Antiviral; SARS-CoV-2; RSV; deuterated form of GS-621763

The tri-isobutyrate ester VV116 can also inhibit RSV replication (EC50 = 1.20 ± 0.32 μM, CC50 = 95.92 ± 9.27 μM, SI = 80, EC90 = 3.08 ± 1.253 μM) in A549 cells, which suggested that the ester moiety of VV116 was susceptible to hydrolysis by cellular enzymes to release the parent nucleoside. Anti-RSV activities of these compounds were also confirmed in HEp-2 and NHBE cells, other permissive cells for RSV. [1]

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Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers that influence measurement during the drug development process. It's possible that the pharmacokinetics and functional range of medications contribute to the concern over mutagenesis [1]. Potential benefits of compounds with delayed generation include: (1) compounds with delayed generation may be able to extend the compound's pharmacokinetic characteristics, which could extend the compound's safety, tolerability, and improved tolerance; and (2) compounds with delayed generation may expand intestinal bioavailability. Deuterated compounds may be able to lessen the amount of first-pass metabolism required in the colon and intestinal wall, which would enable a higher percentage of the medicine to reach high bioavailability levels, which dictate its efficacy at low doses and better tolerability. (3) Enhance the properties of metabolism. Drug safety, drug metabolism (4), and hazardous or reactive metabolite reduction are all potential benefits of metabolites. Deuterated chemicals are harmless and have the potential to lessen or completely eradicate the negative effects of medicinal drugs. (5) Preserve medicinal qualities. According to earlier research, deuterated molecules should maintain a biochemistry similar to that of comparable hydrogen compounds.

Mindeudesivir (VV116) (25, 50 and 100 mg/kg; PO; bid for 4 days) exhibits a stronger activity and decreases the virus titers below the detection limit at 50 mg/kg, also reduces lung injury after RSV infection[1]. VV116 ( 25, 50 and 100 mg/kg; PO; single dosage) exhibits favorable PK properties and good safety profile[1]. Pharmacokinetic Parameters of VV116 (JT001) in Balb/c mice[1]. PO (25 mg/kg) PO (50 mg/kg) PO (100 mg/kg) Tmax (h) 0.42 ± 0.14 0.42 ± 0.14 0.42 ± 0.14 Cmax (ng/mL) 5360 ± 560 11617 ± 3443 24017 ± 6521 AUC0-t (ng/mL·h ) 11461 ± 1013 24594 ± 1059 47799 ± 6545 AUC0-∞ (ng/mL·h) 11534 ± 992 24739 ± 1028 48014 ± 6696 MRT0-∞ (ng/mL·h) 2.25 ± 0.32 2.15 ± 0.26 2.2 8 ± 0.53 Tmax ( h) 2.30 ± 1.10 3.27 ± 1.92 4.25 ± 0.53 Animal Model: Balb/c mice[1] Dosage: 25, 50 and 100 mg/kg Administration: PO; single dosage (Pharmacokinetics Analysis) Result: Exhibited favorable PK properties and good safety profile. Animal Model: Balb/c mice (6-8 weeks; intranasally infected with 4 × 10^6 FFU of RSV)[1] Dosage: 25, 50 and 100 mg/kg Administration: PO; bid for 4 days Result: Exhibited a stronger activity and decreased the virus titers below the detection limit at 50 mg/kg, also reduced lung injury after RSV infection.

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Considering the potent effect of Mindeudesivir (VV116) inhibiting RSV in vitro, we further tested the effect of VV16 against RSV in a mouse model. Ribavirin, the off-label used drug to treat RSV in the clinic, was employed as a control. To this end, 6–8-week Balb/c mice were intranasally infected with 4 × 106 FFU of RSV per mouse (day 0), and were then treated with VV116 (25, 50, and 100 mg/kg) or ribavirin (50 and 100 mg/kg) bis in die (b.i.d.) (supplementary Fig S3). Our previous study indicated that both viral load and pathology reached high in RSV infected mice at day 4 post infection (p.i.), and hence at this time point, mice were killed, and lungs were fetched. Viral RNA level in the lung was measured with quantitative RT-PCR and virion load was measured with immunoplaque assay (Fig. 1e). Of note, the low dose of VV116 (25 mg/kg) displayed a comparable antiviral effect to that of 100 mg/kg of ribavirin, which decreased the viral RNA copies and the infectious tilters by ~1.5 log10 and ~2.0 log10, respectively (Fig. 1e). The medium dose (50 mg/kg) of VV116 exhibited a stronger activity and decreased the virus titers below the detection limit (Fig. 1e). We also evaluated the lung pathology of the challenged mice by histochemical analysis. After RSV infection, mice treated with vehicle displayed severe inflammation with alveolar inflammatory patches. By contrast, only slight lung infiltration was observed in mice treated with VV116, demonstrating that VV116 treatment can reduce lung injury after RSV infection [1].

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The PK study in Balb/c mice showed that Mindeudesivir (VV116) had a linear PK profile in doses of 25 to 100 mg/kg (Fig. 1c, supplementary Table S6). Because of the first-pass metabolism of the esterase-sensitive prodrug, VV116 was not detected in mouse plasma even at 100 mg/kg. Following oral administration, the blood concentration of the parent nucleoside X1 quickly reached Cmax within 0.5 h, and at the dose of 25 mg/kg, the mean Cmax reached 5360 ng/ml (18.4 µM, Fig. 1c, supplementary Table S6, S7), which was much higher than the EC90 value in vitro. X1 had a short elimination half-life (2.3–4.25 h, supplementary Table S6), which supported a twice-daily dosing regimen. The ester prodrug form of VV116 was designed not only for improving oral adsorption but to circumvent the liver-targeting issue of the nucleoside phosphoramidate prodrugs. The preclinical tissue distribution study revealed that X1 was widely distributed in SD rat tissues,5 and a favorable distribution of X1 was also observed in Balb/c mice with the concentration of X1 in the lung being about half of that in the liver (Fig. 1d, supplementary Table S8). With respect to the therapeutic window of VV116, the 14-day repeated dose oral toxicity study in rats revealed a NOAEL (No-Observed-Adverse-Effect-Level) of 200 mg/kg, at which the AUC0–t of X1 reached a value of 85151 ng h/ml (Supplementary Table S9), ~3.5-folds of that at the dose of 50 mg/kg in mice.[1]

Antiviral activities and cytotoxicity measurement [1]
A549, HEp-2 or NHBE cells were plated into 48 well-plate and incubated overnight. Upon reaching 80% cell confluence, cells were infected RSV A2 (at an MOI of 2 in A549, MOI of 0.5 in HEp-2, and MOI of 1 in NHBE cells) for 2 h after cells were incubated for 1 h with varying concentrations of drugs. Then, the virus-drug mixture was removed, and cells were cultured with drug-containing medium. At 48 h post inoculation, total RNAs were extracted from cells and then in reverse transcription using PrimeScript RT reagent Kit with gDNA Eraser. For determining the viral copies, absolute quantitative RT-PCR was performed with TB Green® Premix Ex TaqTM II. RSV A2 F fragment was quantified with primers 5’-CGAGCCAGAAGAGAACTACCA-3’; and 5’-CCTTCTAGGTGCAGGACCTTA-3’.
Cell viability was performed in 96-well plate with triplicate for each concentration. All drugs were diluted 2 times with 9 gradients starting at 500 micromores in maintenance medium (DMEM containing 2% FBS). After 48 h incubation, the supernatant was removed, and 10 μL WST-8 (2-(2-methoxy-4-(phenyl)-3-(4-(phenyl) to 5 (2, 4-sulpho benzene) -2 h-tetrazolium monosodium salt) in maintenance medium was added in medium. Plates were measured at 450 nm wavelength using spectrophotometer (BioTek) after 2 h incubation, and cell viability was calculated.

Cell viability assay
Cell Types: A549 (infected with RSV) [1]
Tested Concentrations: 0-1000 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Inhibited RSV replication in A549 cells, EC50 is 1.20±0.32 μM and CC50 is 95.92. ± 9.27 μM, selectivity index (SI) 80.

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Cells and viruses [1]
All the cells used in this study were cultured in humidified incubator under 37℃ with 5% CO2. Human laryngeal epidermoid carcinoma (HEp-2) cells, Vero E6 cells, and A549 cells were grown in Dulbocco’s Medified Eagle Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum. Normal human bronchial epithelial (NHBE) cells were maintained in Bronchial Epithelial Cell Growth Medium (BEGM) with all provided supplements in the BulletKit. RSV A2 strain was grown in HEp-2 cells. At 3 or 4 days post-infection, viruses were collected from infected cells. Briefly, RSV infected cells were repeated freezing and thawing 3 times, then the cells were centrifuged at 1000 rpm for 10 min at 4℃. Afterwards, the supernatant was collected and stored at -80℃ until used. Viral titer of RSV A2 was determined in Vero E6 cells by immunoplaque assay as described previously1. All the RSV A2 infection experiments were carried out in biosafety level-2 (BSL-2) laboratory.

In vivo efficacy of Mindeudesivir (VV116) against RSV in mice [1]
Specific pathogen-free (SPF) female Balb/c mice at the age of 6–8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in an SPF environment under standard conditions. All mouse experiments were approved by the ethics committee of the Wuhan Institute of Virology, Chinese Academy of Science (permit number WIVA25202113). Thirty mice (5 animals per group, 6 groups) were anaesthetized with isoflurane and challenged with 4×106 FFU of RSV A2 intranasally (i.n.). The mice were given drugs by intragastric administration. Treatments were commenced in 1 h post infection and continued for 4 days. Mice in the control group were given the solvent (40% PEG 400+10% HS 15+50% ultrapure water (v:v:v)). The mice were euthanized on the 4th day after challenge and their lungs were collected. The weight of the mice was recorded daily.
The left lung was fixed in tissue fixative solution, embedded, sectionized and stained with H&E to observe the pathological changes of lung tissue. After weighing the right lung, add 400μL PBS into the tube, grind it with a grinding instrument. One part of the grinding tissue was used to determine the virus titer, and the other part was used to determine virus copy number by extracted RNA from the tissue supernatant using viral DNA/RNA extraction Kit (TaKaRa, 9766). The determination of viral titers and subsequent treatment of the RNA obtained were the same as above.

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Pharmacokinetic study of Mindeudesivir (VV116) in ICR mice, Balb/c mice, and SD rats [1]
ICR mice (N = 3 for each group, male) were fasted for 12 h before dosing (only for the oral administration). Mindeudesivir (VV116) dissolved in DMSO-enthanol-PEG300-saline (5/5/40/50, v/v/v/v) was administered intravenously at 5.0 mg/kg, and orally at 25 mg/kg. At 5 min, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h post-dosing, blood samples were collected from the jugular vein or the submandibular vein into EDTA-K2 tubes, and immediately mixed with acetonitrile (20 µL blood + 80 μL acetonitrile). The concentrations of analytes in the blood were analyzed by LC-MS/MS.
A total of nine Balb/c mice (N = 3 for each group, male) were divided into three groups, and fasted for 12 h before dosing. The three groups received oral dose of Mindeudesivir (VV116) dissolved in 40%PEG400+10% Kolliphor® HS15+50% ultrapure water at 25 mg/kg, 50 mg/kg and 100 mg/kg, respectively. At 5 min, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h post-dosing, blood samples were collected from the jugular vein or the submandibular vein into EDTA-K2 tubes, and immediately mixed with acetonitrile (20 µL blood + 80 μL acetonitrile). The concentrations of analytes in the blood were analyzed by LC-MS/MS.
SD rats (N = 3 for each group, male) were fasted for 12 h before dosing (only for the oral administration). The test compound (Mindeudesivir (VV116) or VV116-H) was administered intravenously at 5.0 mg/kg dissolved in DMSO-enthanol-PEG300-saline (5/5/40/50, v/v/v/v), and administered orally at 30 mg/kg dissolved in 40%PEG400+10% Kolliphor® HS15+50% ultrapure water. At 5 min, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h post-dosing, blood samples were collected from the jugular vein into EDTA-K2 tubes. Serum samples were obtained following general procedures and the concentrations of analytes in the supernatant were analyzed by LC-MS/MS.

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Tissue distribution study of Mindeudesivir (VV116) in Balb/c mice [1]
A total of thirty Balb/c mice were divided into five groups (3 animals/sex/group). VV116 was intragastrically administered at 100 mg/kg dissolved in 40%PEG400+10% Kolliphor® HS15+50%. At 0 (not administered), 0.25, 2, 6, and 24 h post-dosing, the five groups of mice were anesthetized, respectively. Blood samples were collected, and tissues including liver and lung were harvested. Tissue samples were individually homogenized, and blood samples were processed as above. The concentrations of X1 in liver, lung and blood were analyzed by LC-MS/MS.

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Genetic toxicity assay [1]
The Ames test, the rat micronucleus assay, and the chromosome aberration test were conducted according to NMPA and ICH guidelines.
The Ames test was conducted to determine the mutagenicity of Mindeudesivir (VV116) using histidine-dependent Salmonella typhimurium (TA97a, TA98, TA100, TA1535) and tryptophan-dependent Escherichia coli (WP2). The experiment was carried out by plate permeating method under the -S9 non-metabolic and +S9 metabolic activation conditions. There were 6 dose groups for Mindeudesivir (VV116) (5, 50, 150, 500, 1500 and 5000 µg/dish under each condition) with the negative control (DMSO) and positive controls (ICR191, 2-nitrofluorene, sodium azide, 2-aminofluorene and methyl methanesulfonate). Under the conditions of -S9 and +S9, the average numbers of revertant colonies in the positive control group of each strain were at least twice that of the negative control group. The numbers of revertant colonies of each strain in all VV116 dose groups were less than twice that of the negative control group, and did not show dose-dependent increase. The result showed that VV116 was not mutagenic to histidine-dependent Salmonella typhimurium and tryptophan-dependent Escherichia coli.
The chromosome aberration test was conducted to evaluate whether Mindeudesivir (VV116) had the effect of inducing chromosome damage in Chinese hamster lung (CHL) cells by determining the aberration rate (excluding chromosome gap) under the -S9 and +S9 conditions. CHL cells were exposed to Mindeudesivir (VV116) without S9 for 4 h at the concentrations of 10, 20, 35, 40, 43, 45 and 48 μg/mL (-S9/4h group), or 24 h at the concentrations of 5, 10, 20, 25, 30, 35 and 40 μg/mL (-S9/24h group). In the presence of S9 mix, CHL cells were treated with VV116 for 4 h at the concentrations of 10, 25, 50,100 and 150 μg/mL (+S9/4h group). Meanwhile, negative (DMSO), and positive control groups (Mitomycin C and cyclophosphamide monohydrate) were set up. Based on the cytotoxicity of VV116, three doses of each group were chosen for chromosome aberration analysis. The positive compounds obviously induced chromosome aberrations compared with the negative control. For the -S9/4h group of VV116, the chromosome aberration rates at the concentrations of 20, 35 and 40 µg/mL were 0.0%, 0.3% and 0.0%, respectively; For the -S9/24h group, the rates at the concentrations of 10, 25 and 30 µg/mL were 1.0%, 0.3% and 0.3%, respectively. And for the +S9/4h group, the rates at the concentrations of 20, 50 and 150 µg/mL were 1.3%, 0.7% and 0.3%, respectively. The chromosome aberration rates of all Mindeudesivir (VV116) groups were within the background range, and showed no statistical difference compared with that of the negative control group. The result indicated that VV116 had no effect of inducing chromosome aberration in CHL cells.
The micronucleus assay in rats was conducted to evaluate whether Mindeudesivir (VV116) has the effect of inducing any increase of micronucleated polychromatic erythrocytes in rat bone marrow. Groups of male and female SD rats (5 animals/sex/group) received oral doses of VV116 at 0 (vehicle control), 100 (low), 200 (mild) and 500 mg/kg/d (high) for 14 days. The animals were sacrificed within 24 h after the last dose. Bone marrow smears were prepared for examining the ratio of polychromatic erythrocyte/(polychromatic erythrocyte + normochromatic erythrocyte) (PCE/(PCE + NCE)) and the micronucleus rate of polychromatic erythrocytes (MnPCE/PCE). The result showed that the PCE/(PCE + NCE) ratios of the female animals of the vehicle group, the low, the mild, and the high dose VV116 group were 0.65, 0.57, 0.58 and 0.58, respectively. For the male animals, the ratios were 0.62, 0.64, 0.66 and 0.60, respectively. VV116 did not show obvious bone marrow toxicity in rats. The assay was valid as the average micronucleus rates were 1.4‰ and 0.7‰ for the female and male rats in the vehicle group, respectively, which were within the historical range. The micronucleus rates of the female animals of the three VV116 groups were 1.2‰, 1.0‰ and 0.7‰, respectively, and for the male animals, the rates were 0.3‰, 0.7‰ and 0.3‰, respectively. There was no effect of any dose of VV116 on the micronucleus rate compared to the negative control. VV116 did not have the effect of inducing the increase of micronucleated polychromatic erythrocytes in rat bone marrow up to 500 mg/kg/d for 14 days.

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Toxicokinetics of Mindeudesivir (VV116) in SD rats [1]
Groups of male and female SD rats (4 animals/sex/group) received repeated oral doses of Mindeudesivir (VV116) (dissolved in 40%PEG400+10% Kolliphor® HS15+50% ultrapure water) at 100 (low), 200 (mild) and 500 mg/kg/d (high) for 14 days. At day 1 and day 14, blood samples were collected from the jugular vein into EDTA-K2 tubes at various time points post-dose. Plasma samples were obtained following general procedures and the concentrations of analytes in the samples were analyzed by LC-MS/MS.

VV116 (JT001) is an oral nucleoside analogue candidate for SARS-CoV-2. Three Phase I studies aimed to evaluate the safety, tolerability, and pharmacokinetics of single and multiple escalation oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. The three studies were initiated sequentially: Study 1 (Single-Ascendance Study, SAD), Study 2 (Multiple-Ascendance Study, MAD), and Study 3 (Food Effects Study, FE). A total of 86 healthy subjects were enrolled. Subjects took either VV116 tablets or a placebo as instructed. Blood samples were collected at predetermined time points for pharmacokinetic analysis. The VV116 metabolite 116-N1 was detected in plasma, and its concentration was calculated for the calculation of pharmacokinetic parameters. In the single-dose (SAD) range of 25–800 mg, AUC and Cmax increased approximately dose-proportionately. T1/2 was within 4.80–6.95 hours. In multiple-dose administration (MAD), the cumulative ratios of Cmax and AUC indicated a slight accumulation of the drug after repeated administration of VV116. In standard meal administration (FE), the standard meal had no effect on the Cmax and AUC of VV116. No serious adverse events occurred in the study, and no subjects withdrew from the study due to adverse events. Therefore, VV116 demonstrated satisfactory safety and tolerability in healthy subjects, which supports continued investigation of VV116 in COVID-19 patients. [2]

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Pharmacokinetic studies in Balb/c mice showed that Mindeudesivir (VV116) had linear pharmacokinetic characteristics in the dose range of 25 to 100 mg/kg (Figure 1c, Supplementary Table S6). Due to the first-pass metabolism of the esterase-sensitive prodrug VV116, it was not detected in mouse plasma even at doses up to 100 mg/kg. Following oral administration, the plasma concentration of the parent nucleoside X1 rapidly reached Cmax within 0.5 hours, with a mean Cmax of 5360 ng/ml (18.4 µM, Fig. 1c, Supplementary Tables S6 and S7) at a dose of 25 mg/kg, significantly higher than the in vitro EC90 value. The short elimination half-life of X1 (2.3–4.25 h, Supplementary Table S6) supports a twice-daily dosing regimen. The VV116 ester prodrug was designed not only to improve oral absorption but also to circumvent the liver-targeting issues associated with nucleoside phosphoramide prodrugs. Preclinical tissue distribution studies showed that X1 was widely distributed in SD rat tissues⁵, and good distribution was also observed in Balb/c mice, with lung X1 concentrations approximately half that in the liver (Fig. 1d, Supplementary Table S8). Regarding the therapeutic window of VV116, a 14-day repeated-dose oral toxicity study in rats showed that its NOAEL (no adverse reaction observed) was 200 mg/kg, at which point the AUC_0-t of X1 reached 85151 ng·h/ml (Supplementary Table S9), which is about 3.5 times that of the 50 mg/kg dose in mice^[1].

Safety [2]
No deaths, serious adverse events (SAEs), grade 3 or higher adverse events (AEs), or adverse events leading to drug discontinuation/interruption were reported in the three studies. All adverse events resolved without any treatment or intervention.
Study 1: Single-Elevation Dose Study
The number of subjects (incidence rates) of adverse events in the 25, 200, 400, 800, and 1200 mg dose groups and the placebo group were 2 (50%), 3 (50%), 3 (50%), 3 (50%), 0 (0%), and 5 (50%), respectively (Table 7). No dose-related adverse event occurrence was observed. In the single-escalation dose study, the incidence of adverse events was lower in subjects treated with Mindeudesivir (VV116) than in subjects treated with placebo (39.3% vs. 50%). Except for one case of grade 2 neutropenia, all adverse events were CTCAE grade 1 in severity. The dose escalation termination criteria were not met during the dose escalation period. The most common drug-related adverse events were sinus bradycardia, shortened PR interval on ECG, and elevated serum bilirubin.
Study 2: Multiple Dose Escalation Study
The number of subjects (incidence) of adverse events in the 200 mg, 400 mg, and 600 mg dose groups and the placebo group were 3 (33.3%), 5 (55.6%), 6 (66.7%), and 5 (55.6%), respectively (Table 8). The incidence of adverse events in subjects treated with Mindeudesivir (VV116) was comparable to that in subjects treated with placebo (51.9% vs. 55.6%). The occurrence of adverse events was dose-related. Except for one subject in the placebo group who experienced one of three cases of grade 2 nausea, the severity of adverse events was generally mild (CTCAE grade 1). The most common drug-related adverse events were elevated serum uric acid, dry mouth, urinary crystals, and nausea. Two subjects in the 400 mg dose group experienced grade 1 elevations in transaminases (elevated alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase), for a total of three cases. The elevations in transaminases were transient and resolved spontaneously.
Study 3: Food Effects Study
The number of subjects (incidence) of adverse events after fasting, after a standard meal, and after a high-fat meal were 0 (0%), 2 (16.7%), and 4 (33.3%), respectively. Two subjects experienced first-degree atrioventricular block after consuming a standard meal, while four subjects experienced positive urine bacteria test, urinary crystals, elevated blood pressure, and first-degree atrioventricular block after consuming a high-fat meal. All adverse events were grade CTCAE 1 in severity.
Other Safety Assessments
In Study 3, only one subject in the 400 mg dose group experienced a transient, mild increase in thyroid-stimulating hormone (TSH), which resolved spontaneously without treatment. No clinically significant abnormalities were found in sex hormone testing, ophthalmological examination, or thyroid ultrasound.

[1]. Oral remdesivir derivative VV116 is a potent inhibitor of respiratory syncytial virus with efficacy in mouse model. Signal Transduct Target Ther. 2022;7(1):123. Published 2022 Apr 16.

[2]. Safety, tolerability, and pharmacokinetics of VV116, an oral nucleoside analog against SARS-CoV-2, in Chinese healthy subjects. Acta Pharmacol Sin. 2022;1-9.

[3]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216.

[4]. Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice. bioRxiv [Preprint]. 2021 Sep 17:2021.09.13.460111.

[5]. Design and development of an oral remdesivir derivative VV116 against SARS-CoV-2. Cell Res. 2021 Sep 28;31(11):1212–1214.

Nucleoside antiviral drugs have a high genetic barrier to drug resistance because they target the highly conserved catalytic center of viral polymerase. VV116 has been shown to be effective against a variety of SARS-CoV-2 variants. Its good pharmacokinetic properties and safety make it a promising oral antiviral drug for the treatment of COVID-19. The in vivo efficacy in this study also provides strong evidence for the potential efficacy of VV116 in the treatment of RSV infection. Clinical studies of VV116 should be considered to alleviate RSV infection. [1] Mindrayvir (VV116) is a nucleoside analog prodrug used to treat COVID-19. Rifampin (RDV) was the first drug approved by the FDA for the treatment of COVID-19, and it is also a nucleoside analog. Compared with RDV, VV116 showed better in vitro antiviral activity and selectivity. In addition, VV116 can be administered orally and has good oral bioavailability, which is more convenient for COVID-19 patients than intravenous RDV. [2]
Mindexvir (VV116) is rapidly hydrolyzed to the metabolite 116-N1 after oral administration. 116-N1, not the parent drug VV116, was detected in plasma, and pharmacokinetic parameters were calculated based on this. Peak plasma concentrations of 116-N1 were rapidly reached after oral administration (median Tmax 1.00–2.50 h). In single-dose escalation studies, AUC and Cmax increased approximately dose-proportional across the dose range of 25–800 mg. However, no significant changes were observed when the dose increased from 800 mg to 1200 mg (AUC_0-t: 25886 vs. 28057 h·ng/mL; C_max: 2796 vs. 3086 ng/mL), suggesting that drug absorption may have reached saturation. Drug solubility is an important factor affecting drug absorption. When the drug reaches its maximum concentration (saturation solubility) at the absorption site, the drug absorption reaches its maximum value. It is speculated that the limited solubility of VV116 may be the reason for the drug absorption saturation. Within 0-72 hours after administration, the excretion fraction of 116-N1 in urine was 53.6%, while the excretion fraction of 116-N1 and VV116 in feces was 5.25%, indicating that VV116 is mainly excreted by the kidneys in the form of the metabolite 116-N1. [2] In the single escalation dose study, the mean half-life (t1/2) of Mindeudesivir (VV116) was 4.80–6.95 hours, suggesting that a twice-daily dosing regimen can be used for clinical treatment. Therefore, in the multiple escalation dose study, a regimen of twice-daily dosing (12 hours apart) was adopted for 5.5 days (days 1–6). The cumulative ratios of AUC and Cmax indicate that VV116 accumulates slightly after continuous administration. After multiple 200 mg doses on days 5 and 6, the trough concentration of 116-N1 was in the range of 242–345 ng/mL (Table 5), which is higher than the EC90 of 116-N1 against the omicron variant in preclinical anti-SARS-CoV-2 trials (186.5 ng/mL). Therefore, a dosing regimen of 200 mg or more twice daily can maintain effective antiviral drug concentrations and is recommended for use in subsequent clinical studies in COVID-19 patients. [2]
The median Tmax under fasting, standard meal, and high-fat meal conditions were 1.50, 3.00, and 2.50 hours, respectively, indicating that eating can prolong the time to peak concentration. Compared with fasting, the geometric mean ratio (GMR) of Cmax (90% CI) under both standard and high-fat meal conditions was in the range of 80%–125%; the GMR (90% CI) of AUC under standard meal conditions was also in the range of 80%–125%, but AUC0-t and AUC0-∞ increased slightly by 26.32% and 24.67% respectively under high-fat meal conditions. Since food intake has no effect on the Cmax of minoxidil (VV116), while a high-fat diet slightly increases AUC, it is recommended that VV116 be taken on an empty stomach or after a meal in the treatment of COVID-19. [2]
In single-dose escalation studies, no obvious dose-related trend was observed, and the proportion of subjects reporting adverse events in the placebo group (50.0%) was higher than that in the minoxidil (VV116) group (39.3%). Except for one case of grade 2 neutropenia, all adverse events were CTCAE grade 1. In multiple dose escalation studies, the incidence of adverse events in the VV116 group was comparable to that in the placebo group (51.9% vs. 55.6%). The incidence of adverse events was slightly dose-dependent. Only one subject in the 400 mg dose group reported elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST). All adverse events (AEs) in subjects treated with VV116 were grade 1 and resolved without treatment. No serious adverse events occurred during the study, and no subjects withdrew from the study due to adverse events. Preclinical animal toxicology studies have suggested that VV116 may be toxic to the eyes, thyroid, and gonads. In this study, we performed ophthalmological examinations, thyroid function tests, thyroid ultrasound examinations, and sex hormone tests on healthy subjects before and after VV116 administration. No significant toxicity was observed in these organs. Overall, VV116 demonstrated a satisfactory safety profile in healthy subjects across the three studies. [2]
Hepatotoxicity was the major adverse reaction (ADR) of RDV, manifested as elevated transaminases. In the Phase I clinical study (GS-US-399-5505 study), subjects received a loading dose of 200 mg of RDV followed by a dose of 100 mg for up to 9 days. Grade 1 or 2 transient ALT elevations were observed in 9 out of 20 subjects (45%). Elevated transaminases were also reported as the most common adverse reaction in COVID-19 patients treated with RDV. In the multiple-dose escalation study of mindiva (VV116), only 1 out of 27 subjects (3.7%) experienced a Grade 1 transient ALT elevation, which resolved spontaneously upon discontinuation of VV116. This is likely due to the high liver targeting of RDV, with a liver/blood concentration ratio approximately 21 times that of VV116. Four hours after a single intravenous injection of 10 mg/kg [14C]RDV, the liver/blood concentration ratio of RDV (calculated as 14C-GS-5734 equivalent) was 57.8; while two hours after a single oral administration of 30 mg/kg VV116, the liver/blood concentration ratio of VV116 (calculated as major metabolite 116-N1) was only 2.8. Although the risk of hepatotoxicity of VV116 is lower than that of RDV, liver function will continue to be monitored in a subsequent Phase II study of VV116 in COVID-19 patients. [2] Conclusion [2] Mindeudesivir (VV116) showed satisfactory safety and tolerability in healthy subjects. After oral administration of VV116, plasma drug concentrations of 116-N1 rapidly reached peak (median Tmax 1.00–2.50 hours). In the dose range of 25–800 mg, AUC and Cmax increased in approximately dose-proportional manner, while drug absorption saturation was likely reached at a dose of 800 mg. Standard meals had no effect on the Cmax and AUC of VV116. Effective antiviral concentrations were achieved with multiple doses, twice daily at dose levels of 200–600 mg. [2] In conclusion, the safety data and pharmacokinetic characteristics from these studies support continued investigation of VV116 in COVID-19 patients. Despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, the COVID-19 pandemic remains uncontrolled, highlighting the need to develop highly effective antiviral drugs. Breakthrough infections are becoming increasingly common in patients with weakened immunity following infection and vaccination, while treatment options remain limited. Furthermore, the emergence of SARS-CoV-2 variants and their potential to evade monoclonal antibody therapy underscore the need to develop second-generation oral antiviral drugs that target highly conserved viral proteins for rapid application in outpatient settings. In this article, we demonstrate the in vitro antiviral activity and in vivo therapeutic effects of GS-621763. GS-621763 is a highly bioavailable oral prodrug of GS-441524, the parent nucleoside analog of remdesivir, and targets a highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic characteristics observed after oral administration of GS-621763 translated into dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic doses of GS-621763 significantly reduced viral load, alleviated lung pathological damage, and improved lung function in a COVID-19 mouse model. A direct comparison of GS-621763 with monospiravir, an oral nucleoside analog antiviral drug currently undergoing human clinical trials, indicates that the two drugs have similar efficacy. These data suggest that oral remdesivir prodrug treatment significantly improves the prognosis of SARS-CoV-2 infected mice. Therefore, GS-621763 supports exploring the use of oral remdesivir prodrug GS-441524 for the treatment of human COVID-19. [4]

C24H30DN5O7

502.54

502.228

C, 57.36; H, 6.42; N, 13.94; O, 22.29

2647442-33-7

GS-621763;2647442-13-3; 2647442-33-7; 2779498-79-0 (HBr)

163358784

White to off-white solid powder

2.3

1

11

11

36

887

4

[2H]C1=C2C(=NC=NN2C(=C1)[C@]3([C@@H]([C@@H]([C@H](O3)COC(=O)C(C)C)OC(=O)C(C)C)OC(=O)C(C)C)C#N)N

RVSSLHFYCSUAHY-QXMJNOOVSA-N

InChI=1S/C24H31N5O7/c1-12(2)21(30)33-9-16-18(34-22(31)13(3)4)19(35-23(32)14(5)6)24(10-25,36-16)17-8-7-15-20(26)27-11-28-29(15)17/h7-8,11-14,16,18-19H,9H2,1-6H3,(H2,26,27,28)/t16-,18-,19-,24+/m1/s1/i7D

(2R,3R,4R,5R)-2-(4-amino-5-deuteropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate)

VV116; VV 116; VV-116; JT001; JT-001; JT 001;

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 : ~100 mg/mL ( ~198.98 mM ) Ethanol : ~100 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.)