HIV-1(EC50=0.03-6.92 nM);HIV-2(EC50=0.018-0.02 nM)
HIV-1 and HIV-2 reverse transcriptase (RT); viral RNA-dependent RNA polymerase (RdRp).

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]

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Azvudine is a highly potent nucleoside reverse transcriptase inhibitor (NRTI). It shows potent inhibition against HIV-1 and HIV-2 in vitro. Against HIV-1, the EC50 ranges from 0.03 to 6.92 nM, and against HIV-2, the EC50 ranges from 0.018 to 0.025 nM. Notably, it exhibits picomolar potency against the HIV-1IIIB and HIV-1RF wild-type strains (EC50 30-110 pM). Azvudine is also effective against various drug-resistant HIV strains, including those resistant to protease inhibitors (PIs) and NRTIs. For instance, the EC50 values against the multi-drug resistant HIV-174V strain is 0.36 nM. In addition to HIV, azvudine has potent anti-HBV activity. In a cell model, it suppressed the secretion of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) with EC50 values of 0.037 microM and 0.044 microM, respectively, and reduced intracellular and extracellular HBV DNA levels by >90%. The compound also inhibits the replication of both wild-type and lamivudine-resistant HBV clinical isolates. Azvudine's triphosphate form acts as a chain terminator, incorporating into the growing viral DNA or RNA chain and preventing further elongation.

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].

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In vivo efficacy of azvudine has been demonstrated in animal models of HIV and HBV. In a murine model of HIV-1 infection (using humanized mice), treatment with azvudine resulted in a significant reduction of viral load and protection against CD4+ T cell depletion, showing superior efficacy compared to the standard-of-care drug azidothymidine (AZT). In a duck hepatitis B virus (DHBV) model, oral administration of azvudine significantly reduced serum DHBV DNA levels. The clinical antiviral efficacy of azvudine was confirmed in human trials for HIV, HBV, and COVID-19. For COVID-19, a Phase 3 clinical trial showed that oral azvudine reduced viral load and improved clinical outcomes in patients with mild to moderate COVID-19. For HIV, it is approved as part of a combination therapy. For HBV, it has shown potential in early-phase trials. These results confirm that azvudine is an orally active and broad-spectrum antiviral agent.

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]

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The inhibitory activity of azvudine triphosphate against HIV reverse transcriptase (RT) is measured using an in vitro polymerase assay. Recombinant HIV-1 RT is incubated in a reaction buffer (e.g., 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mg/mL BSA) with a heteropolymeric template/primer, such as poly(rA) oligo(dT) or a specific RNA/DNA hybrid. The reaction is initiated by adding a mixture of dNTPs, including radiolabeled dTTP (e.g., [3H]-dTTP), and varying concentrations of azvudine triphosphate. After incubation at 37degC for 30-60 minutes, an aliquot of the reaction mixture is spotted onto DE81 filter paper discs. The filters are washed extensively with 2x SSC buffer to remove unincorporated radiolabeled dNTPs. The incorporated radioactivity is then measured by liquid scintillation counting. The IC50 value for inhibition of RT activity is calculated. The selectivity of azvudine for viral RT over host DNA polymerases (e.g., DNA polymerase alpha, beta, gamma) is similarly assessed using appropriate templates and primers. For the RdRp inhibition assay for SARS-CoV-2, a similar assay is used, employing recombinant SARS-CoV-2 nsp12-nsp7-nsp8 complex as the polymerase and a specific RNA template-primer, with the active form of azvudine being azvudine triphosphate.

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.

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The cellular antiviral activity of azvudine is evaluated using a viral replication assay. For HIV, human T-cell lines (e.g., MT-4, C8166) or peripheral blood mononuclear cells (PBMCs) are infected with HIV-1 (e.g., IIIB or RF strain) at a multiplicity of infection (MOI) of 0.01-0.1 in the presence of various concentrations of azvudine (typically ranging from 0.001 nM to 1 uM). After 4-7 days of incubation at 37degC, the extent of viral replication is assessed by measuring the reverse transcriptase (RT) activity in the culture supernatant or by quantifying the HIV-1 p24 antigen (a core protein) via ELISA. The EC50 is calculated as the concentration that reduces RT activity or p24 production by 50% relative to the untreated infected control. Cytotoxicity is assessed in parallel in uninfected cells using a viability assay (e.g., MTT or CellTiter-Glo), and the CC50 (50% cytotoxic concentration) is determined. The selectivity index (SI = CC50/EC50) is then calculated. For HBV, HepG2.2.15 cells (which stably replicate HBV) are treated with various concentrations of azvudine for 9 days. The extracellular and intracellular HBV DNA is extracted and quantified by real-time PCR. The levels of HBsAg and HBeAg in the culture medium are measured by ELISA. The EC50 values for reducing these markers are then determined.

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.

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The in vivo efficacy of azvudine is evaluated in the standard murine HIV-1 model using NOD/SCID mice engrafted with human hematopoietic stem cells (humanized mice). For HIV-1 infection, the humanized mice are infected intraperitoneally with HIV-1 (e.g., 2,000 TCID₅0). One week post-infection, mice are randomized into treatment groups (n=10 per group). Azvudine is formulated in a vehicle such as sterile water or 0.5% methylcellulose and administered orally by gavage at doses ranging from 0.5 to 10 mg/kg, once or twice daily, for 4-6 weeks. Control groups include a vehicle-treated infected group and a group treated with a positive control drug (e.g., AZT at 100 mg/kg). Blood samples are collected via retro-orbital puncture at baseline and weekly. Plasma viral load is quantified by real-time PCR. At the end of the study, mice are euthanized, and spleens and lymph nodes are harvested. The percentage of CD4+ and CD8+ T cells in the blood and tissues is analyzed by flow cytometry. The HIV-1 viral load in tissues is also quantified. For HBV, a similar model using HBV-transgenic mice is employed. The duck HBV (DHBV) model is also used. One-day-old ducklings are infected intravenously with DHBV. Two weeks post-infection, ducks are treated orally with azvudine at various doses for 10 days. Serum DHBV DNA is measured by PCR.

The pharmacokinetic (PK) properties of azvudine have been well-characterized in preclinical species and in humans. In rats and dogs, after oral administration, azvudine is rapidly absorbed, with a time to peak concentration (Tmax) of 0.5-2 hours. The absolute oral bioavailability is moderate to high. The plasma elimination half-life (t1/2) is relatively short, typically 1-2 hours in rodents and 2-4 hours in larger animals. In humans, after oral administration of the approved dose for HIV, the half-life is approximately 2-3 hours. The active triphosphate metabolite is retained intracellularly with a much longer half-life (over 100 hours in peripheral blood mononuclear cells), which supports once-daily dosing. Azvudine is primarily excreted unchanged in the urine. It has low protein binding and a moderate volume of distribution, indicating good tissue penetration.

Azvudine has been evaluated extensively in preclinical toxicology studies. The compound has an acceptable safety profile at therapeutic doses. The primary dose-limiting toxicity observed in animal studies is bone marrow suppression (leading to anemia and neutropenia), which is an on-target effect of NRTIs due to mitochondrial toxicity. This is typically observed at high-dose exposures. Azvudine is less toxic to mitochondria than some older NRTIs (like AZT). In a 4-week oral toxicity study in rats, the no-observed-adverse-effect-level (NOAEL) was established. Azvudine was not genotoxic in the standard Ames test and showed no carcinogenic potential in long-term studies. In clinical trials for HIV and COVID-19, azvudine was generally well-tolerated. The most common adverse events were mild and included gastrointestinal symptoms (nausea, diarrhea) and transient elevations in liver enzymes.

[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 being investigated in the clinical trial NCT04668235 (Safety and clinical efficacy study of azvudine in COVID-19 patients (SARS-CoV-2 infection)). Mechanism of action: Azvudine is a nucleoside reverse transcriptase inhibitor that is effective against HIV, HBV, and HCV. Some studies have also shown that it can regulate the expression of proteins such as P-glycoprotein (P-gp), MRP2, and BCRP; in one study, it also enhanced P-gp activity. In 2020, this compound was used in several clinical trials to treat mild and common COVID-19. Azvudine is a novel nucleoside reverse transcriptase inhibitor with antiviral activity against human immunodeficiency virus, hepatitis B virus, and hepatitis C virus. This article reports the in vitro activity of azvudine against HIV-1 and HIV-2, and its resistance characteristics, when used alone or in combination with other antiretroviral drugs. Azvudine exhibited potent inhibitory activity against both HIV-1 (EC50 range 0.03 to 6.92 nM) and HIV-2 (EC50 range 0.018 to 0.025 nM). When used in combination with six approved anti-HIV drugs, it showed synergistic effects on C8166 cells and peripheral blood mononuclear cells (PBMCs). In combination therapy studies, azvudine was used at concentrations 1000-fold or 500-fold lower than the other drugs. Azvudine also showed potent inhibitory activity against nucleoside reverse transcriptase inhibitor (NRTI) resistant strains (L74V and T69N). Although the M184V mutation resulted in a 250-fold decrease in sensitivity, azvudine remained active at nanomolar concentrations. In in vitro resistance induction assays, the frequency of the M184I mutation increased with increasing induction time, indicating that M184I is a key mutation for azvudine treatment. As a control, lamivudine treatment resulted in a higher frequency of M184I/V mutations and a higher incidence of M184V at the same induction time. Molecular modeling analysis showed that the steric hindrance of mutant M184I was more significant than that of M184V due to the azide group in azivudine. Current data suggest that azivudine has the potential to complement existing anti-HIV drugs. M184I should be the key mutation; however, azivudine remains effective against HIV-1LAI-M184V at nanomolar concentrations. [2]

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The novel nucleoside analog FNC is effective against both wild-type and lamivudine-resistant clinical isolates of HBV. HBV infection is a major global public health problem. The clinical limitations of current antiviral drugs for the treatment of hepatitis B virus (HBV), such as lamivudine, have led to the rapid emergence of drug-resistant strains during long-term antiviral therapy. Therefore, there is an urgent need for new antiviral drugs to prevent or delay the selection of drug-resistant HBV mutants. A novel cytidine analog, FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), has recently been shown to potently inhibit the replication of human HBV and duck HBV (DHBV) both in vitro and in vivo. This study aimed to evaluate the in vitro antiviral activity of FNC against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. Methods: HBV DNA was extracted from serum samples collected before lamivudine treatment and at viral breakthrough, and amplified by PCR. The amplicon was cloned into a novel expression vector, pHY106, which initiates the intracellular HBV replication cycle after cell transfection. Drug susceptibility testing was performed after transfecting the cloned amplicon into HepG2 cells. Intracellular HBV DNA levels were determined using quantitative real-time PCR, and the effective concentration (EC50) required to inhibit HBV replication by 50% was calculated. Results: FNC inhibited the replication of wild-type and lamivudine-resistant HBV clinical isolates in a dose-dependent manner, with mean ± standard deviation EC50 values of 0.12 ± 0.01 μM and 0.27 ± 0.01 μM, respectively. Conclusion: FNC is a potential antiviral drug that can effectively combat wild-type and lamivudine-resistant HBV clinical isolates, and therefore deserves further evaluation in the treatment of HBV infection. [3]

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Azvudine/FNC is a novel nucleoside analog that can inhibit the proliferation and tumor growth of various human cancer cells. Inhibiting cellular DNA synthesis is a strategy to block cancer cell division. Nucleoside analogs can be incorporated into DNA and terminate the extension of DNA chains. Currently, a number of nucleoside analogs have been successfully used as anticancer drugs. FNC, namely 2'-deoxy-2'-β-fluoro-4'-azidocytidine, is a novel cytidine analog that shows potent activity against hepatitis C virus (HCV). To investigate the therapeutic potential of FNC in human cancer, we studied its activity against a variety of cancer cells in vitro and in vivo. [4]

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Azvudine hydrochloride is approved in China for the treatment of HIV-1 infection (brand name: FNC) and has received conditional approval for the treatment of novel coronavirus pneumonia (COVID-19) in adults. It is a first-in-class nucleotide analogue featuring a unique combination of an azido group at the 4' position and a fluorine atom at the 2' position of the ribose ring. This structure confers high potency and a favorable resistance profile. The active triphosphate form acts as a chain terminator during RNA or DNA synthesis and also functions as a weak inhibitor of viral RNA-dependent RNA polymerase (RdRp), which is the primary mechanism for its anti-SARS-CoV-2 activity. Azvudine is given orally, making it convenient for outpatient use. It is not yet approved in other countries (USA, Europe, Japan). Ongoing clinical trials are evaluating its potential in other viral infections, such as hepatitis B. The approval for COVID-19 is based on a small, open-label trial, and further studies are required to fully establish its efficacy in larger, well-controlled trials. As with all NRTIs, there is a potential for the development of resistance with long-term use, and its use is contraindicated in pregnant women due to potential embryotoxicity in animal studies.

C9H12CLFN6O4

322.68078327179

322.059

1333126-31-0

Azvudine;1011529-10-4

54579399

Light yellow to khaki solid powder

4

7

3

21

533

4

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

MKMLHJHSIBILJH-DBSFTZRASA-N

InChI=1S/C9H11FN6O4.ClH/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);1H/t5-,6-,7+,9+;/m0./s1

4-amino-1-[(2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one;hydrochloride

Azvudine HCl; RO-0622; FNC; RO0622; Azvudine (hydrochloride); Azvudine hydrochloride; 1333126-31-0; 4-amino-1-[(2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one;hydrochloride; CHEMBL1822611;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : ~125 mg/mL (~387.38 mM)

Solubility in Formulation 1: 14.29 mg/mL (44.29 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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