FIPV ( EC50 = 0.78 μM ); RNA-dependent RNA polymerase (RdRp)

In vitro activity: The cells exhibit normal growth and appearance across all GS-441524 concentrations; however, they are unable to absorb the fluorescent dye CellTox Green after 24 hours. Hence, the cytotoxic concentration at 50% (CC50) is greater than 100 μM. It is calculated that GS-441524 has an effective concentration of 50% (EC50) of 0.78 μM[1].

---

Combined application of GC376 and GS441524 has an enhanced ability to inhibit SARS-CoV-2 in Vero E6 cells [5]
We evaluated the inhibitory efficacy of GC376, GS441524 and the combined application of GC376 and GS441524 (molar ratio: 1:1) (GC376 + GS441524) on the replication of live virus (SARS-CoV-2: HRB26 and HRB26M) in Vero E6 cells. We first tested the cellular cytotoxicity of these compounds in vitro. GC376 and GS441524 did not produce obvious cytotoxicity at concentrations up to 250 μM in Vero E6 cells (CC50 > 250 μM; Supplementary Figure 3). Our results showed that GC376 and GS441524 were efficacious against HRB26, with 50% inhibitory concentration (EC50) values of 0.643 ± 0.085 μM and 5.188 ± 2.476 μM, respectively (Figure 2A, B). GC376 and GS441524 were also efficacious against HRB26M, with EC50 values of 0.881 ± 0.109 μM and 5.047 ± 2.116 μM, respectively (Figure 2D, E). Our results showed that the ability of GC376 to inhibit viral (HRB26 and HRB26M) replication was better than GS441524 when the agents were applied alone. We observed that the GC376 + GS441524 more effectively inhibited HRB26 and HRB26M replication than single treatment, with EC50 values of 0.531 ± 0.162 μM and 0.369 ± 0.068 μM, respectively (Figure 2C, F). In the replication process of coronavirus, Mpro is one of the first nsps processed by the polyprotein, and other replication-related proteins, such as RdRp, can be produced with the participation of Mpro and PLP proteases [8,9]. This phenomenon may be the reason why GC376 is better than GS441524, and their combined application may result in a synergistic effect because these agents target different proteins involved in virus replication.

---

In vitro antiviral activity of GS441524 and GC376 in patients infected with feline infectious peritonitis virus [6]
To evaluate the therapeutic efficacy of oral GC376 and GS441524, we investigated the antiviral activity of these two drugs in feline kidney (CRFK) cells. GC376 and GS441524 were tested for their ability to inhibit FIPV-rQS79 and FIPV II multiplication using CCK-8 assays. Our laboratory previously constructed a recombinant virus designated FIPV-rQS79, which has been demonstrated to cause 100% mortality in vivo(Delaplace et al., 2021; Wang et al., 2021). We confirmed the antiviral activity of GC376 and GS441524 against FIPV-rQS79 and FIPV II. CRFK cells were typically cultured with drug and virus for 48 h. Both GC376 and GS441524 showed effects against FIPV II, with EC50 values of 0.9 μM and 2.142 μM, respectively (Fig. 1 A and D). The EC50 values previously reported for FIPV were 0.78 μM (Murphy et al., 2018) and 0.3 μM (Pedersen et al., 2018), respectively. GC376 and GS441524 also inhibited FIPV-rQS79, with EC50 values of 1.239 μM and 2.52 μM (Fig. 1B and E), respectively. The possible cytotoxicity of GC376 and GS441524 were determined by CCK-8 assay. Neither of the compounds showed obvious cytotoxicity at any of the concentrations up to 100 μM in CRFK cells (Fig. 1 C and F). Anti-FIPV potency in infected cells treated with GC376 and GS441524 at serial dilutions was verified using an immunofluorescence assay 12 h after infection (Fig. 1 G). Compared with positive controls, no obvious pathological changes were observed when cells were incubated with 1.25 μM GS441524 or 2.5 μM GC376 (Fig. 1 G) for 24 h after FIPV-rQS79 and FIPV II infection. Our results indicated that GS441524 and GC376 effectively inhibited FIPV-rQS79 and FIPV II infection in CRFK cells.

The lymphocyte counts and rectal temperatures of all ten treated cats quickly return to pre-infection levels, as do the levels of the two asymptomatic cats. As of now, more than eight months after infection, none of the ten cats who received one or two treatments have changed. Some cats experience a brief "stinging" reaction after receiving an injection within ten seconds of the compound being administered. Unusual posturing, licking at the injection site, and/or vocalizations that persist for about 30 to 60 seconds following injection are signs of localized and temporary pain. Certain animals exhibit more noticeable injection reactions than others, and these reactions vary from injection to injection and become less pronounced over time[1].
GS-441524 is found in serum at 1000-fold higher concentrations than Remdesivir in NHP after receiving Remdesivi (IV injection) for a 7-day period of treatment[3].

---

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[5].

---

Oral GS441524 and GC376 were effective in the FIPV model [6]
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.

---

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 [6]
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.

---

Compared with oral or subcutaneous GC376, GS441524 exhibited more advantages in pharmacokinetic parameters [6]
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.

---

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 [5].

There are several methods for measuring the RdRP Enzymatic Activity of inhibitors as detailed below: [4]
Biochemical RdRP Enzyme Activity Assays
(1) Polymerase Elongation Template Element (PETE) Assay for RdRP
Because RdRP catalyzes the incorporation of NTPs during RNA elongation, a PETE assay can be developed to detect the elongation activity of RdRP.46 In this assay approach, an oligonucleotide at the 5′ end of an RNA template is labeled with a fluorescent probe for fluorescence polarization (FP) measurements. The polarization signal from the fluorescent probe increases as its mobility becomes low following the elongation of the newly synthesized complementary RNA chain by RdRP. Inhibition of RdRP activity by a compound reduces the FP signal as the elongation of the complementary RNA chain stops.
(2) Fluorescence-Based Alkaline Phosphatase–Coupled Polymerase Assay (FAPA)
The FAPA approach includes a modified nucleotide analog in the substrate system during RNA synthesis by RdRP. As the polymerase reaction proceeds, incorporation of modified nucleotide analog results in the release of the fluorophore, allowing detection. For example, a modified nucleotide analog (2-[2-benzothiazoyl]-6-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) incorporated into the growing RNA chain was catalyzed by RdRP, resulting in a by-product of BBT, pyrophosphate (PPi). The BBTPPi subsequently was reacted with alkaline phosphatase to produce a highly fluorescent BBT anion.
(3) Fluorometric RdRP Activity Assay
Fluorophores have been extensively used for the detection of RNA and DNA. In this fluorometric RdRP activity assay, fluorophores are used to detect dsRNA formation from the ssRNA template (Fig. 3C). One application of this assay was to screen the inhibitors of hepatitis C virus (HCV) RdRP.51 By using a poly(C) RNA template, HCV RdRP catalyzed the primer-independent synthesis of dsRNA that was detected by fluorescent dye PicoGreen.51 PicoGreen was originally developed to quantify dsDNA, but it was subsequently found to also preferentially bind dsRNA instead of ssRNA.51 This assay can be easily adapted to compound screening for RdRP inhibitors for many types of viruses. In addition to PicoGreen, other fluorophores have also been used to distinguish dsRNA from ssRNA, and they are useful for this type of RdRP assay.
(4) Scintillation Proximity Assay (SPA)
SPA has also been used in RdRP enzyme assays for HTS. This assay relies on the incorporation of radioactive nucleotides to the newly synthesized RNA chain catalyzed by RdRP using a biotinylated primer-template in the presence of 3H-GTP. Application of streptavidin-coupled SPA detection beads in this radioactive enzyme assay enables homogeneous assay detection that avoids the labor-intensive filtration and washing steps from the original radioactive NTP incorporation assay. Because they are radioactive assays, however, specific safety precautions and waste handling are required that may be inconvenient and require enhanced safety protocols. Therefore, most radioactive assays have been replaced by fluorometric assays in recent years.

GS-441524 is treated with 100, 33.3, 11.1, 3.7, or 1.2 μM for a 24-hour period on CRFK cells in order to assess its toxicity[1].

Evaluation of antiviral activity in Vero e6 cells[5]
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.

---

Cytotoxicity of GS441524 and GC376 in CRFK cells[6]
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:
Cell viability = [OD (compound) - OD (blank)] / [OD (control) - OD(blank)] × 100%·
The EC50 values were calculated using GraphPad Prism software version 8.0.2.

---

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.

---

Indirect immunofluorescence assay (IFA)[6]
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.

Cats: Three days after unambiguous clinical evidence of FIP (days 12-19 post infection), the 10 cats that showed disease signs were split into two groups and treated with either 5 mg/kg (Group A; n=5) or 2 mg/kg (Group B; n=5) GS-441524 SC q24 h. The two cats that do not show any symptoms of the disease act as controls for normal rectal temperature and blood lymphocyte counts[1].

---

In vivo toxicity study of GC376 and GS441524[5]
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.

---

In vivo antiviral study of GC376 and GS441524[5]
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.

---

Pharmacokinetics study of GC376 and GS441524 in BALB/c mice and SD rats[5]
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.

---

Pharmacokinetic studies of GC376 and GS441524 in cats [6] 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.

---

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 [6].

Absorption, Distribution and Excretion
GS-441524 has been found to transport poorly into cells compared to remdesivir.

---

Metabolism / Metabolites
GS-441524 is phosphorylated 3 times to form the active nucleoside triphosphate.

---

Pharmacokinetics of GS441524 and GC376 by different routes of administration and with different dosages [6]
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.

---

Pharmacokinetics study of GC376 and GS441524 alone or in combination [5]
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).

GC376 and GS441524 did not produce obvious cytotoxicity at concentrations up to 250 μM in Vero E6 cells (CC50 > 250 μM; Supplementary Figure 3). [5]

---

The possible cytotoxicity of GC376 and GS441524 were determined by CCK-8 assay. Neither of the compounds showed obvious cytotoxicity at any of the concentrations up to 100 μM in CRFK cells (Fig. 1 C and F).[6]

[1]. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet Microbiol. 2018 Jun;219:226-233.

[2]. What Do We Know About Remdesivir Drug Interactions? Clin Transl Sci. 2020 May 13;10.1111/cts.12815.

[3]. Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment. ACS Med. Chem. Lett. 2020.
[4]. https://journals.sagepub.com/doi/full/10.1177/2472555220942123

[5]. The preclinical inhibitor GS441524 in combination with GC376 efficaciously inhibited the proliferation of SARS-CoV-2 in the mouse respiratory tract. Emerg Microbes Infect. 2021 Dec;10(1):481-492.

[6]. Better therapeutic effect of oral administration of GS441524 compared with GC376. Vet Microbiol. 2023 Aug:283:109781.

GS-441524 is a C-nucleoside analog that is (2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile substituted by a 4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl group at position 2. It is the active metabolite of remdesivir and exhibits a broad range of inhibitory activity against various RNA viruses including HCV, parainfluenza and SARS-CoV. It has a role as a drug metabolite, an antiviral agent and an anticoronaviral agent. It is a pyrrolotriazine, a nitrile, a C-nucleoside and an aromatic amine.
GS-441524 is an adenosine nucleotide analog antiviral, similar to [remdesivir]. This molecule was patented in 2009. In vitro studies of GS-441524 have determined it has a higher EC50 than remdesivir against a number of viruses, meaning GS-441524 is less potent. GS-441524 continues to be studied in the treatment of Feline Infectious Peritonitis Virus, a coronavirus that only infects cats.

---

Mechanism of Action
GS-441524 is phosphorylated 3 times to form the active nucleoside triphosphate, which is incorporated into the genome of virions, terminating its replication.

---

These results indicated that the utilization efficiency of GS441524 in vivo is significantly higher than that of GC376. This finding may be one of the reasons for the poor ability of GC376 to inhibit SARS-CV-2 in vivo. The previous results showed that GC376 targeting the FIPV 3CLpro could effectively reduce the virus load in the macrophages from the ascites of cats with the duration of antiviral treatment [17,19]. Therefore, we speculate that GC376 can effectively inhibit the proliferation of coronaviruses (FIPV and SARS-CoV-2), but cannot quickly clear the virus from infected tissues. During continuous administration, GC376 needs to maintain an effective concentration for a long time to inhibit virus proliferation in the infected tissue. However, compared with GS441524, GC376 could be quickly cleared in the BALB/c mice and SD rats. Besides, the nasal turbinate and lung are the main target organs for SARS-CoV-2 proliferation, there is a large amount of SARS-CoV-2 in these tissues. Therefore, it is difficult for GC376 to completely inhibit the proliferation of SARS-CoV-2 in the mice nasal turbinates and lungs.

Furthermore, we found that the combined application of GC376 and GS441524 extended T1/2 from 1.51 ± 0.16 h to 1.67 ± 0.24 h and the residence time of GS441524 (MRT0−t from 2.07 ± 0.42 h to 2.37 ± 0.73 h) in SPF BALB/c mice (Figure 5C and Table 1). Similarly, the PK results showed that the combined application of GC376 and GS441524 extended T1/2 from 3.80 ± 1.17 h to 5.13 ± 2.56 h and the residence time of GS441524 (MRT0−t from 4.50 ± 1.11 h to 6.03 ± 1.37 h) in SPF SD rats (Figure 5F and Table 2). Moreover, the PK study results showed that GC376 reached Cmax earlier (Tmax = 0.25 h in mice and T max = 1.40 ± 0.49 h in SD rats) than GS441524 (Tmax = 0.55 ± 0.24 h in mice and T max = 3.40 ± 1.20 h in SD rats) to produce a synergistic effect (Figure 5C, F). When these agents were combined, GC376 was the first drug to inhibit SARS-CoV-2 replication. After the plasma concentration of GC376 decreased, GS441524 reached its Cmax (Figure 5C, F and Tables 1 and 2) to produce a continuous inhibition of SARS-CoV-2 proliferation and maintain the effective concentration for a longer time. This phenomenon may explain why the combined application of GC376 and GS441524 was better than single application alone.

In summary, we assessed the efficacy of GC376 and GS441524 to inhibit SARS-CoV-2 replication using a mouse-adapted virus infection model. Importantly, we found that intranasal administration of GS441524 and GC376 + GS441524 significantly prevents the replication of virus in the upper respiratory tract, and the efficacy of GC376 + GS441524 to inhibit the viral replication in the lower respiratory tract significantly better than that of GS441524. Combined i.n. and i.m. administration of GS441524 and GC376 + GS441524 effectively protected mice against HRB26M infection in the upper and lower respiratory tracts, but GC376 alone failed to block the proliferation of SARS-CoV-2 in mice. Compared with GC376 and GS441524 alone, the dosage of GC376 + GS441524 is halved, these results showed an additive effect of the combined application of the Mpro and RdRp inhibitors, so it should be developed and considered for future clinic practice. [5]

---

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.[6]

C12H13N5O4

291.2627

291.096

C, 49.48; H, 4.50; N, 24.04; O, 21.97

1191237-69-0

1355149-45-9 (GS-441524 triphosphate); 2378280-82-9 (HCl); 1809249-37-3 (Remdesivir); 1355050-21-3; 1809249-37-3; 2378280-83-0 (sulfate);1355357-49-1; 2647442-13-3

44468216

White to off-white solid powder

1.84±0.1 g

-1.4

4

8

2

21

456

4

O1[C@H](CO)[C@H]([C@H]([C@]1(C#N)C1=CC=C2C(N)=NC=NN12)O)O

BRDWIEOJOWJCLU-LTGWCKQJSA-N

InChI=1S/C12H13N5O4/c13-4-12(10(20)9(19)7(3-18)21-12)8-2-1-6-11(14)15-5-16-17(6)8/h1-2,5,7,9-10,18-20H,3H2,(H2,14,15,16)/t7-,9-,10-,12+/m1/s1

(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile

Remdesivir metabolite; GS-441524; GS-5734 metabolite; GS 441524; GS441524; GS5734 metabolite; GS 5734 metabolite; Remdesivir-metabolite; GS-5734-metabolite; GS5734-metabolite; GS 5734-metabolite; 2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile; EVO984; EVO-984; (2R,3R,4S,5R)-2-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile;

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 : ~120 mg/mL (with ultrasonic)
Water : Insoluble

Solubility in Formulation 1: 10 mg/mL (34.33 mM) in 5% ethanol, 30% propylene glycol, 45% PEG 400, 20% water (pH 1.5 with HCI) (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

---

Solubility in Formulation 2: ≥ 2.75 mg/mL (9.44 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (7.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (7.14 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

Solubility in Formulation 5: ≥ 2.08 mg/mL (7.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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