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
111 or 55.5 mg/kg
i.m.

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In vivo toxicity study of GC376 and GS441524[3]
The 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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In vivo antiviral study of GC376 and GS441524[3]
Firstly, 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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Pharmacokinetics study of GC376 and GS441524 in BALB/c mice and SD rats[3]
Healthy 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.

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Pharmacokinetic studies of GC376 and GS441524 in cats [4] A 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.

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Healthy 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].

Pharmacokinetics of GS441524 and GC376 by different routes of administration and with different dosages [4]
To determine the oral dose of GC376 and GS441524, we tested the pharmacokinetics of the two drugs given by different administration routes (including subcutaneous, oral and intravenous injection) in healthy adult cats. The concentration-time curves of GS441524 and GC376 after each different administration route are shown in Fig. 2 A and C. The pharmacokinetic samples were assessed using LC-MS-MS to detect the concentrations. According to the results, oral GS441524 has the same area under the curve as that given subcutaneously, indicating that changing the method of administration did not affect drug absorption; thus, GS441524 can be administered orally at the doses reported in the literature. In contrast, the absorption of oral GC376 was significantly lower than when given by subcutaneous administration; thus, oral GC376 may require higher doses.
Due to the low solubility of GS441524, we hypothesized that the reduced drug solubility would affect drug absorption. To test this hypothesis, we evaluated the pharmacokinetic differences between oral GS441524 and GC376 given as a powder or in solution. The results showed that compared with liquid GS441524, GS441524 powder had significantly lower drug absorption; however, there was no significant difference in drug AUC when comparing oral and subcutaneous administration of GS441524 (Fig. 2 G and H). Conversely, compared with liquid GC376, there was no change in the drug absorption of GC376 powder; however, there was a significant difference in drug absorption when oral and subcutaneous administrations of GC376 were compared (Fig. 2E and F). In this study, the solution was used as the primary dosage form for subsequent animal tests.

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Pharmacokinetics study of GC376 and GS441524 alone or in combination [3]
To further examine the potential of GC376 and GS441524, we evaluated their pharmacokinetic (PK) properties in SPF BALB/c mice and SD rats following i.m. administration 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. In mice, the PK results showed that GC376 and GS441524 were rapidly absorbed after i.m. administration, and the peak plasma level was reached 0.22 ± 0.07 h and 0.80 ± 0.24 h after injection, respectively (Figure 5A, B and Table 1). Because the i.m. administered dose of GC376 was approximately 1.7-fold that of GS441524, we found that the maximum detected plasma drug concentration (Cmax) of GC376 (46.70 ± 10.69 μg/ml) was approximately 1.2-fold that of GS441524 (39.64 ± 2.93 μg/ml) (Table 1). However, the value of the area under the curve (AUC0−t) of GS441524 (AUC0−t = 106.82 ± 16.79) was approximately 1.9-fold that of GC376 (AUC0−t = 55.29 ± 11.26). Meanwhile, we observed that the clearance rate of GC376 (CL/F, 1985 ± 485 ml/h/kg) in plasma was approximately 3.1-fold that of GS441524 (CL/F, 639 ± 119 ml/h/kg) (Table 1). Besides, the PK results in SD rats showed that the Tmax of GC376 and GS441524 were 1.30 ± 0.60 h and 2.00 ± 1.10 h, respectively (Figure 5D, E and Table 2). Compared with mice, the utilization efficiency of GS441524 in vivo is significantly higher than that of GC376 in SD rats. We found that the maximum detected plasma drug concentration (Cmax) of GC376 (12.56 ± 1.90 μg/ml) was approximately 2.5-fold lower than GS441524 (30.96 ± 8.40 μg/ml) (Table 2). The value of the area under the curve (AUC0−t) of GS441524 (AUC0−t = 183.33 ± 64.36) was approximately 2.0-fold higher than 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-fold 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 stops the cleavage and activation of functional viral proteins required for replication and transcription in host cells. The compound is known as a direct acting antiviral (DAA) for coronaviruses, and was initially developed using structure-guided design to combat MERS-Cov infections. In addition, GC-376 displays potent activity against various coronaviruses such as feline, ferret, and mink Mpro coronavirus. A prodrug of [GC-373], GC-376 has recently been used to test inhibition of SARS-Cov-2 Mpro in vitro, and results show potent inhibition of this target. These results suggest that GC-376, and its metabolites may have therapeutic potential for Covid-19.

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FIP is a fatal feline disease caused by FIPV. Two drugs (GS441524 and GC376) target FIPV and have good therapeutic effect when administered by subcutaneous injection. However, subcutaneous injection has limitations compared with oral administration. Additionally, the oral efficacy of the two drugs has not been determined. Here, GS441524 and GC376 were shown to efficiently inhibit FIPV-rQS79 (recombination virus with a full-length field type I FIPV and the spike gene replaced with type II FIPV) and FIPV II (commercially available type II FIPV 79-1146) at a noncytotoxic concentration in CRFK cells. Moreover, the effective oral dose was determined via the in vivo pharmacokinetics of GS441524 and GC376. We conducted animal trials in three dosing groups and found that while GS441524 can effectively reducing the mortality of FIP subjects at a range of doses, GC376 only reducing the mortality rate at high doses. Additionally, compared with GC376, oral GS441524 has better absorption, slower clearance and a slower rate of metabolism. Furthermore, there was no significant difference between the oral and subcutaneous pharmacokinetic parameters. Collectively, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using a relevant animal model. We also verified the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical drug use. Furthermore, the pharmacokinetic data provide insights into and potential directions for the optimization of these drugs.[4]

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FIP caused by FIPV threatens feline health. GS441524 and GC376 have effective for FIPV by inhibiting virus replication, subcutaneous injection as administration way has limitations, oral administration has its advantages, including patient compliance, convenience, cost, and ease of storage (Shriya S. Srinivasan, 2022). Oral administration is expected to be a new method for FIP treatment. Thus, our study demonstrates the efficacy of oral GS441524 and GC376 against lethal recombinant FIPV-rQS79 in vivo and in vitro. First, we demonstrated that these two drugs can effectively inhibit two kinds of FIPV viruses in cell culture. The two drugs both had broad-spectrum antiviral effects against type FIPV-rQS79 and type FIPV II in vitro. Pharmacokinetics (PK) is closely related to pharmacodynamics. Testing pharmacokinetics can determine drug processes relevant to cats (absorption, distribution, metabolism and excretion) (Asif et al., 2005). Through a pharmacokinetic study, we found that compared with GS441524, GC376 was metabolized faster. Compared with GS441524, GC376 also had a faster plasma elimination half-life and a shorter MRT. Compared with subcutaneous injection, oral administration of GC376 significantly increased the overall clearance rate and apparent distribution volume (P < 0.0001). GC376 is a covalent peptidomimetic inhibitor, and it may be modified from a peptide inhibitor, so there are more unstable chemical bonds. Previous studies have also shown that the bisulfite adducts readily revert to the aldehyde forms in water, which are readily epimerized and form the active inhibitory stereoisomer (Vuong et al., 2021). The above two reasons suggest that the use of GC376 may lead to unsatisfactory clinical results. However, when using GS441524, there were no significant differences in these PK parameters when comparing the subcutaneous and oral routes. GS441524 exhibited favorable PK parameters, and GS441524 given by either subcutaneous injection or oral administration led to the same area under the curve (same bioavailability). This study is different from previous reports in humans and mice because oral bioavailability varies greatly in different species, with F values of 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). The results indicated that metabolic differences are one of the reasons for the differences in the effects of these drugs in vivo. These results preliminarily explain why GS441524 has better efficacy than GC376 in vivo. Solubility is one of the factors affecting drug absorption, because of the low solubility of GS441524, we suspect that preparation factors also play a role in oral absorption. The results show that compared with liquid GS441524 given orally, GS441524 powder given orally had significantly lower drug absorption; however, there was no significant difference in drug absorption when comparing oral and subcutaneous administrations of GS441525. Conversely, compared with liquid GC376 given orally, GC376 powder given orally did not alter drug absorption. Therefore, solubility affects GS441524 absorption but not GC376 absorption.

In vivo study, we found that oral GS441524 has efficacy regardless of the dose, but oral GC376 only has efficacy at the high dose (150 mg/kg). Although the two drugs have good inhibitory effects in vitro, the effects of the two drugs are significantly different in vivo.

Drugs can be introduced into the body through different routes, including enteral, parenteral, and topical routes. Each of the different routes of administration has a specific purpose, advantages and disadvantages. Fundamentally, the accessibility of the respective target site of the drugs and the effectiveness of drug treatment are both strongly dependent on the route of administration.

Among the various routes of administration, oral dosing has attracted the most attention because of its advantages, including patient compliance, convenience, cost, and ease of storage, transport and administration (Mignani et al., 2013). Although oral administration is the optimal method for small molecules, there are some limitations to its application. Compared with other routes, the mechanism of drug absorption following oral administration is more complex and influenced by many factors (for example gastrointestinal motility, gastric emptying rate and presence of food). Orally administered drugs must overcome the harsh acidic environment of the stomach and be able to be dissolved in GI fluid and remain stable among dynamic intestinal microbiota; additionally, these drugs must evade degradative enzymes that can penetrate the viscous mucus barrier and efflux pumps to achieve therapeutic bioavailability (Srinivasan et al., 2022). Overcoming these barriers is difficult. Apart from the oral route, the drug can be injected subcutaneously through the small blood vessels under the skin and into the circulatory system to exert its effect; thus, this method of administration relatively quickly achieves efficacy, but it also has a stimulating effect, and the route is painful for animals. Additionally, the production costs and quality requirements for injection solutions are high. These pathway differences lead to differences in the absorption and metabolism of drugs.

Therefore, we should choose a better route of drug administration based on various factors. At the same time, pharmacokinetics provides guidance for clinical drug use. The pharmacokinetic data can also be used to provide ideas for drug structure optimization. We can further reduce the existing disadvantages of the compound through structural modification and hopefully develop more advantageous anti-FIPV compounds. For example, the structure of GC376 was optimized by Liu et al., who found that additional modification of the benzyl group may lead to a stronger bond or even additional hydrogen bonds with Mpro. Compound NK-0163 has the advantage of a long half-life in critical tissues such as the lung, although this halogen substitution may have altered its pharmacokinetics (Liu et al, 2022). Quan et al. reported that a series of potent α-ketoamide-containing compounds, specifically Y180, had superior bioavailability in rodents and nonrodents (Quan et al., 2022).

Previous studies also have reported that GS441524 has good antiviral activity and has the potential to be given by oral administration. However, the unfavorable oral PK prevented its further development into an oral drug (Li et al., 2022). To address this issue, Wei et al. reported a series of GS441524 analogs with modifications on the base or the sugar moiety, as well as some prodrug forms, in which 3′-isobutyryl ester 5a, 5′-isobutyryl ester 5c, and isobutyryl ester 5 g hydrobromides have better oral bioavailability than GS441524 (F=71.6%, 86.6% and 98.7%, respectively)(Wei et al., 2021). The above modifications of GC376 and GS441524 can be further tested, it may be improved the therapeutic effect of GC376 and GS441524 drugs on patients with FIPV.

In conclusion, this study is the first to report that oral GS441524 and GC376 can effectively treat FIPV infection in an animal model. Our research demonstrated that oral dosing can be used to replace subcutaneous injections, although we still need to solve the problems that exist with new approaches or new methods. Overall, GS441524 and GC376 completely inhibited FIPV-rQS79 and FIPV II replication in CRFK cells. Our study also verified the effect of oral GC376 and GS441524 treatment and confirmed the oral availability of GS441524 and GC376. Through PK studies, we determined the absorption, distribution, metabolism and excretion of the two drugs in cats. Additionally, the PK results further explained the reason for the differences in efficacy between the two drugs in vivo and provided insights into and directions for drug optimization and transformation.[4]

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COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus. Source: Nat Commun. 2020; 11: 4417.

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The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice. Overall outcome of clinical symptoms and survival upon SARS-CoV-2 challenge were not improved in mice treated with GC-376 compared to controls. The treatment with GC-376 slightly improved survival from 0 to 20% in mice challenged with a high virus dose at 105 TCID50/mouse. Most notably, GC-376 treatment led to milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls in mice challenged with a low virus dose at 103 TCID50/mouse. This was particularly the case in the brain where a 5-log reduction in viral titers was observed in GC-376 treated mice compared to vehicle controls. This study supports the notion that GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection and that the K18-hACE2 mouse model is suitable to study 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.)