The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies

Abstract

Feline infectious peritonitis (FIP) is a common and highly lethal coronavirus disease of domestic cats. Recent studies of diseases caused by several RNA viruses in people and other species indicate that antiviral therapy may be effective against FIP in cats. The small molecule nucleoside analog GS-441524 is a molecular precursor to a pharmacologically active nucleoside triphosphate molecule. These analogs act as an alternative substrate and RNA-chain terminator of viral RNA dependent RNA polymerase. We determined that GS-441524 was non-toxic in feline cells at concentrations as high as 100 uM and effectively inhibited FIPV replication in cultured CRFK cells and in naturally infected feline peritoneal macrophages at concentrations as low as 1 uM. We determined the pharmacokinetics of GS-441524 in cats in vivo and established a dosage that would sustain effective blood levels for 24 h. In an experimental FIPV infection of cats, GS-441524 treatment caused a rapid reversal of disease signs and return to normality with as little as two weeks of treatment in 10/10 cats and with no apparent toxicity.

Keywords: Cell culture; EC50; Experimental infection; FIP virus (FIPV); Feline infectious peritonitis (FIP); GS-441524; Laboratory cats; Nucleoside analog; Pharmacokinetics; Tri-phosphorylation.

Fig. 1

A. Molecular structure of triphosphorylated GS-441524. B. CRFK cell cultures infected with FIPV-79-1146 showing typical CPE and crystal violet staining pattern. Infected cells were treated with 3 uM (row a), 2 uM (row b), 1 uM (row c), 0.75 uM (row d), 0.5 uM (row e), 0.25 uM (row f), 0 uM GS-441524 (row g); untreated and uninfected cells (row h). C. A nonlinear regression analysis utilizing these data determined the EC50 for GS-441524 to be 0.78 uM.

Fig. 2

GS-441524 inhibits FIPV associated replication in feline cells. A. Viral RNA expression as measured by qRT-PCR (y axis) in FIPV infected CRFK cells treated with 0 to 50 uM of GS-441524 (x axis). B. Feline peritoneal macrophages derived from ascitic fluid of a naturally FIPV-infected cat and treated with 10 uM GS-441524 have a 1000-fold reduction in viral copy number relative to untreated cells (p < 0.0001). C. Intracellular concentrations of GS-441524 (O) and its triphosphate form (Δ) following incubation of CRFK cells with 1 uM GS-441524 for 3 days. The intracellular level of tri-phosphorylated GS-441524 was sustained at approximately 1 pM/million cells. D. Plasma pharmacokinetic profiles of GS-441524 were similar in cats treated subcutaneously or intravenously with 5 mg/kg of GS-441524. E. The intracellular triphosphate concentration in PBMC was 8–20-fold greater in cats treated IV or SC with GS-441524 than the 0.78 uM concentration necessary to inhibit FIPV replication in CRFK cells by 50%.

Fig. 3

GS-441524 treatment results in FIP remission in experimentally FIPV infected cats. Ten of 12 SPF cats experimentally infected with FIPV showed no clinical signs of FIP for 10–17days after challenge-exposure (dashed line). At that time, they developed fever and lymphopenia (*). Two cats (16–115 and 16-118) did not become ill (dashed line). A two-week treatment course (black line) with either 5 mg/kg (group A) or 2 mg/kg (group B) of GS-441524 was instigated within 2–3 days of disease onset and resulted in rapid remission of disease signs (grey line). A recurrence of clinical signs after four and six weeks in two of the 10 treated cats, 16–116 (Group A) and 16–127 (Group B) necessitated a second 2-week course of treatment (black line). All cats have remained disease free 10 months later at the time of writing.

Fig. 4

Virus-associated fever is rapidly normalized by GS-441524 treatment in experimentally FIPV-infected cats. Ten of 12 SPF cats experimentally infected with FIPV developed hyperthermia (>102.5 °F) 10–17 days after FIPV inoculation (cats 16–113, 16–116, 16–119, 16–123, 16–124, 16–127, 16–128, 16–129, 16–130). Two cats (16-115 and 16-118) were presumably resistant to disease and were not treated. Fevers responded rapidly to GS-441524 treatment regardless of dosage.

Fig. 5

Virus-associated lymphopenia is normalized by GS-441524 treatment in experimentally FIPV-infected cats. Eleven of 12 SPF cats experimentally infected with FIPV developed lymphopenia (less than or equal to 1700 cells/ul blood (--------) within 10–17 days after FIPV inoculation (cats 16–113, 16–116, 16–119, 16–123, 16–124, 16–127, 16–128, 16–129, 16–130). Cat 16–118, which was one of two cats that resisted infection, did not manifest lymphopenia, while a second resistant cat (16–115) had mild lymphopenia. Lymphocyte counts returned rapidly to pre-disease levels, regardless of dosage.