Main Research Findings

1) Doses of 4.0 mg/kg of GS-441524 was found to be an effective form of treatment for FIP in feline test subjects.

2) Administration of GS-441425 has the potential to inhibit the virus that causes feline infectious peritonitis in cats and feline tissue cultures.

 

Selected Data

1) Gilead Sciences provided samples of the peptide GS-441524 in a pure and highly stable powdered form. The research team then diluted the peptide to a concentration of either 10 or 15 mg/ml in various solvents, including: 20% water (pH 1.5 HCl),30% propylene glycol, 45% PEG 400, or 10 or 15 mg/ml in 5% ethanol. All mixtures were placed in sterile injection bottles and agitated until suspended, followed by 5-20 minutes in a sonicated water bath until clear. The diluted samples of the peptides were refrigerated and used for experimental treatment within 3-4 weeks of preparation.

It is important to note that the study did not include a control group. The decision to exclude a control group was based on preparatory in vivo and in vitro studies that indicated treatment with GS-441524 is more safe and effective than receiving no treatment at all. Additionally, there was no control group included in the experiment as the research team concluded there was no effective treatment that the peptide could be compared against [1].

The study conducted by Pedersen et. al was conducted under protocols approved by the Clinical Trial Review Board of the Veterinary Medical Teaching Hospital Clinical trials Committee, the University of California, Davis, and the Institutional Animal Care and Use Committee. All diseased feline subjects included in the experiment were obtained from shelters or cat fosters/rescues that required the cats to be owned or adopted in order for treatment to occur with consent of the owner. Cats were recruited for the experiment from the owners or veterinarians looking for new treatment and early access to an antiviral drug trial.

Test subjects were diagnosed with FIP based on routine laboratory results, characteristic signs and symptoms, clinical histories, and observation of swelling in the thoracic or abdominal regions. While diagnosis based on immunohistochemistry or RT-PCR assessments were preferred by the research team, they were not required for admittance into the experimental trial. Cats were excluded from the trial if they were exhibiting signs of obvious neurological or macular degeneration as this could affect the ability of GS-441524 to pass through the blood-brain or blood-eye barriers, respectively. At the start of the treatment period, 31 cats were recruited into the study. 26 of the subjects were treated at UC Davis, while the remaining 5 received treatment from their local veterinarian [1].

Early tissue cultures and pharmacokinetic studies led the research team to initially set the dosage regimen for the peptide at 2.0 mg/kg, given every 24 hours via subcutaneous injection. The treatment protocol was followed for 12 weeks and was extended by one or more weeks based on whether or not the test subjects still had abnormal levels of serum protein. If the treatment had to be extended or if the cats had a relapse of FIP symptoms, the dosage was increased from 2.0 mg/kg to 4.0 mg/kg. At the start of the trial all test subjects were weaned off of any non-essential treatments and medication and were housed at UC Davis to be monitored by the research team. Every 12 hours the temperature, appetite, activity, defecation, and urination of the cats were recorded. Blood samples were also collected at 1-3 day intervals in order to keep track of any changes in hematocrit, bilirubin, white blood cell count, and total protein [1].

Typically within 3-5 days the test subjects experienced favorable responses to treatment, at which point they were discharged to their owner. All owners were given instructions as to how to properly administer subcutaneous doses of GS-442124, as well as record temperature, body weight, urination, defecation, activity, and appetite. The cats were seen by their local veterinarian on a monthly basis where a complete blood count and serum chemistry panel were routinely performed to monitor any observable changes in bilirubin, total protein, hematocrit, and white blood cell count [1].

2) Feline infectious peritonitis virus (FIPV) is a complex, infectious disease belonging to the Coronoavirdae family, species Alphacoronavirus I, and subspecies feline coronavirus. Initial research found that GC376, a 3C-like protease inhibitor, was the first highly effective experimental treatment for FIPV. Because the FIPV is considered a positive single stranded RNA virus, new research has emerged examining the efficacy of treatment with GS-441524 considering that the peptide has been found to exhibit potent antiviral activity against various RNA viruses. Additionally, a phosphoramidate prodrug of GS-441524 has been shown to inhibit the replication of RNA viruses such Ebola virus, Lassa fever virus, and respiratory syncytial virus. Based on previous research, the team of Murphy et. al hypothesized that GS-441524 has the potential to treat FIP in cats experimentally induced with the virus, as well as inhibit replication of the virus in vitro [2].

GS-441524 was provided to the research team by Gilead Sciences. The Feline Research Laboratory breeding colony of the Feline Nutrition Center at UC Davis purposefully bred specific pathogen free (SPF) cats for this experiment. Twelve of the cats were used for experimental infection and treatment purposes, while six cats were used to evaluate the pharmacokinetic profile of the peptide. For the in vitro portion of the study, Crandell-Rees feline kidney (CRFK) cells were propagated using a mixture 25 ml of DMEM and 10% FBS. This preparation was followed by infecting the cells with serotype II FIPV at 70% confluency and 48 hours of incubation. After incubation the culture was centrifuged to remove any debris; the remaining supernatant was used to infect CRFK cells grown in 24 well plates [2].

Visual and colorimetric inhibition of CPE and suppression of viral RNA expression by qRT-pCR were used to quantify whether GS-441524 could effectively inhibit replication of the FIP virus. The visual method included growing CRFK cells in well tissue culture plates, followed by infection of the FIPV at 70% confluency. The peptide was then applied to the culture plates in concentrations ranging from 0.05 uM to 100 uM. After 48 hours the plates were washed to remove any unattached cells while the remaining attached cells were fixed and stained for further inspection. Additional tissue culture wells were either infected with the virus alone or left uninfected and assigned as positive and negative controls, respectively.

In order to determine the ability of GS-441524 to inhibit viral RNA assays, CRFK cells were cultured in six well plates and infected with FIPV after 24 hours. The well plates were then treated with doses of the peptide of either 50, 10, 1.0, 0.1, or 0 uM, followed by culturing over a 20 hour period. After 20 hours a cell scraper was used to gently remove any adherent, unattached cells. The existing medium and culture supernatant were then frozen and thawed to be assessed by qRT-pCR [2].

Laboratory cats were then utilized in a pharmacokinetic study of GS-441524 in order to assess the metabolism and toxicity of the peptide. Treatments were prepared by dissolving 12.5 mg/ml of GS-441524 in 5% ethanol, 30% propylene glycol, 20% water, and 45% PEG 400, followed by the addition of HCl to adjust the pH to 1.9. Six laboratory cats were then randomly assigned to either Group A or Group B. Group A was intravenously administered 5 mg/kg of the peptide, while Group B was subcutaneously administered 5 mg/kg of the peptide. Cats were then monitored for signs of toxicity over the course of 5 days. Blood samples were obtained via venipuncture at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after treatment was administered. Immediately after collection the samples were placed on ice and centrifuged. The resulting isolated plasma was snap frozen and shipped to Gilead Sciences Incorporated for further testing [2].

The research team also observed the effects of GS-441524 in cats that were experimentally infected with the “cat-passaged” serotype I strain of FIPV [2]. 2-3 weeks after the intraperitoneal infection, symptoms began manifesting in 80% of the test subject in the form of fever, lymphopenia, jaundice, and abdominal swelling. If no interventions were taken the severity of the virus would significantly increase within 1-2 weeks, and eventually prove to be fatal. Five cats were randomly assigned into Group A and received a 5 mg/kg dose of the peptide, daily. Another five cats were randomly assigned into Group B and received a 2 mg/kg dose of the peptide, daily, over the course of two weeks. Within an average of 3 days the course of the disease in each test subject was apparent, and any cats experiencing recurrent viral symptoms had their treatment period extended by two weeks. All test subjects included in the study were observed for a minimum of 8 months post-treatment for recurrence of the virus [2].

 

Discussion

1) Out of the 31 cats included in the study, 26 of them presented with effusive FIP, while the remaining five presented with non-effusive FIP. Four of the test subjects were either euthanized or died in the first 2-5 days of the experiment due to disease complications. After 26 days of treatment, a fifth cat was also euthanized due to a lack of response to the peptide. 3 cats were given two week respites after 4 or 8 weeks where treatment was paused due to adverse skin reactions that made it difficult for the owners to properly administer subcutaneous injections. Apart from the brief respites there were no interruptions in the treatment. The research team of Pedersen et. al thought it was important to note that subject CT53 received 12 weeks of treatment rather than 8 due to a second relapse in FIP symptoms marked by a rise in serum levels of urea and symmetric dimethylarginine [1].

Figure 1: Time scale of treatment and clinical outcomes for all 31 test subjects.

12 weeks of treatment with GS-441524 results in significant improvements in the 26 cats that completed the full protocol. Within 12-36 hours any instance of fever was typically resolved, while appetite, activity, and weight improved on a daily basis. Additionally, starting 10-14 days after the start of treatment, any swelling that was observed in the abdomen disappeared within 1-2 weeks. Jaundice was also resolved over a 2-4 week period, and any signs of ocular disease began to improve within 24-48 hours. Any cats experiencing dyspnea as a result of thoracic effusion had any pleural effusions removed by their veterinarians prior to the experiment. However, any residual instances of dyspnea rapidly dissipated after only 7 days of treatment with the peptide. After 2 weeks, key parameters such as total white blood cells, lymphocyte count, serum protein, serum globulin, and serum albumin, were still being closely monitored, however, according to the owners, at this point in time all 26 of the test subjects appeared “normal” or close to normal [1].

Figure 2: Average changes in body temperature over the first 5 days of treatment with GS-441524.

Out of the 26 cats that completed the full 12 week experimental treatment period, 18 of them did not require any further treatment. However, the remaining 8 cats suffered disease relapses that occurred an average of 23 days post-treatment. The research team thought it was important to mention that out of the 8 cats that experienced disease relapse, 3 of them had temporary breaks throughout the initial treatment period, while the other 5 required an extension of their primary treatment. 2 out of the 8 relapses were of a neurological nature and accompanied with a high fever, posterior ataxia, and incoordination. The other 6 cats that relapsed experienced fever, decreased activity levels, and anorexia. Following the disease relapse in 8 of the test subjects, the research team increased the dose of GS-441524 from 2.0 mg/kg to 4.0 mg/kg. All 8 cats responded favorably to the increased dosage and 25/26 cats that were treated “achieved a sustained remission of FIP” [1].

The simplest way of determining the efficacy of treatment was measured by changes in body weight. Both during and after treatment weight gains of 20-120% occurred in all test subjects. CBCs and chemistry profiles were also used to mark significant changes. Results of these tests reported that cats with elevated white blood cell counts decreased to normal levels within 2 weeks of treatment with the peptide. Packed cell volume (PCVs) reflected cases of mild to severe anemia at the start of the trial, however PCVs returned to normal levels within 6-8 weeks of treatment. That being said, monitored levels of white blood cells allowed the research team to observe short term effects of treatment, while monitoring PCVs allowed them to gather more accurate information regarding long term treatment progress [1].

Figure 3: Average white blood cell count of the test subjects over a 12 week treatment period

Figure 4: Average packed cell volume (PCV) of the test subjects over a 12 week treatment period.

Prior to treatment, the test subjects presented with high total serum protein concentrations, high serum globulin, and low serum albumin levels. After 8-10 weeks of treatment with GS-441524, serum protein values significantly improved with the most dramatic increase occurring 3 weeks into treatment. The research team thought it was important to note that this improvement of serum protein levels occurred simultaneously with the rapid resolution of abdominal swelling. Serum globulin levels were initially shown to rise and peak within the first 3 weeks of treatment, followed by a gradual drop to a maximum value of 4.5 g/dl by the 9th week of the experiment. Finally, serum albumin levels of the 26 test subjects started out very low but slowly increased until normal levels were achieved 8 weeks after the start of treatment [1].

Figure 5: (A) average serum globulin levels over 12 weeks of treatment, and (B) average albumin levels over 12 weeks of treatment

2) Results of the in vitro portion of the study conducted by Murphy et. al found that very low concentrations of GS-441524 was able to effectively inhibit replication of FIPV. The inhibitory aspects of the peptide were measured through the use of crystal violet staining and qRT-PCR in the CRFK cells infected with the virus. Complete inhibition of FIPV was seen when dosages of 50 uM and 10 uM of GS-441524 were applied to the infected cells. Partial inhibition occurred after a 1.0 uM dose of the peptide was applied and no inhibition occurred with lower concentrations of the compound.

The pharmacokinetic portion of the study assessed how GS-441524 is metabolized in infected animals. While both cats were treated with 5 mg/kg of the peptide, one group received the treatment intravenously while the other group received treatment via subcutaneous injection. Overall, the resulting plasma pharmacokinetic profiles were similar between both groups, however, the cats subcutaneously treated with GS-441524 experienced an 8-20 times higher concentration of intracellular triphosphate levels in PBMCs. Due to variation in the sample these changes were not considered significant by the research team [2].

In order to examine an effective dosage regimen of GS-441524 for cats with FIP, the test subjects were intraperitoneally treated with a strain of FIPV that has been well-characterized by previous experimental studies. Twelve laboratory cats were exposed to the virus and their responses to GS-441524 were closely monitored by the research team. Within 10-18 days of infection ten out of the twelve cats were experiencing clinical signs related to FIP, while the other two cats remained healthy. Symptoms of FIP were initially signaled by hyperthermia and lymphopenia; the virus quickly progressed and the subjects began to experience depression, anorexia, and hyperbilirubinemia. There were no observed changes in rectal temperature and levels of lymphocytes in the two asymptomatic cats; these cats were used as control subjects [2].

Figure 6: Temperature of the twelve test subjects over the course of the experimental treatment period

Figure 7: Levels of lymphocytes in the twelve test subjects over the course of the experimental treatment period

After 10 out of the twelve cats developed viral symptoms, all 10 cats were split into two groups and subcutaneously administered either 5 mg/kg or 2 mg/kg of GS-441524, starting three days after there was unequivocal evidence of FIP. All ten cats that received treatment experienced a rapid response to the peptide. All rectal temperatures and lymphocyte levels returned to baseline at the same level as the two asymptomatic cats serving as control subjects. Two out of the ten cats experienced a recurrence of symptoms at four and six weeks post-treatment. They were treated with GS-441524 for a second time with a nearly identical response to that of the initial treatment. As of 8 months after the end of the experiment, all ten of the test subjects remained healthy with no disease recurrence regardless of whether they were treated with the peptide once or twice. Additionally, there were no significant signs of toxicity other than injection site irritation lasting for approximately 30-60 seconds [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019 Apr;21(4):271-281. doi: 10.1177/1098612X19825701. Epub 2019 Feb 13. PMID: 30755068; PMCID: PMC6435921.

[2] Murphy BG, Perron M, Murakami E, Bauer K, Park Y, Eckstrand C, Liepnieks M, Pedersen NC. 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. doi: 10.1016/j.vetmic.2018.04.026. Epub 2018 Apr 22. PMID: 29778200; PMCID: PMC7117434.

 

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