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 drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 (single ascending-dose study, SAD), Study 2 (multiple ascending-dose study, MAD), and Study 3 (food-effect study, FE). A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25-800 mg. T1/2 was within 4.80-6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.[2]

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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]

Safety [2]
No deaths, serious adverse event (SAE), AEs of Grade 3 or above, or AEs leading to drug discontinuation/interruption were reported throughout the three studies. All AEs were recovered without any treatment or intervention.

Study 1: single ascending-dose study
The number (incidence) of subjects experiencing AEs for 25, 200, 400, 800, and 1200 mg dose group and placebo group was 2 (50%), 3 (50%), 3 (50%), 3 (50%), 0 (0), and 5 (50%), respectively (Table 7). No relation with dose was observed for the AE occurrence. The incidence of AE for subjects administered Mindeudesivir (VV116) in total was lower than those administered placebo (39.3% vs. 50%) in a single ascending-dose study. The severity of AEs was CTCAE Grade 1 with the exception of one case of Grade 2 neutropenia. The dose-escalation termination criteria were not met during dose escalation. The most common drug-related AEs were sinus bradycardia, shortened electrocardiogram PR, and increased blood bilirubin.

Study 2: multiple ascending-dose study
The number (incidence) of subjects experiencing AEs for 200 mg, 400 mg, 600 mg dose group and placebo group was 3 (33.3%), 5 (55.6%), 6 (66.7%), and 5 (55.6%), respectively (Table 8). The incidence of AE for subjects administered Mindeudesivir (VV116) in total was comparable with those administered placeboes (51.9% vs. 55.6%). AE occurrence was detected to be related to dose. Besides one subject in the placebo group experienced three cases of Grade 2 nausea, the severity of AEs was generally mild (CTCAE Grade 1). The most common drug-related AEs were increased blood uric acid, dry mouth, presence of crystal urine, and nausea. Three cases had increased transaminases (increased alanine aminotransferase, increased aspartate aminotransferase, and increased gamma-glutamyltransferase) with Grade 1 observed in two subjects of 400 mg dose group. Transaminase increase was transient, and recovered spontaneously.

Study 3: food-effect study
The number (incidence) of subjects experiencing AEs under fasting condition, fed condition with a standard meal, fed condition with a high-fat meal was 0 (0), 2 (16.7%), and 4 (33.3%). Two subjects under fed condition with a standard meal were observed atrioventricular block with first degree, while four subjects under the fed condition with a high-fat meal experienced positive results in urine bacterial test, presence of crystal urine, increase in blood pressure, and atrioventricular block with first degree. All AEs were CTCAE Grade 1 in severity.

Other safety assessments
Only one subject in 400 mg dose group of Study 3 experienced mild increase in transient blood thyroid-stimulating hormone, which recovered spontaneously without any treatment. No clinically significant abnormality was discovered in sex hormone test, ophthalmological examination, and thyroid B ultrasound test.

[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 agents have a high genetic barrier to resistance since they target the highly conserved catalytic center of viral polymerase, and VV116 has been found to be effective against different SARS-CoV-2 variants. The favorable PK properties and good safety profile make it to be a very promising oral antiviral for treating COVID-19. Herein, the in vivo efficacy study also provided strong evidence for potential therapeutic usage of VV116 against RSV infection. The clinical studies of VV116 should be considered to mitigate RSV infection.[1]

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Mindeudesivir (VV116) is a prodrug of nucleoside analog, intended for the treatment of COVID-19. RDV is the first FDA-approved drug for the treatment of COVID-19, which is also a nucleoside analog. Compared with RDV, VV116 exhibits better in vitro antiviral activity and selectivity. In addition, VV116 could be administered orally and has favorable oral bioavailability, that is more convenient for COVID-19 patients than intravenous administration of RDV. [2]

Mindeudesivir (VV116) was hydrolyzed rapidly to its metabolite 116-N1 after oral administration. 116-N1, instead of the prototype drug VV116, was detected in plasma, and calculated for the PK parameters. Peak plasma 116-N1 concentration reached quickly after oral administration (median Tmax 1.00–2.50 h). In the single ascending-dose study, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg. However, the parameters did not show significant change with dose escalation from 800 to 1200 mg (AUC0-t: 25886 vs. 28057 h·ng/mL; Cmax: 2796 vs. 3086 ng/mL), indicating the probability of drug absorption saturation. Drug solubility is an important factor affecting the drug absorption and maximum drug absorption occurs when the drug has maximum concentration (saturation solubility) at the site of absorption. It was suspected that limited solubility of VV116 might be the reason for drug absorption saturation. The fractional excretion of 116-N1 in urine was 53.6% in 0–72 h after administration, while that of 116-N1 and VV116 in feces was 5.25%, which indicated that VV116 was principally excreted through kidney in the form of metabolite 116-N1. [2]

The mean t1/2 of Mindeudesivir (VV116) was 4.80–6.95 h in the single ascending-dose study, suggesting twice-daily dosing in the clinical treatment. Thereby, continuous twice-daily dosing (12 h apart) for 5.5 days (days 1–6) was adopted in the multiple ascending-dose study. The accumulation ratio of AUC parameters and Cmax indicated a slight accumulation of VV116 after continuous dosing. The trough concentrations of 116-N1 following multiple administration of 200 mg at day 5 and day 6 were within 242–345 ng/mL (Table 5), which were above the EC90 (186.5 ng/mL) of 116-N1 against the omicron variant in a preclinical anti-SARS-CoV-2 assay. Therefore, the dosage regimen of 200 mg BID and above can continuously maintain the effective antiviral concentration, and is recommended for subsequent clinical studies in patients with COVID-19. [2]

The median Tmax under fasting, standard meal and high-fat meal condition was 1.50, 3.00, and 2.50 h, respectively, indicating that fed condition could prolong the time to the peak. Compared with fasting condition, the GMR (90% CIs) of Cmax under fed condition with both standard meal and high-fat meal was within the equivalent range 80%–125%; the GMR (90% CIs) of AUC for standard meal was also within the range 80%–125%, however for high-fat meal, AUC0-t and AUC0-∞ slightly increased by 26.32% and 24.67%, respectively. Since food intake has no effect on Cmax of Mindeudesivir (VV116), while high-fat meal slightly increases AUC, it is recommended that VV116 could be taken under fasting condition or fed condition with regular meal in the treatment of COVID-19. [2]

In the single ascending-dose study, there was no apparent dose-related trend, with a greater proportion of subjects reporting AEs following administration of placebo (50.0%) than following administration of Mindeudesivir (VV116) (39.3%). The severity of AEs was CTCAE Grade 1 with the exception for one case of Grade 2 neutropenia. In the multiple ascending-dose study, the incidence of AEs in the VV116 group was comparable with that in the placebo group (51.9% vs. 55.6%). AE occurrence was slightly dose-related. Only 1 subject in 400 mg dose group reported one case of increased alanine aminotransferase and increased aspartate aminotransferase, respectively. All AEs in subjects administered VV116 were Grade 1 in severity, and were recovered without any treatment. No serious adverse event occurred throughout the study, and no subject withdrew from the study due to AE. In the preclinical animal toxicology study, it was discovered that VV116 might have toxicity on eyes, thyroid, and gonads. In our studies, ophthalmology examination, thyroid function, thyroid B ultrasound, and sex hormone tests were performed on healthy subjects before and after VV116 administration. No obvious toxicity was observed in the above organs. Overall, VV116 demonstrated satisfactory safety profiles in healthy subjects throughout the three studies. [2]

Hepatotoxicity is the primary adverse drug reaction (ADR) of RDV, manifested as transaminase elevation. In phase I clinical study (Study GS-US-399-5505), subjects received one loading dose of 200 mg RDV followed by 100 mg for up to 9 days, transient ALT elevation of Grade 1 or 2 was observed in 9 of 20 subjects (45%). Transaminase elevation has also been reported as the most frequent ADR in patients with COVID-19 who received RDV. In the multiple ascending-dose study of Mindeudesivir (VV116), only 1 of 27 subjects (3.7%) experienced transient ALT elevation of Grade 1, which recovered spontaneously after VV116 termination. This can be explained by the high targeting capability of RDV to the liver and its liver/blood concentration ratio is about 21 times that of VV116. The liver/blood concentration ratio of RDV (calculated by equivalents 14C-GS-5734) after a single intravenous administration of 10 mg/kg [14C]RDV at 4 h was 57.8, while the ratio of the VV116 (calculated by major metabolite 116-N1) after a single oral dose of 30 mg/kg VV116 to rat at 2 h was only 2.8. Despite the lower risk of hepatotoxicity of VV116 compared to RDV, monitoring for the hepatic function will continue in the subsequent phase II study of VV116 in COVID-19 patients. [2]

Conclusions [2]
Mindeudesivir (VV116) exhibited satisfactory safety and tolerability in healthy subjects. Peak plasma drug concentration of 116-N1 reached quickly after oral administration of VV116 (median Tmax 1.00–2.50 h). AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg, while drug absorption saturation was probably achieved at the dose of 800 mg. Standard meal had no effect on Cmax and AUC of VV116. Effective antiviral concentration was achieved at dose levels between 200 and 600 mg BID following multiple administration. [2]
In conclusion, the safety data and PK profile from these studies support the continued investigation of VV116 in patients with COVID-19.

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The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the 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 profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans. [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.)