PEDV 3CLpro (IC50 = 1.11 μM); TGEV (IC50 = 0.15 μM); FIPV (IC50 = 0.2 μM); PTV (IC50 = 0.15 μM); MNV-1 (IC50 = 5.3 μM); 229E (IC50 = 0.15 μM); MHV (IC50 = 1.1 μM); BCV (IC50 = 0.6 μM)

GC376 and Molnupiravir exhibit additive activity against SARS-CoV-2 at 72 hours and synergistic activity against SARS-CoV-2 at 48 hours.Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro[2].

GC376, a dipeptidyl bisulfite adduct salt, it is an inhibitor of 3CLpro (3C-like protease) with potent antiviral and coronavirus activity, notably against SARS-CoV.The unprecedented coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious threat to global public health. Development of effective therapies against SARS-CoV-2 is urgently needed. Here, we evaluated the antiviral activity of a remdesivir parent nucleotide analog, GS441524, which targets the coronavirus RNA-dependent RNA polymerase enzyme, and a feline coronavirus prodrug, GC376, which targets its main protease, using a mouse-adapted SARS-CoV-2 infected mouse model. Our results showed that GS441524 effectively blocked the proliferation of SARS-CoV-2 in the mouse upper and lower respiratory tracts via combined intranasal (i.n.) and intramuscular (i.m.) treatment. However, the ability of high-dose GC376 (i.m. or i.n. and i.m.) was weaker than GS441524. Notably, low-dose combined application of GS441524 with GC376 could effectively protect mice against SARS-CoV-2 infection via i.n. or i.n. and i.m. treatment. Moreover, we found that the pharmacokinetic properties of GS441524 is better than GC376, and combined application of GC376 and GS441524 had a synergistic effect. Our findings support the further evaluation of the combined application of GC376 and GS441524 in future clinical studies[3].

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Oral GS441524 and GC376 were effective in the FIPV model [4]
Based on the GS441524 and GC376 PK analysis data, we selected various oral dosing regimens. In this phase, we evaluated the antiviral activity of different oral doses of GS441524 (5 mg/kg, 10 mg/kg and 20 mg/kg) and GC376 (15 mg/kg, 100 mg/kg and 150 mg/kg) against FIPV-rQS79 in vivo. Cats were randomly assigned to eight groups (n = 3) for oral inoculation with FIPV-rQS79. We inoculated the cats with FIPV-rQS79 at a dose of 105 TCID50. All cats inoculated with virus showed symptoms including fever and dramatic weight loss at the time of GS441524 treatment. After inoculation with the virus, the animal body weight gradually decreased, and after treatment, the body weight gradually increased, as shown in Fig. 3A. After treatment intervention, fever symptoms in the cats were significantly reduced, and their body temperatures gradually returned to the normal range, as shown in Fig. 3B. The clinical scores of the patients increased significantly after inoculation, and after GS441524 treatment, the clinical scores decreased gradually; specifically, the patients in the group treated with GS441524 had milder symptoms than the patients in the positive control group (Fig. 3C). During the observation period, the infected cats in all groups treated with GS441524 (5 mg/kg, 10 mg/kg and 20 mg/kg) had significantly improved survival rates compared with infected cats in the untreated control group (P < 0.05). GS441524 doses of 20 mg/kg and 10 mg/kg provided the greatest protection, with 100% protection. A GS441524 dosage of 5 mg/kg/day provided 66% protection (Fig. 3D). The results showed that 5 mg/kg GS441524 given orally was effective, although it did not completely protect patients with FIPV infection.

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Thirty days of GS441524 treatment at three dosages and GC376 at a dosage of 150 mg/kg PO q 24 h prevented FIP-associated mortality in the FIPV-rQS79 infection animal model [4]
Twenty cats were exposed to FIPV-rQS79, and their response to GS441524 and GC376 treatment was monitored after the disease signs appeared (Fig. 9). Approximately one week after inoculation with the virus, most cats had clinical symptoms. GS441524 and GC376 by different dose treatment 30 days, some of cats return healthy, but four of them treated by oral administration had disease recurrence at five to six weeks posttreatment, and two of them presented with severe neurological symptoms characterized by unequal pupil size and inability to control the hind legs. Except for the cats with recurrent disease, the remaining cats treated once remained normal after two months.

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Compared with oral or subcutaneous GC376, GS441524 exhibited more advantages in pharmacokinetic parameters [4]
To illustrate the differences in the effect of oral treatments with GC376 or GS441524, the pharmacokinetic parameters of GS441524 in this study are shown in Table 1, Table 2. Compared with GC376, GS441524 had a longer clearance time. When the GC376 plasma concentration after subcutaneous injection reached Cmax (18000 ηg/mL), it took approximately 8 h for levels to drop below the effective concentration. But GS441524 after subcutaneous injection was 2000 ηg/mL, and approximately 12 h after dosing, the concentration was reduced to a level below the effective concentration. Although GC376 has a higher AUC (0-∞) than GS441524, its mean residence time (MRT) is also relatively short, indicating that it is more easily metabolized than GS441524.

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses[1].

Evaluation of antiviral activity in Vero e6 cells[3]
Cell viability was determined using the Cell Titer-Glo kit following the manufacturer’s instructions. Briefly, Vero E6 cells were seeded in 96-well plates with opaque walls. After 12–16 h, the indicated concentrations of GC376 (0, 1, 5, 10, 50, 100, 500 µM), GS441524 (0, 1, 5, 10, 50, 100, 500 µM) and GC376 + GS441524 (0, 0.5, 2.5, 5, 25, 50, 250 µM) were added for 24 h. Cell Titer-Glo reagent was added to each well, and luminescence was measured using a GloMax 96 Microplate Luminometer.

Antiviral activity experiment was determined following a previous method. Briefly, Vero E6 cells were pretreated with the indicated concentrations of GC376 (0, 0.5, 1, 2, 4, 6, 8, 10 µM), GS441524 (0, 0.5, 1, 2, 4, 6, 8, 10 µM) and GC376 + GS441524 (0, 0.25, 0.5, 1, 2, 3, 4, 5 µM) or with vehicle solution (12% sulfobutylether-β-cyclodextrin, pH 3.5) alone for 1 h. The cells were then infected with HRB26 or HRB26M at an MOI of 0.005 and incubated for 1 h at 37°C. The cells were washed with PBS, and virus growth medium containing the indicated amounts of GC376, GS441524 and GC376 + GS441524 or vehicle solution alone was added. The supernatants were collected at 24 h p.i. for viral titration by a PFU assay in Vero E6 cells. Relative viral titres were calculated on the basis of the ratios to the viral titres in the mock-treated counterparts. The data were analyzed using GraphPad Prism 7.0. The results are shown as the mean values with standard deviations of three independent experiments.

In 96-well clear flat-bottom plates, Vero E6 cells (3000 cells/well) were seeded and incubated for 24 hours at 37°C with 5% CO₂. Following incubation, a multiplicity of infection (MOI) of 0.1 was used to infect cells. Adsorption of SARS-CoV-2 was allowed to occur for one hour at 37°C. After the virus inoculum was removed, the cells were covered with media that contained molnupiravir (0.62–50 μM), nirmatrelvir (0.62–50 μM), and GC376 (0.21–16.7 μM) diluted three times. Every plate contained mock-infected cells, infected positive controls (SARS-CoV-2 alone), and negative controls (compounds alone). Following 48 and 72 hours of incubation at 37°C with 5% CO₂, the viability of the cells was assessed using the MTT reduction assay.

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Cytotoxicity of GS441524 and GC376 in CRFK cells[4]
CRFK cells were seeded into a 96-well plate and grown in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS). When the cells had formed monolayers, the medium was replaced by 2% FBS and different concentrations of GC376 (0.3125 µM, 0.625 µM, 1.25 µM and 2.5 µM) or GS441524 (0.3125 µM, 0.625 µM, 1.25 µM and 2.5 µM). DMEM containing 0.4% DMSO was used as the blank control. The cells were incubated for 48 h at 37 °C under an atmosphere containing 5% CO2 and then washed twice with phosphate buffered saline (PBS). FBS-free MEM (100 µL) and CCK-8 (10 µL) were then added, and the cells were incubated at 37 °C for 1–4 h. A FLUOstar Omega was used to read the optical density (OD) at 450 nm. Cell viability was calculated using the following equation:

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Cell viability = [OD (compound) - OD (blank)] / [OD (control) - OD(blank)] × 100%·

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The EC50 values were calculated using GraphPad Prism software version 8.0.2.

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The antiviral effects of GC376 and GS441524 against FIPV-rQS79 and FIPV II in vitro, (0.01 MOI) were assessed; different concentrations of GC376 or GS441524 were added to 96-well plates containing monolayers of CRFK cells, and the cells were incubated at 37 °C under an atmosphere containing 5% CO2 for 28 h. Each drug concentration was tested in five replicate wells, and 0.4% DMSO was used as the blank control. EC50 values were determined by CCK-8 assay.

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Indirect immunofluorescence assay (IFA)[4]
Immunofluorescence analysis of FIPV N protein expression during FIPV infection and in GS441524- and GC376-treated CRFK cells was performed by seeding cells on glass cover slips and allowing growth until they reached 50% membrane fusion. Briefly, CRFK cell monolayers on cover slips were inoculated with FIPV II or FIPV-rQS79 (MOI =0.01) for 24 h at 37 °C. After washing with PBS, we fixed CRFK cells in 4% paraformaldehyde for 20 min, followed by permeabilization in 0.3% Triton-X-100 for 30 min at room temperature and blocking with 5% BSA for 30 min at 37 °C. After washing with PBS, we incubated the cells with anti-FIPV N monoclonal antibody overnight at 4 °C. After washing with PBS Tween-20 (PBST) 3 times, we then incubated the cells with goat anti-rabbit 488 (1:1000) for 1.5 h at 37 °C. We followed this incubation with another incubation for 15 min with DAPI (1:1000) at room temperature. The triple-stained cells were then washed three times with PBST, and we captured images for analysis under high magnification.

Female BALB/c mice
\n111 or 55.5 mg/kg
\ni.m.

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\nIn vivo toxicity study of GC376 and GS441524[3]
\nThe toxicity studies were performed in 4- to 6-week-old female BALB/c mice. BALB/c mice were assigned to four groups (five mice per group), one mock group (i.m. administration of solvent) and three i.m. administered groups: GC376 (40 mM/l, 100 µl), GS441524 (40 mM/l, 100 µl) and GC376 + GS441524 (20 mM/l, 100 µl), respectively. Mice in the mock and experimental groups were weighed daily for 15 days. In addition, blood samples were collected at 0, 5, 10 and 15 days after administration. Various blood chemistry values or blood cell counts were performed at Wuhan Servicebio Biological Technology Co., Ltd. The data were analyzed using GraphPad Prism 7.0.

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\n\nIn vivo antiviral study of GC376 and GS441524[3]
\nFirstly, groups of six 4- to 6-week-old female BALB/c mice were treated i.m. with a loading dose of GC376 (40 or 8 mM/l, 100 µl), GS441524 (40 or 8 mM/l, 100 µl) and GC376 + GS441524 (20 or 4 mM/l, 100 µl), followed by a daily maintenance dose. Alternatively, mice were treated intranasally with a single treatment (GC376, 20 mM/l, 50 µl; GS441524, 20 mM/l, 50 µl; GC376 + GS441524, 10 mM/l, 50 µl) or a combination of GC376 (20 mM/l, 50 µl, i.n. and 40 mM/l, 100 µl, i.m.), GS441524 (20 mM/l, 50 µl, i.n. and 40 mM/l, 100 µl, i.m.) and GC376 + GS441524 (10 mM/l, 50 µl, i.n. and 20 mM/l, 100 µl, i.m.), followed by a daily maintenance dose. As a control, mice were administered vehicle solution (12% sulfobutylether-β-cyclodextrin, pH 3.5) daily. One hour after administration of the loading dose of GC376, GS441524 and GC376 + GS441524 or vehicle solution, each mouse was inoculated intranasally with103.6 PFU of HRB26M in 50 μl. Three mice from each group were euthanized on days 3 and 5 p.i. The nasal turbinates and lungs were collected for viral detection by qPCR and PFU assay according to previously described methods]. The amount of vRNA for the target SARS-CoV-2 N gene was normalized to the standard curve from a plasmid containing the full-length cDNA of the SARS-CoV-2 N gene. The assay sensitivity was 1000 copies/ml. The data were analyzed using Microsoft Excel 2016 and GraphPad Prism 7.0.

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\n\nPharmacokinetics study of GC376 and GS441524 in BALB/c mice and SD rats[3]
\nHealthy SPF BALB/c mice (7-8 weeks) and SD rats (4-6 weeks) were used in a single-dose PK study. At time point zero, the BALB/c mice and SD rats of groups A, B and C (each group including twenty BABL/c mice or five SD rats) received i.m. injections of GC376 (111 mg/kg), GS441524 (67 mg/kg) and GC376 + GS441524 (55.5 + 33.5 mg/kg), which are the same doses according to in vivo antiviral study. The blood was collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h and placed in a precooled polypropylene centrifuge tube containing 3.0 µl of 40% EDTAK2. Then, the whole blood was centrifuged at 7800 g/min for 10 min at 4°C. Plasma was collected and stored in a freezer at −80°C. Plasma drug concentration was analyzed using LC-MS/MS. Pharmacokinetic parameters were calculated using WinNonlin software (version 6.4), and a non-atrioventricular model was used for data fitting. The data were analyzed using Microsoft Excel 2016 and GraphPad Prism 7.0.\n

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\n\nPharmacokinetic studies of GC376 and GS441524 in cats [4]\nA pharmacokinetic (PK) study was performed in laboratory cats to determine the efficacy of oral GS441524 and GC376. GS441524 was dissolved at a concentration of 12 mg/mL in 5% ethanol, 30% propylene glycol, 45% PEG 40%, and 20% water and adjusted to pH 1.9 with concentrated HCl. All animals were randomly divided into the following three groups: A (n ≧ 3; IV administration), B (n ≧ 3; SC administration), and C (n ≧ 3; PO oral administration). At time point zero, Group A cats were administered 5 mg compound/kg body weight intravenously, while Group B cats received 5 mg compound/kg subcutaneously. Serial 0.5 mL whole blood samples in EDTA were obtained from the radial vein of the forelimb from each cat at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h. After collection, blood samples were immediately placed on ice and centrifuged at 5000 rpm for 5 min. The isolated plasma was pipetted into a 1.5 mL microcentrifuge tube and frozen at − 80 °C for further analysis of free GS441524. All samples were assessed using LCsingle bondMS-MS to detect the concentrations.\n

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\nHealthy cats (1–3 years old, 2.0–4.5 kg) were randomly assigned to eight groups (four animals per group) once they had been confirmed to be virus-free by a neutralizing antibody test. Meanwhile, we also measured their liver and kidney functions, and the test results were normal, ensuring that the test cats had the ability to absorb and metabolize drugs normally. The effect of GC376 or GS441524 oral administration was investigated first in FIP. After viral infection, animals with FIPV-rQS79 in the treatment groups received oral doses of GS441524 (20 mg/kg, 10 mg/kg or 5 mg/kg) or GC376 (150 mg/kg, 100 mg/kg or 15 mg/kg) in PBS (500 µL). The control group received the same volume of PBS. On day 0, the cats were infected by oral administration of FIPV-rQS79 (105 × TCID50) in DMEM (1000 µL). The therapeutic effect of GC376 and GS441524 after the onset of infection was examined next. Clinical signs and survival rates of the animals were monitored as described previously, and cat health was assessed every day [4].

Pharmacokinetic Study of GS441524 and GC376 under Different Routes of Administration and Doses [4]
To determine the oral doses of GC376 and GS441524, we tested the pharmacokinetics of healthy adult cats after administration via different routes of administration (including subcutaneous, oral, and intravenous). Figures 2A and 2C show the concentration-time curves of GS441524 and GC376 after different routes of administration, respectively. The drug concentrations in the pharmacokinetic samples were detected by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that the area under the curve for oral administration of GS441524 was the same as that for subcutaneous administration, indicating that changing the route of administration does not affect drug absorption; therefore, GS441524 can be administered orally at the doses reported in the literature. In contrast, the absorption rate of oral GC376 was significantly lower than that of subcutaneous administration; therefore, oral GC376 may require a higher dose.
Due to the low solubility of GS441524, we hypothesized that the decrease in drug solubility would affect drug absorption. To test this hypothesis, we evaluated the pharmacokinetic differences between orally administered GS441524 and GC376 administered in powder or solution form. The results showed that the absorption rate of GS441524 powder was significantly lower than that of liquid GS441524; however, there was no significant difference in the drug AUC between oral and subcutaneous administration of GS441524 (Figure 2 G and H). Conversely, the absorption rate of GC376 powder did not change compared to liquid GC376; however, a significant difference was found between oral and subcutaneous administration of GC376 (Figure 2 E and F). In this study, the solution formulation was used as the primary formulation for subsequent animal studies.

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Pharmacokinetic Study of GC376 and GS441524 alone or in combination [3]
To further investigate the potential of GC376 and GS441524, we evaluated their pharmacokinetic (PK) characteristics in SPF BALB/c mice and SD rats after intramuscular injection of GC376 (111 mg/kg), GS441524 (67 mg/kg), and GC376 + GS441524 (55.5 + 33.5 mg/kg), the same doses as in the in vivo antiviral studies. In mice, pharmacokinetic results showed that both GC376 and GS441524 were rapidly absorbed after intramuscular injection, with peak plasma concentrations reached at 0.22 ± 0.07 h and 0.80 ± 0.24 h, respectively (Figures 5A, B and Table 1). Since the intramuscular dose of GC376 was approximately 1.7 times that of GS441524, we found that the maximum plasma drug concentration (Cmax) of GC376 (46.70 ± 10.69 μg/ml) was approximately 1.2 times that of GS441524 (39.64 ± 2.93 μg/ml) (Table 1). However, the area under the curve (AUC0−t) of GS441524 (AUC0−t = 106.82 ± 16.79) was approximately 1.9 times that of GC376 (AUC0−t = 55.29 ± 11.26). Simultaneously, we observed that the plasma clearance rate of GC376 (CL/F, 1985 ± 485 ml/h/kg) was approximately 3.1 times that of GS441524 (CL/F, 639 ± 119 ml/h/kg) (Table 1). Furthermore, pharmacokinetic results in SD rats showed that the time to peak concentration (Tmax) of GC376 and GS441524 were 1.30 ± 0.60 h and 2.00 ± 1.10 h, respectively (Figures 5D, E and Table 2). Compared with mice, the utilization efficiency of GS441524 in SD rats was significantly higher than that of GC376. We found that the maximum plasma drug concentration (Cmax) of GC376 (12.56 ± 1.90 μg/ml) was approximately 2.5 times that of GS441524 (30.96 ± 8.40 μg/ml) (Table 2). The area under the curve (AUC0−t) of GS441524 (AUC0−t = 183.33 ± 64.36) was approximately 2.0 times that of GC376 (AUC0−t = 92.14 ± 9.99). We also observed that the clearance rate of GC376 (CL/F, 1208 ± 122 ml/h/kg) in plasma was approximately 2.9 times that of GS441524 (CL/F, 423 ± 186 ml/h/kg).

[1]. Viruses. 2020 Feb 21;12(2):240.

[2]. Microorganisms. 2022 Jul 21;10(7):1475.

[3]. Emerg Microbes Infect. 2021 Dec;10(1):481-492.

[4]. Vet Microbiol. 2023 Aug:283:109781.

GC-376 is a 3C-like protease (3CLpro, or Mpro) inhibitor that prevents the cleavage and activation of functional viral proteins required for viral replication and transcription in host cells. This compound is a known direct-acting antiviral (DAA) for coronaviruses, originally developed using structure-guided design to combat Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. Furthermore, GC-376 has also shown potent activity against the Mpro of several coronaviruses, such as feline coronavirus, ferret coronavirus, and mink coronavirus. As a prodrug of [GC-373], GC-376 has recently been used in vitro to test its inhibitory effect on SARS-CoV-2 Mpro, showing potent inhibition of this target. These results suggest that GC-376 and its metabolites may have the potential to treat COVID-19. Feline infectious peritonitis (FIP) is a fatal feline disease caused by feline infectious peritonitis virus (FIPV). Two drugs (GS441524 and GC376) targeting feline panleukopenia virus (FIPV) showed good therapeutic efficacy when administered subcutaneously. However, subcutaneous administration has limitations compared to oral administration. Furthermore, the oral efficacy of these two drugs remains undetermined. This study demonstrates that GS441524 and GC376 effectively inhibited FIPV-rQS79 (a recombinant virus whose spike protein gene is composed of a full-length type I FIPV strain, replaced by a type II FIPV strain) and FIPV II (commercially available type II FIPV strain 79-1146) at non-cytotoxic concentrations in CRFK cells. In addition, the effective oral doses of GS441524 and GC376 were determined through in vivo pharmacokinetic studies. We conducted three dose-dependent animal studies and found that GS441524 effectively reduced mortality in the FIP model across a range of doses, while GC376 only reduced mortality at high doses. In addition, compared with GC376, oral GS441524 was better absorbed, cleared more slowly, and metabolized more slowly. Moreover, there was no significant difference in pharmacokinetic parameters between oral and subcutaneous administration. In summary, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using relevant animal models. We also validated the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical use. In addition, the pharmacokinetic data provide insights and potential directions for the optimization of these drugs. [4]

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FIP caused by FIPV threatens the health of felines. GS441524 and GC376 are effective against feline infectious peritonitis virus (FIPV) by inhibiting viral replication, but subcutaneous administration has limitations, while oral administration has advantages such as good patient compliance, convenience, low cost, and easy storage (Shriya S. Srinivasan, 2022). Oral administration is expected to become a new method for the treatment of feline infectious peritonitis (FIP). Therefore, this study validated the efficacy of oral GS441524 and GC376 against lethal recombinant FIPV-rQS79 in vitro and in vivo. First, we demonstrated that both drugs effectively inhibited both FIPV viruses in cell culture. In vitro experiments showed that both drugs had broad-spectrum antiviral activity against FIPV-rQS79 and FIPV II viruses. Pharmacokinetics (PK) is closely related to pharmacodynamics. Pharmacokinetic tests can determine the cat-related drug processes (absorption, distribution, metabolism, and excretion) (Asif et al., 2005). Through pharmacokinetic studies, we found that GC376 was metabolized more rapidly than GS441524. GC376 also had a shorter plasma elimination half-life and shorter mean residence time (MRT) compared to GS441524. Oral administration of GC376 significantly improved total clearance and apparent volume of distribution compared to subcutaneous injection (P < 0.0001). GC376 is a covalent peptide mimic inhibitor, which may be derived from peptide inhibitors and therefore has more unstable chemical bonds. Previous studies have also shown that bisulfite adducts readily convert to aldehydes in water, and the aldehyde form readily undergoes epimerization to form an inhibitory stereoisomer (Vuong et al., 2021). These two reasons suggest that the use of GC376 may lead to undesirable clinical outcomes. However, when using GS441524, there were no significant differences in pharmacokinetic parameters between subcutaneous and oral administration. GS441524 exhibited favorable pharmacokinetic parameters, with the same area under the curve (AUC) (same bioavailability) for subcutaneous or oral administration. This study differs from previous reports in humans and mice because oral bioavailability varies significantly across species: the F-value was 33% in rats, 85% in dogs, and 8.3% in cynomolgus monkeys (Davis et al., 2021; Humeniuk et al., 2020; Li et al., 2022; Wei et al., 2021; Xie and Wang, 2021). These results suggest that metabolic differences are one reason for the differences in the in vivo effects of these drugs. These results preliminarily explain why GS441524 is more effective than GC376 in vivo. Solubility is one of the factors affecting drug absorption. Given the low solubility of GS441524, we hypothesized that formulation factors also affect its oral absorption. The results showed that the absorption of GS441524 powder was significantly reduced when administered orally compared to liquid GS441524; however, there was no significant difference in drug absorption between oral and subcutaneous administration routes. Conversely, oral GC376 powder did not alter drug absorption compared to oral liquid GC376. Therefore, solubility affects the absorption of GS441524 but not GC376. In vivo studies found that oral GS441524 was effective regardless of dose, while oral GC376 was only effective at high doses (150 mg/kg). Although both drugs exhibit good inhibitory activity in vitro, their effects in vivo differ significantly. Drugs can enter the body via multiple routes, including enteral, parenteral, and local administration. Each route has its specific purpose, advantages, and disadvantages. Fundamentally, the accessibility of drugs to their targets and the effectiveness of therapeutic treatment strongly depend on the route of administration. Among various routes of administration, oral administration has attracted considerable attention due to its numerous advantages, including good patient compliance, convenience, low cost, and ease of storage, transportation, and administration (Mignani et al., 2013). Although oral administration is the optimal route of administration for small molecule drugs, its application still has some limitations. Compared to other routes of administration, oral drug absorption mechanisms are more complex and influenced by multiple factors (e.g., gastrointestinal motility, gastric emptying rate, and the presence of food). Orally administered drugs must overcome the highly acidic environment of the stomach, dissolve in gastric juice, and remain stable within the dynamic gut microbiota; furthermore, these drugs must avoid degrading enzymes that can penetrate the viscous mucus barrier and efflux pumps to achieve the required therapeutic bioavailability (Srinivasan et al., 2022). Overcoming these obstacles is no easy task. Besides oral administration, drugs can also exert their effects through subcutaneous injection into the circulatory system; this method of administration has a relatively rapid onset of action but is also irritating and painful for animals. In addition, the production cost and quality requirements for injection solutions are higher. These differences in routes lead to differences in drug absorption and metabolism. Therefore, we should choose a better route of administration based on multiple factors. Meanwhile, pharmacokinetic data provides guidance for clinical drug use. Pharmacokinetic data can also provide insights for drug structure optimization. We can further reduce the existing shortcomings of compounds through structural modification and hopefully develop more advantageous anti-FIPV compounds. For example, Liu et al. optimized the structure of GC376 and found that further modification of the benzyl group could lead to stronger bonds with Mpro, even additional hydrogen bonds. The compound NK-0163 has the advantage of a long half-life in important tissues such as the lungs, although this halogen substitution may alter its pharmacokinetics (Liu et al., 2022). Quan et al. A series of potent α-ketoamide compounds, particularly Y180, have been reported to have excellent bioavailability in both rodents and non-rodents (Quan et al., 2022). Previous studies have also reported that GS441524 has good antiviral activity and potential for oral administration. However, its unfavorable oral pharmacokinetics hinders its further development as an oral drug (Li et al., 2022). To address this issue, Wei et al. reported that a series of GS441524 analogues (with modifications to the base or glycosyl groups) and several prodrug forms, including 3′-isobutyryl ester 5a, 5′-isobutyryl ester 5c, and isobutyryl ester 5g hydrobromide, exhibited superior oral bioavailability compared to GS441524 (71.6%, 86.6%, and 98.7%, respectively) (Wei et al., 2021). Further research is warranted on the modifications to GC376 and GS441524, which may improve their therapeutic efficacy in FIPV patients. In summary, this study is the first to report the effectiveness of oral GS441524 and GC376 in treating FIPV infection in an animal model. Our research suggests that oral administration can replace subcutaneous injection, although new methods or pathways are still needed to address existing challenges. Overall, GS441524 and GC376 completely inhibited the replication of FIPV-rQS79 and FIPV II in CRFK cells. Our study also validated the efficacy of oral administration of GC376 and GS441524 and confirmed the oral bioavailability of GS441524 and GC376. Through pharmacokinetic (PK) studies, we determined the absorption, distribution, metabolism and excretion of the two drugs in cats. In addition, the PK results further explained the reasons for the difference in efficacy between the two drugs in vivo and provided ideas and directions for drug optimization and transformation. [4] The World Health Organization declared COVID-19 a global pandemic on March 11 because it posed a huge threat to global public health. The coronavirus main protease (Mpro, also known as 3CLpro) is crucial for the processing and maturation of viral polyproteins and is therefore considered a highly attractive drug target. This study demonstrates that both the clinically approved anti-hepatitis C virus (HCV) drug Boceprevir and the preclinical inhibitor of feline infectious peritonitis virus (FIPV), GC376, effectively inhibit SARS-CoV-2 virus in Vero cells by targeting Mpro. Furthermore, the combined use of GC376 with remdesivir (a nucleotide analog that inhibits viral RNA-dependent RNA polymerase (RdRp)) produces a significant additive inhibitory effect. Further structural analysis reveals that both inhibitors bind to the catalytic active site of the SARS-CoV-2 protease Mpro, which is their primary inhibitory mechanism. Our findings may provide crucial information for optimizing and designing more effective SARS-CoV-2 virus inhibitors. Source: Nat Commun. 2020; 11: 4417.

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The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most significant global public health emergency of this century. GC376 is an Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. This study evaluated the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 using a K18-hACE2 mouse model. GC-376 treatment was non-toxic to K18-hACE2 mice. Compared to the control group, mice in the GC-376 treatment group did not show overall improvement in clinical symptoms and survival rate after SARS-CoV-2 challenge. After challenge with a high viral dose of 10⁵ TCID₅₀/mouse, GC-376 treatment slightly increased the survival rate from 0% to 20%. Most notably, compared to the vector control group, mice in the GC-376 treatment group showed less tissue damage, lower viral load, lower viral antigen content, and milder inflammatory response after challenge with a low viral dose of 10³ TCID₅₀/mouse. Particularly in brain tissue, the viral titer in the GC-376 treatment group was reduced by five orders of magnitude compared to the vector control group. This study supports the following: GC-376 is a promising lead compound worthy of further development for the treatment of SARS-CoV-2 infection; and the K18-hACE2 mouse model is suitable for investigating antiviral therapies against SARS-CoV-2. Source: Sci Rep. 2021; 11: 9609.

C21H30N3NAO8S

507.5330

507.165

C, 49.70; H, 5.96; N, 8.28; Na, 4.53; O, 25.22; S, 6.32

1416992-39-6

1416992-39-6 (sodium);1417031-79-8 (free acid);

71481119

Off-white to yellow solid powder

4

8

12

34

783

2

CC(C)C[C@@H](C(=O)N[C@@H](CC1CCNC1=O)C(O)S(=O)(=O)[O-])NC(=O)OCC2=CC=CC=C2.[Na+]

BSPJDKCMFIPBAW-JPBGFCRCSA-M

InChI=1S/C21H31N3O8S.Na/c1-13(2)10-16(24-21(28)32-12-14-6-4-3-5-7-14)19(26)23-17(20(27)33(29,30)31)11-15-8-9-22-18(15)25;/h3-7,13,15-17,20,27H,8-12H2,1-2H3,(H,22,25)(H,23,26)(H,24,28)(H,29,30,31);/q;+1/p-1/t15?,16-,17-,20?;/m0./s1

sodium;(2S)-1-hydroxy-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate

GC376 sodium; GC-376; GC 376; GC-376 sodium; GC376; GC376 sodium; H1NMJ5XDG5; (betaS)-alpha-Hydroxy-beta-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidinepropanesulfonic acid sodium salt; Sodium (2S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate; GC-376; sodium;(2S)-1-hydroxy-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate; GC 376 sodium

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 (~197.0 mM)
Ethanol: ~100 mg/mL (~197.0 mM)

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.)