HIV-1(EC50=0.03-6.92 nM);HIV-2(EC50=0.018-0.02 nM)

Azvudine (RO-0622) exhibits robust suppression against HIV-1IIIB and HIV-1RF wild-type, with an EC50 varying between 30 and 110 pM. Azvudine has EC50 values of 6.92, 0.34, and 0.45 nM against HIV-1KM018, HIV-1TC-1, and HIV-1WAN T69N, respectively. The PIs-resistant strains HIV-1L10R/M46I/L63P/V82T/I84V and HIV-1RF V82F/184V, the FIs-resistant strain pNL4-3 gp41 (36G) V38A/N42T, and the NRTIs-resistant strain HIV-174V are all susceptible to azvudine. Azvudine's EC50 values against these resistant strains are, in turn, 0.11, 0.14, 0.37, and 0.36 nM[1].

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In the cell model, Azvudine (FNC) effectively suppressed the secretion of the HBV antigens in a dose-dependent manner, with 50% effective concentration values of 0.037 μM for hepatitis B surface antigen and 0.044 μM for hepatitis B e antigen on day 9. Consistent with the HBV antigen reduction, Azvudine (FNC) also reduced the HBV DNA level by 92.31% and 93.90% intracellularly and extracellularly, respectively. [2]

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Azvudine (FNC) inhibited the replication of both wild-type and lamivudine-resistant HBV clinical isolates in a dose-dependent manner, with mean ±SD EC(50) values of 0.12 ±0.01 μM and 0.27 ±0.01 μM, respectively. Conclusions: Azvudine (FNC) is a potential antiviral agent against both wild-type and lamivudine-resistant HBV clinical isolates, and therefore deserves further evaluation for the treatment of HBV infection.[3]

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Azvudine (FNC) potently inhibited cell proliferation with an IC(50) of 0.95-4.55μM in a variety of aggressive human cancer cell lines including B-cell non-Hodgkin's lymphomas, lung adenocarcinoma and acute myeloid leukemia. Cells treated with FNC exhibited G1 and S cell cycle arrest at high and low dose, respectively, which confirms the mechanism of action of nucleoside analogues. Treatment of B-NHL cell lines with FNC induced apoptosis in a dose and time dependent manner.[4]

In vivo antiviral efficacy[2]
DHBV DNA levels were markedly reduced after treatment with the Azvudine (FNC) at 0.5, 1.0 and 2.0 mg/kg•day dosages. The inhibition rate of Azvudine (FNC) at the dose of 2.0 mg/kg•day reached 91.68% and 81.96%, in duck serum and liver, respectively, on day 10. Furthermore, significant liver histology restoration after FNC treatment was observed, as evaluated by the histopathological analysis.[2]

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In vivo antitumor efficacy[4]
Finally, mouse xenograft models of hepatocarcinoma (H22), sarcoma (S180) and gastric carcinoma (SGC7901) demonstrated that Azvudine (FNC) had significant tumor growth inhibition activity in a dose-dependent manner with low toxicity[4].

Quantification of HBV DNA by fluorescent quantitative PCR[2]
To further confirm the antiviral activity of Azvudine (FNC) in HepG2.2.15 cells, the extracellular and intracellular HBV DNA levels were evaluated by fluorescent quantita- tive (FQ)-PCR. Viral DNA was extracted from the culture supernatant and cells, and then real-time quantitative PCR was performed in Light-Cycler 1.5 using the HBV Fluorescent Quantita- tive PCR Detection Kit according to the manufacturer’s protocol. The cycling programme was as follows: after an initial denaturation (95°C for 2 min), the samples were subjected to 40 cycles of denaturation (94°C for 5 s) and annealing/extension (each at 56°C for 45 s).[2]

Anti-HIV activity in vitro[1]
C8166 cells were infected with different HIV-1 or HIV-2 laboratory strains and resistant strains at different serial concentration compounds with multiplicity of infection (MOI) of 0.075∼0.6. PHA-stimulated PBMCs were incubated with different clinical strains in RPMI-1640 (with 10% FBS, 50 U/ml IL-2 and 2 µg/ml polybrene) at MOI of 0.1. After infection at 37°C in 5% CO2 for 2 hours, C8166 cells were washed three times to remove free viruses and re-suspended by RPMI-1640 (with 10% FBS). 100 µl 4×104 cells (5×105 cells for PBMC) were seeded each well in a 96-well plate with gradient concentration of FNC. The plate was placed in a humidified incubator at 37°C, 5% CO2. 3TC and AZT were used as control. After incubation of 3–7 days, the percentage inhibition of syncytia formation was scored or the level of p24 was measured by ELISA [19] and 50% effective concentration (EC50) were calculated.

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Cytotoxicity assays[1]
Briefly, a serial concentration of FNC was added to a 96-well plate, followed by 100 µl 4×104 C8166 cells (5×105 cells for PBMC). After incubation at 37°C, 5% CO2 for 3 days (7 days for PBMCs), 20 µl MTT was added each well. After incubation for 4 hours, 100 µl supernatant was removed and 100 µl 20%SDS-50%DMF was added. The plate was incubated at 37°C overnight. The optical absorbance was measured by ELISA reader at 570 nm and 630 nm, and 50% cytotoxicity concentration (CC50) was calculated. 3TC and AZT were used as control.

DHBV infection and drug treatment experiment[2]
Each duck, aged 1 day, was injected into its tibial vein with 0.2 ml of serum from ducks with positive DHBV serology on day 3. The drug treatment experiment was carried out 7 days after ducks were infected with DHBV. The DHBV-positive ducks were randomly divided into five groups with 16 ducks in each group. Azvudine (FNC) in differ- ent concentrations and 3TC control were given orally to DHBV-infected ducks, respectively. Five groups were observed: FNC 0.5 mg/kg•day, Azvudine (FNC) 1.0 mg/kg•day, Azvudine (FNC) 2.0 mg/kg•day and 3TC 20 mg/kg•day as a posi- tive control. Normal saline was used as a mock treat- ment for the negative control group. The drugs were given once daily for 10 days continuously. The blood was drawn from the leg vein of all ducks before treat- ment, after medicating for 5 days and 10 days, and after withdrawal of the drug for 3 days. The serum samples and livers were separated and stored at -80°C.

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Measurement of DHBV DNA by FQ-PCR[2]
DHBV DNA was measured on day 0, days 5 and 10 during treatment, and day 13, that is day 3 after ces- sation of treatment on day 10 by FQ-PCR. For DHBV DNA, the DNA was extracted from serum using a DNA Extraction Kit, and FQ-PCR was performed in Light-cycler 1.5 using SYBR Green I. A pair of primers was designed based on the sequences from a previously published report, and used for amplifying the genome of DHBV, and the amplified PCR fragments were then cloned into pMD-18T. Based on the conserved sequences of DHBV S gene, another pair of primers for real-time PCR were designed and used to amplify the recombinant plasmid for constructing the standard curves. Meanwhile, the specificity, sensitivity and repeatability of the assay were tested. A rapid and specific SYBR Green I real-time PCR assay was estab- lished to detect DHBV. DHBV DNA from the serum and liver of experimentally infected ducklings was detected by this assay at different time points as indicated.[2]

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Mouse xenograft studies[4]
All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care. Kunming mice (including male and female, body weight 20 ± 2 g from Shanghai Sikelai Co., Shanghai, China) were injected with 1 × 107 sarcoma (S-180) and hepatoma (H22) cells subcutaneously into the right front flank and divided randomly into several different test groups with 8–10 mice per cohort. One day after implantation of tumor cells, the mice were treated daily by IV or IG with vehicle (saline) or 5-FU (15 mg/kg/day), cisplatin (1.0 mg/kg/day), capecitabine (400 or 600 mg/kg/day) and Azvudine (FNC) (0.5, 1.0, 2.0 mg/kg/day) formulated in saline or distilled water (for capecitabine) for 8 days. Then the mice were sacrificed and the tumors were excised and weighed for evaluating the tumor growth inhibition at 24 h after the end of treatment. BALB/c nu/nu mice were provided by Shanghai Sikelai Co. and human gastric cancer cells (SGC7901) were subcutaneously implanted in the right hind back using 200 μl of a 1 × 107 cell/ml suspension in PBS. When tumors reached an average diameter of 5–8 mm, mice were weighed, randomized by tumor size, assigned to the various study groups, and treated with vehicle (saline), capecitabine (600 mg/kg/day), or Azvudine (FNC) (0.5, 1.0, 2.0 mg/kg/day) by IG daily for 20 days. After treatment, mice were sacrificed and the tumors were excised and weighed for evaluating the tumor growth inhibition. All results are represented as mean ± SEM of eight or ten animals.

[1]. Azvudine, a novel nucleoside reverse transcriptase inhibitor showed good drug combination features and better inhibition on drug-resistant strains than lamivudine in vitro. PLoS One. 2014 Aug 21;9(8):e105617.

[2]. Novel nucleoside analogue FNC is effective against both wild-type and lamivudine-resistant HBV clinical isolates. Antivir Ther. 2012;17(8):1593-9.

[3]. Antiviral activity of FNC, 2'-deoxy-2'-β-fluoro-4'-azidocytidine, against human and duck HBV replication. Antivir Ther. 2012;17(4):679-87.

[4]. FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells. J Biol Chem. 2008 Jan 25;283(4):2167-75.

Azvudine is under investigation in clinical trial NCT04668235 (Study on Safety and Clinical Efficacy of AZVUDINE in COVID-19 Patients (Sars-cov-2 Infected)).

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Mechanism of Action
Azvudine is a nucleoside reverse transcriptase inhibitor that acts against HIV, HBV, and HCV. Some studies also show that it is able to modulate the expression of proteins like P-glycoprotein (P-gp), MRP2, and BCRP; in one instance, it was also able to increase the activity of P-gp. In 2020, the compound was tested in numerous clinical trials for the treatment of mild and common COVID-19.

C9H11FN6O4

286.2198

286.082

C, 37.77; H, 3.87; F, 6.64; N, 29.36; O, 22.36

1011529-10-4

Azvudine hydrochloride;1333126-31-0;Azvudine-13C,15N2,d2

24769759

White to off-white solid powder

-0.21

3

7

3

20

533

4

F[C@]1([H])[C@]([H])(N2C(N=C(C([H])=C2[H])N([H])[H])=O)O[C@](C([H])([H])O[H])([C@@]1([H])O[H])N=[N+]=[N-]

KTOLOIKYVCHRJW-XZMZPDFPSA-N

InChI=1S/C9H11FN6O4/c10-5-6(18)9(3-17,14-15-12)20-7(5)16-2-1-4(11)13-8(16)19/h1-2,5-7,17-18H,3H2,(H2,11,13,19)/t5-,6-,7+,9+/m0/s1

4-amino-1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one

Azvudine; FNC; RO-0622; RO 0622; RO0622; Azvudine; 1011529-10-4; RO-0622; 4'-C-azido-2'-deoxy-2'-fluoro-beta-D-arabinocytidine; 4-amino-1-[(2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one; RO 0622; 4-amino-1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one; Azvudine?;

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)

DMSO : 57~125 mg/mL ( 199.14~436.73 mM )
Water : ~57 mg/mL
Ethanol : ~57 mg/mL

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.)