue transmission season and a sponsor-investigator review concluding insufficient potency to warrant further investigation. [3] Negative Trial: Despite promising in vitro activity and achievement of target plasma concentrations, balapiravir showed no measurable antiviral or clinical efficacy in dengue patients treated within 48 hours of symptom onset. [3] Possible Explanations for Lack of Efficacy: (1) Insufficient plasma concentrations (Cmin may need to be higher); (2) timing of treatment may be too late given that viremia declines rapidly due to host immune response; (3) inefficient intracellular phosphorylation in relevant target cells; (4) protein binding or distribution issues. [3] Study Power: Post-hoc simulations estimated the study had 80% power to detect a 0.25 log10 reduction in viremia per day in the high-dose group. The lack of any observable trend suggests true effect, if any, is smaller than this. [3] Significance: Although balapiravir was not effective, this trial establishes a framework for future antiviral treatment trials in dengue and provides a clinically evaluated benchmark molecule. [3] |
| Molecular Formula |
C9H12N6O5
|
|---|---|
| Molecular Weight |
284.232
|
| Exact Mass |
284.086
|
| CAS # |
478182-28-4
|
| PubChem CID |
457388
|
| Appearance |
White to off-white solid powder
|
| LogP |
-0.2
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| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
20
|
| Complexity |
529
|
| Defined Atom Stereocenter Count |
4
|
| SMILES |
C1=CN(C(=O)N=C1N)[C@H]2[C@@H]([C@@H]([C@](O2)(CO)N=[N+]=[N-])O)O
|
| InChi Key |
ODLGMSQBFONGNG-JVZYCSMKSA-N
|
| InChi Code |
InChI=1S/C9H12N6O5/c10-4-1-2-15(8(19)12-4)7-5(17)6(18)9(3-16,20-7)13-14-11/h1-2,5-7,16-18H,3H2,(H2,10,12,19)/t5-,6+,7-,9-/m1/s1
|
| Chemical Name |
4-amino-1-[(2R,3R,4S,5R)-5-azido-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one
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| Synonyms |
R-1479 R1479 R 1479
|
| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~351.83 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.80 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.80 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (8.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5183 mL | 17.5914 mL | 35.1828 mL | |
| 5 mM | 0.7037 mL | 3.5183 mL | 7.0366 mL | |
| 10 mM | 0.3518 mL | 1.7591 mL | 3.5183 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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