825701

JFM

Journal of Feline Medicine and SurgeryPedersen et al

Original Article

Efficacy and safety of the nucleosideanalog GS-441524 for treatmentof cats with naturally occurring
feline infectious peritonitis

Journal of Feline Medicine and Surgery
2019, Vol. 21(4) 271­–281© The Author(s) 2019
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DOI: 10.1177/1098612X19825701
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This paper was handled and processedby the American Editorial Office (AAFP)
for publication in JFMS

Niels C Pedersen1 , Michel Perron2, Michael Bannasch1,
Elizabeth Montgomery1, Eisuke Murakami2,
Molly Liepnieks3 and Hongwei Liu3

Abstract

Objectives The aim of this study was to determine the safety and efficacy of the nucleoside analog GS-441524 forcats suffering from various forms of naturally acquired feline infectious peritonitis (FIP).
Methods Cats ranged from 3.4–73 months of age (mean 13.6 months); 26 had effusive or dry-to-effusive FIP and five
had non-effusive disease. Cats with severe neurological and ocular FIP were not recruited. The group was started
on GS-441524 at a dosage of 2.0 mg/kg SC q24h for at least 12 weeks and increased when indicated to 4.0 mg/kg
SC q24h.
Results Four of the 31 cats that presented with severe disease died or were euthanized within 2–5 days and a
fifth cat after 26 days. The 26 remaining cats completed the planned 12 weeks or more of treatment. Eighteen ofthese 26 cats remain healthy at the time of publication (OnlineFirst, February 2019) after one round of treatment,
while eight others suffered disease relapses within 3–84 days. Six of the relapses were non-neurological and two
neurological. Three of the eight relapsing cats were treated again at the same dosage, while five cats had the
dosage increased from 2.0 to 4.0 mg/kg q24h. The five cats treated a second time at the higher dosage, including
one with neurological disease, responded well and also remain healthy at the time of publication. However, one of
the three cats re-treated at the original lower dosage relapsed with neurological disease and was euthanized, while
the two remaining cats responded favorably but relapsed a second time. These two cats were successfully treateda third time at the higher dosage, producing 25 long-time survivors. One of the 25 successfully treated cats was
subsequently euthanized due to presumably unrelated heart disease, while 24 remain healthy.
Conclusions and relevance GS-441524 was shown to be a safe and effective treatment for FIP. The optimum
dosage was found to be 4.0 mg/kg SC q24h for at least 12 weeks.
Keywords: Nucleoside analog; GS-441524; feline infectious peritonitis; FIP; field trial
Accepted: 28 December 2018

Introduction
Drugs that inhibit virus replication have become mainstays in the treatment of acute and chronic RNA andDNA virus infections of people.1 However, interest in
antiviral drugs for infections of animals has been muchslower to develop. This is especially true for cats,
which suffer from several chronic viral infections
resembling those in people. The infectious agentsinclude feline leukemia and immunodeficiency viruses(FeLV and FIV, respectively),2 feline herpesvirus

1Center for Companion Animal Health, School of Veterinary

Medicine, University of California, Davis, CA, USA
2Gilead Sciences, Foster City, CA, USA
3Department of Pathology, Microbiology and Immunology, Schoolof Veterinary Medicine, University of California, Davis, CA, USA
Corresponding author:
Niels C Pedersen DVM, PhD, Center for Companion Animal
Health, School of Veterinary Medicine, University of California,
213 CCAH Bldg, One Shields Avenue, Davis, CA 95616, USA
Email: ncpedersen@ucdavis.edu

272(FHV),3 virulent systemic calicivirus4,5 and a coronavirus causing feline infectious peritonitis (FIPV).6 FeLV
and FIV infections have been controlled with testing,isolation and/or vaccination. FHV-associated disease
was the first feline viral infection to incorporate anantiviral for treatment.3 Highly fatal systemic calicivirus affects only a small number of cats. FIPV infectionis the best candidate for antiviral drug development asvaccines are ineffective, multi-cat environments makeprevention extremely difficult and it kills 0.3–1.4% ofcats around the world.7–9
The emergence of exotic diseases such as Ebola,
Middle East respiratory syndrome and severe acute respiratory syndrome in people has prompted intensive
research into drugs that will inhibit RNA virus replication. One of the most promising antiviral drugs against
emerging RNA viruses is the adenosine nucleosidemonophosphate prodrug GS-5734 (Remdesivir; Gilead
Sciences). GS-5734 has been effective in preventing
experimental Ebola in rhesus monkeys,10 and inhibiting
both epidemic and zoonotic coronaviruses in tissue
culture and in mouse infection models.11 These promising findings prompted initial research on GS-5734 andits parent nucleoside GS-441524 against FIPV infectionof cats.12 GS-441524 and GS-5734 were found to havecomparable EC50 (1.0 µM) and CC50 (>100 µM)
values against FIPV in cat cells. Therefore, it was
decided to concentrate on the less chemically complex
GS-441524 for further testing with laboratory cats. A­pharmacokinetic study in two laboratory cats demonstrated sustained and effective plasma levels of
GS-441524 over 24 h f­ollowing a single dose given subcutaneously (SC) or intravenously (IV). These results
were then extended to 10 laboratory cats with experimentally induced ­abdominal effusive feline infectious
peritonitis (FIP).12 This study showed GS-441524 to behighly effective against experimental FIP and opened
the way for the present field trial.
The goal of this study was to demonstrate the safety
and efficacy of GS-441524 in the treatment of cats withnaturally occurring FIP. Small-molecule drugs such as
GS-441524 are <900 daltons in weight and around 1 nm in
size, and can easily enter cells and interact with key target
molecules. Unlike published substances or drugs that
inhibit FIPV by hampering cellular processes usurped byviruses for their replication,13,14 small molecules like
GS-441524 interfere directly with replicative processes
encoded by the virus.12,15

Materials and methods
Drug preparation
GS-441524 was provided by Gilead Sciences as a pureand highly stable powder and diluted to a concentrationof 10 or 15 mg/ml in 5% ethanol, 30% propylene
glycol, 45% PEG 400, 20% water (pH 1.5 with HCI). The

Journal of Feline Medicine and Surgery 21(4)mixture was placed in sterile 50 ml glass injection bottles,
agitated until in suspension, and then placed in a sonicated water bath for 5–20 mins until clear. The diluted
drug was refrigerated and used within 3–4 weeks.
Study design
This study was conducted under protocols 19336 and19863 approved by the Institutional Animal Care and
Use Committee and the Clinical Trial Review Board of
the Veterinary Medical Teaching Hospital (VMTH)
Clinical Trials Committee, University of California(UC), Davis. Institutional rules precluded the use of
diseased cats obtained directly from shelters or kitten
foster/rescue groups, thus requiring that all cats belegally owned/adopted and treated under specific conditions and with owner consent (supplementary material). The study did not incorporate a control group
because there is no effective treatment to compare itagainst. A placebo treatment group was not included,
given that preparatory in vitro and in vivo studies indicated that GS-441524 would be safe and more effective
than no treatment.12
Case selection and disease confirmation
Cats with FIP were recruited from owners or their veterinarians seeking current treatment options or access to an
earlier antiviral drug trial.6 The initial diagnosis of FIP
was based primarily on characteristic signalment, clinical histories and disease signs, routine laboratory test
results, and examination of abdominal or thoracic effusions. A more definitive diagnosis based on RT-PCR or
immunohistochemistry was desirable but not essential
for inclusion. Cats with obvious ocular or neurologicaldisease were discouraged to enter the trial because ofconcerns over the ability of antiviral drugs, including
GS-441524, to penetrate the blood–brain or blood–eyebarriers.12,15
Thirty-one cats and their owners were ultimately
recruited (Table 1). Owners or representatives of
26 cats came to UC Davis for initial treatment, and fiveowners and their cats (CT59, CT73, CT76, CT78, CT80)
were treated by their local veterinarian. Cats seen at UC
Davis were re-evaluated and their FIP diagnosis­reconfirmed based on signalment, clinical history, physical examination, prior laboratory test results and a repeatof complete blood count (CBC), serum protein and effusion analyses. Thoracic or abdominal effusions from catswith wet FIP were confirmed positive for FIPV 7b RNA
by RT-PCR.16 Cats with signs of non-effusive FIP were
further tested by abdominal and thoracic ultrasonography for primary lesions. Ocular disease was confirmedby the ophthalmology service of the VMTH, UC Davis.
Neurological status in cases with possible ­central nervous system disease signs was evaluated by the VMTH
neurology service.

Pedersen et al
Treatment regimen
The initial dosage regimen for GS-441524 was 2.0 mg/kg
SC q24h, based on earlier tissue culture experiments andpharmacokinetic studies in laboratory cats.12 The minimum treatment period was 12 weeks based on experienceswith the 3CL protease inhibitor GC376 against naturally
occurring FIP.6 Treatment was extended one or more
weeks in cats that still had abnormal serum protein values.
The dosage was increased during later stages of the trialfrom 2.0 to 4.0 mg/kg in cases where treatment had to beextended or when disease relapses occurred. Owners weresent a new supply of drug every 4 weeks in the form of
preloaded 1 or 3 ml Luer lock syringes with 1 inch 22 G
Luer hub needles. Syringes were stored in the refrigerator
and warmed to room temperature prior to injection.
Injections were spaced across the dorsum from 2 cm
behind the shoulder blades to the mid-lumbar area andone-half the distance down the adjacent chest and flanks.
Monitoring during initial treatment period
Cats were taken off all non-essential treatments such as
antibiotics, corticosteroids, interferons, pentoxifylline,
non-steroidal anti-inflammatory drugs or pain relief
medication at the time of entry into the trial. They werethen monitored every 12 h during their stay at UC Davisfor temperature, appetite, activity, urination and defecation. Blood was also taken at 1–3 day intervals to evaluate hematocrit, total protein, bilirubin, white blood cellcount and white cell differential count.
Ascites samples were collected at entry and one ormore day intervals for as long as possible and tested
for the levels of FIPV 7b RNA transcripts by quantitative (q)RT-PCR (IDEXX Molecular Diagnostics).16
Immunohistochemistry for the FIPV nucleocapsid protein was performed on formalin-fixed tissue sections
from five cats that were necropsied.
Monitoring initial and long-term responseto treatment
Cats were discharged to their owners when a significant
favorable response to treatment was noted, usually
within 3–5 days. Owners were instructed during thistime on how to properly give subcutaneous injections of
the drug and encouraged to continue daily logs on bodytemperature, activity, appetite, defecation and urination, and weekly body weight measurements. A CBC
and serum chemistry panel were done at monthly intervals by local veterinarians or during visits to the VMTH,
and any abnormal signs or behaviors were to be noted
and promptly reported. Euthanasia, when required,
was usually conducted by the owner’s veterinarian or,when possible, at UC Davis. Bodies were sealed in plastic bags, immediately refrigerated, and shipped within
2 days or less in insulated containers with ice-packs to
UC Davis by overnight express mail. Necropsies were273
performed by one of the authors (ML) in the Anatomic
Pathology Service of the School of Veterinary Medicine,
UC Davis. The owner’s request for the final dispositionof the body was honored.

Results
Signalment and disease presentation
Thirty-one cats ranging from 3.4–73 months of age (mean
13.6 months) were enrolled in the trial (Table 1). Eighteen
cats were domestic short- or longhairs and 13 were pedigreed from 10 different breeds (Table 1). The domestic
cats were adopted from kitten/foster rescue organizations (n = 13), formal shelters (n = 2) or obtained asneighborhood strays (n = 3). The study included 14
females (seven intact; seven spayed) and 17 males (oneintact; 16 castrated).
Twenty-six of the 31 cats presented with effusive FIP(six thoracic, 20 abdominal). Five cats presented with
non-effusive FIP; four of them (CT59, CT64, CT73, CT78)
with disease localized to the abdomen (mesenteric andileo/cecal/colic lymph nodes) and one (CT79) to thechest (lung, hilar lymph nodes) (Table 1). Four other cats
had indications of earlier non-effusive FIP that had progressed to the effusive form (CT57, CT65, CT67, CT71)(Table 1). Gross signs of ocular disease consistent with
underlying FIP was confirmed by ophthalmoscopic
examination in three of the 31 cats (CT56, CT65, CT71).
Two cats (CT71, CT80) were either reluctant or no longer
able to jump to higher places, suggestive of neurologicalinvolvement.
Treatment outcome
Four cats were euthanized (CT62, CT72, CT75) or died
(CT56) within the first 2–5 days because of severe disease and other complications, and a fifth cat was euthanized (CT54) after 26 days owing to a lack of treatment
response (Figure 1). Treatment periods were not interrupted except for three cats that were given 2 week respites at week 4 (cat CT80) or week 8 (cats CT53, CT71)
because of problems with giving injections and skinreactions (Figure 1). Cat CT53 was treated after a second
relapse for 8 rather than 12 weeks because of a rise inblood urea and serum levels of symmetric dimethylarginine (SDMA).
The clinical response of the 26 cats that completed atleast 12 weeks of treatment was dramatic. Fever usuallyresolved within 12–36 h (Figure 2), concurrent with amarked daily improvement in appetite, activity levels
and weight gain. Abdominal effusions rapidly disappeared over a 1–2 week period starting around 10–14
days post-treatment. Cats with thoracic effusions wereusually dyspneic upon presentation to private veterinarians, prompting removal of pleural effusions prior tocoming to UC Davis. Residual dyspnea and thoracic
effusion responded rapidly to treatment and were noJournal of Feline Medicine and Surgery 21(4)274Table 1 List of 31 cats enrolled in the trial, including laboratory designation, name given by owner, breed, clinical formof feline infectious peritonitis (FIP), and date of diagnosis
Cat ID Name

Date of birth

Breed

Sex

OriginDate of diagnosis

FIP form

CT52
CT53
CT54
CT55
CT56
CT57
CT58
CT59
CT60
CT61
CT62
CT63
CT64
CT65
CT66
CT67
CT68
CT69
CT70
CT71
CT72
CT73
CT74
CT75
CT76
CT77
CT78
CT79
CT80
CT81
CT82

9 Jan 2017
2 Aug 2016
11 Jul 2016
26 Jun 2016
1 Jul 201631 Oct 201617 Apr 201611 Apr 201125 Jul 2016
1 Jul 2016
10 Mar 2016
6 Nov 2016
27 Jun 2016
14 Mar 2016
7 Jun 2016
2 Mar 2016
8 May 201616 Jun 2015
1 Aug 201429 Jun 201625 Feb 2017
8 Aug 2015
4 Mar 201731 Mar 201720 Jul 2016
15 Mar 2017
1 Mar 2016
21 Sep 201617 Oct 2016
8 Sep 2016
8 Apr 2016

Savannah
DLH
Siberian
DSH
DSH
DSH
Ragdoll
DLH
DSH
DSH
DSH
DSH
DSH
Exotic SH/Persian
DSH
Ragdoll
Maine CoonSnowshoe
DSH
DSH
British SH
DSH
Siberian
DSH
Exotic SH
Siberian
DSH
Himalayan
DLH
Persian
DSH

F
MC
F
MC
MC
FS
F
MC
MC
MC
MC
MC
MC
F
FS
FS
MC
MC
MC
MC
F
FS
FS
F
M
FS
MC
F
MC
FS
MC

Breeder
RescueBreeder
RescueShelter
RescueBreeder
Stray
RescueRescueRescueRescueRescueBreeder
Shelter
Breeder
RescueShelter
RescueStray
Breeder
Shelter
Breeder
RescueBreeder
Breeder
RescueBreeder
Stray
Breeder
Rescue24 Apr 2017
5 May 201715 Apr 201715 Apr 2017
12 May 2017
8 May 2017
25 May 201710 Apr 201720 May 201729 May 201730 May 2017
3 Jun 201722 May 201711 Jun 201714 Jun 201720 Jun 201722 Jun 201722 Jun 201725 Jun 201720 Jun 201720 Jun 201721 Jun 2017
7 Jul 201710 Jul 201728 Jun 2017
3 Jul 201722 Jul 201718 Jul 201725 Jul 2017
1 Aug 2017
6 Aug 2017

Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal non-effusive
Abdominal effusive
Thoracic effusive
Abdominal effusive
Abdominal effusive
Abdominal non-effusive
Thoracic effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Thoracic effusive
Abdominal effusive
Thoracic effusive
Abdominal effusive
Abdominal non-effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal effusive
Abdominal non-effusive
Thoracic non-effusive
Abdominal effusive
Thoracic effusive
Abdominal effusive

Luna
Ice Bear
Charolett
Dempsey
Mudsa
Boone
Justyna
Bubba
Joey
HudsonLuca
Bao Bao
Cedrick
Mona
Squeekers
DoubleTuckerman
Danny
Tolstoy
AmadeusBella
Siersha
Maive
Lucy
Pie
Mila
Polly
Oona
FezzikJewelkatTiko

F = female intact; DLH = domestic longhair; MC = male castrated; DSH = domestic shorthair; FS = female spayed; SH = shorthair; M = male intact

longer apparent after 7 days. Jaundice slowly resolved
over 2–4 weeks, in parallel with decreasing hyperbilirubinemia. Signs of ocular disease began to clear within
24–48 h and were no longer apparent outwardly or byophthalmoscopic examination by 7–14 days. Enlarged
mesenteric and ileo/cecal/colic lymph nodes slowly
decreased in size over the course of the treatment. All 26
cats appeared outwardly normal or near normal in theestimation of the owners after 2 weeks of treatment. The
emphasis of treatment after 2 weeks was on monitoring
several blood test parameters. Key values included
packed cell volume (PCV), total white blood cells, absolute lymphocyte count, total serum protein, serum globulin, serum albumin and albumin:globulin (A:G) ratio.
Eighteen of 26 cats that completed at least 12
weeks of uninterrupted primary treatment required
no further treatment. However, eight other cats suffered disease relapses within 3–84 days (average 23days) (Figure 1). This group included the three catsthat had temporary breaks in their initial treatment(CT53, CT71, CT80) and five cats (CT53, CT57, CT60,
CT68, CT73) that had required extended primary
treatment (Figure 1). Disease relapses in 2/8 cats
(CT57, CT71) were clearly of a neurological nature
with high fever and severe posterior ataxia and incoordination, while disease relapses in the remaining
six cats consisted mainly of fever, anorexia and lackof activity. Only one cat (CT60) had an obvious
abdominal effusion upon relapse. One cat (CT57) was
euthanized 2 weeks after suffering a relapse withneurological signs that failed to respond to a second
round of treatment.
A decision was made to increase the dose of GS-441524
from 2.0 to 4.0 mg/kg in eight of the cats, either because
treatment had to be extended (CT77, CT80), because they
suffered one (CT60, CT68, CT71, CT73) or two relapses

Pedersen et al

275

Figure 1 Time scale for the treatment and clinical outcome of 31 cats that were enrolled in the GS-441524 field trial. The periodof treatment is indicated by a solid line (2 mg/kg dose) or a broken line (4 mg/kg dose). Asterisks indicate the point of relapse.
The treatment end date for the cats that achieved a sustained clinical remission is given in parenthesis. The time point andcause of death is indicated by a cross(CT53, CT63), or because the relapse was neurological(CT71). All eight cats responded well to the higher dosage
regimen.
In total, 25/26 cats treated for 12 weeks or longer
achieved a sustained remission of FIP, although one ofthem subsequently died of an unrelated heart problem(see ‘necropsy findings’). The longest survivors at thetime of publication (OnlineFirst, February 2019) ended
treatment in August 2017 and the shortest in May 2018,
all beyond the longest time for relapses to occur (ie, 84

days after stopping treatment). The 24 surviving catswill be carefully monitored for any return of diseasesigns and periodically tested for total protein, globulin, albumin and A:G ratios for the first year. Less
intense monitoring will be maintained for the remainder of the cats’ lives. Owners were cautioned to avoid
unnecessary stresses on their cats for the first 3 months,
although four cats (CT52, CT58, CT65, CT79) were
spayed and one cat (CT76) castrated withoutcomplications.

276

Journal of Feline Medicine and Surgery 21(4)Figure 2 Mean (solid line) and 1 SD (dashes) of bodytemperatures for the first 5 days of GS-441524 treatment.
The normal temperature range for cats is 100–102.5°F(37.7–39.1°C). Temperatures dropped into normal range
within 12–36 h of starting treatment

Favorable treatment response indicators
The simplest long-term measure of treatment efficacy
was body weight. Weight gains of 20–120% occurred
during and following treatment, even in cats 1 year ofage and older at disease onset. Younger cats also
appeared to grow in stature at an increased rate, as independently noted by owners. These post-treatment surges
in growth indicated that FIP was subclinical in many of
the cats for some time prior to diagnosis and had affected
growth. CBCs and a chemistry profile also proved helpful in monitoring the later effects of treatment andobserving for possible drug toxicities.
CBCs
Cats presented with elevated white blood cell counts,
which dropped to normal levels within the first 2 weeks
of treatment (Figure 3a). Lymphopenia was noted at thetime of entry and resolved over the first week of treatment (Figure 3b). A mild to moderately severe anemia
was observed at entry, as reflected by the packed cell volume (PCV) (Figure 4). PCVs did not return to normal
levels until after 6–8 weeks of treatment. Therefore, absolute total white cell and lymphocyte counts were only ofvalue during the first week of treatment, while the PCV
gave a more accurate picture of treatment progress overthe first 8 weeks.
Changes in serum proteins
Cats with FIP frequently presented with higher than normal total serum protein concentration, high serum globulin, low serum albumin levels and a low A:G ratio(Figures 5–7). Abnormal serum protein values improved
progressively and reached normal levels after 8–10­
weeks of treatment (Figures 5–7). The level of total protein was the least informative, as indicated by the weakR2 value (0.1883) for the trend line (Figure 5). However, adramatic and transient rise in total protein levels

Figure 3 (a) Mean white blood cell count with standard error
for 26 cats that completed the primary treatment regimen of
12 or more weeks. (b) Mean absolute blood lymphocyte countwith standard error for 26 cats that completed at least 12
weeks of treatment

Figure 4 Mean packed cell volume (PCV) with standard error
for 26 cats that completed at least 12 weeks of treatment. The
dotted line indicates the trend of rising PCV over timeoccurred 3 weeks into treatment (Figure 5). It was associated with an increase in serum globulins (Figure 6a) andoccurred at a time when abdominal effusions were rapidly resolving.

Pedersen et al

Figure 5 Mean serum total protein levels and standard error
for 26 cats that completed at least 12 weeks of treatment

277

Figure 7 Mean albumin:globulin (A/G) ratios and standard
error for 26 cats that completed at least 12 weeks of treatment

The levels of albumin in serum of the 26 cats treated
for at least 12 weeks were usually low (⩽3.2 g/dl) at thetime treatment was started (Figure 6b). Albumin levels
then increased slowly and reached normal levels at 8
weeks. The trend line for this rise in albumin had astrong R2 value (0.79), making serum albumin levels, aswell as PCV, a good indicator of treatment progress. As
is expected, the A:G ratio showed an equally strong
trendline over time and reached a level above 0.70 ataround week 8 of treatment (Figure 7).
Decrease in viral RNA levels in cells from ascitic
fluid associated with treatment
Sequential ascites samples were collected from eight cats
over the first 2–9 days of antiviral treatment and tested
for levels of viral RNA by qRT-PCR (Table 2). Wholeeffusions, or their cell fractions, were the most reliable
sources of FIPV RNA. Viral RNA levels decreased, often
to undetectable levels, by 2–5 days in 7/8 cats. Only one
cat (CT54) failed to show a significant drop in viral RNA
levels over a 9 day period.
Side effects observed during and after treatment
Focal injection site reactions Injection site reactions were

Figure 6 (a) Mean serum globulin levels and standard error
for 26 cats that completed at least 12 weeks of treatment.
(b) Mean serum albumin levels and standard error for 26 catsthat completed at least 12 weeks of treatment

Plasma globulin levels rose during the first 3 weeks oftreatment, peaked, and then slowly dropped to a maximum reference value of 4.5 g/dl or lower by week 9(Figure 6a). Although globulin levels over time appeared
to indicate the status of treatment, the low R2 value(0.3621) made this a less reliable indicator of treatment
progress.

of two types, and whether they were caused by the drug,
diluent or both was not determined. Immediate pain
reactions were manifested by vocalization, occasional
growling and postural changes lasting for 30–60 s. These
initial reactions lessened in severity over time as owners
became more adept at administering the injection andcats became more adjusted to the routine. Sixteen of 26
treated cats manifested injection site reactions (Table 3).
Reactions were most common during the first 4 weeks
and progressed to open sores only in 7/16 cats. Ulcerations healed within 2 weeks by clipping surrounding
hair and gently cleaning the wound with a cotton ballsoaked in one-part household hydrogen peroxide andtwo-parts water twice a day. Only three cats had noticeable scars at injection sites.

Journal of Feline Medicine and Surgery 21(4)278Table 2 Levels of feline infectious peritonitis virus 7b RNA
transcripts in whole ascites or in the cellular fraction of
ascitic fluid during initial treatment with GS-441524
Sample ID Days oftreatment

Sample type

Copies of viral
RNA/mlCT52

Ascites
Ascites
Ascites
Ascites
Ascites
Ascites
Ascites
Ascites
Ascites
Ascites
Cells
Cells
Cells
Cells
Cells
Ascites
Ascites
Ascites
Ascites
Ascites
Ascites

9.44 × 104
UND
8.49 × 105
6.97 × 104
2.44 × 103
2.07 × 103
6.46 × 104
5.96 × 103
1.53 × 103
UND
6.51 × 106
3.39 × 105
9.08 × 106
4.75 × 105
2.50 × 105
5.47 × 104
3.93 × 103
4.10 × 103
UND
1.13 × 104
UND

CT54

CT62

CT74
CT75

CT77
CT80
CT82

0
3
0
2
4
7
9
0
2
8
0
2
0
3
4
0
2
0
2
0
5

UND = undetectable

Table 3 Injection site reactions in 16 of 26 cats treatedwith GS-441524 for 12 weeks or longer
Cat ID

Superficial lesions

Open sores

Scars

CT53
CT58
CT60
CT61
CT63
CT64
CT65
CT66
CT68
CT71
CT73
CT74
CT76
CT78
CT79
CT82

3
1
0
5
2
1
9
3
4
5
7
3
10
7
2
2

1
0
0
0
2
0
1
2
0
1
1
1
0
0
0
0

0
0
2
0
0
1
1
0
0
0
0
0
0
0
0
0

Most lesions were superficial, involving the epidermis, and required notreatment, while some progressed to open sores that healed within 2
weeks with topical treatment. Some reactions left small permanent scars

Systemic drug reactions GS-441524 treatment over a total

period of 12–30 weeks was remarkably safe. No longterm abnormalities were observed in CBC values(Figures 3 and 4). Tests for liver and kidney function andlevels of amylase/lipase remained normal during andafter treatment (supplementary figures S1–S3). The only
exception was cat CT53, which had a progressive rise inblood urea nitrogen (BUN) to 35 mg/dl (reference interval [RI] 16–37 µg/dl) and sudden rise in SDMA (20 µg/dl) (RI 0–14 µg/dl) at 8 weeks into a third round of treatment with the higher 4 mg/kg dosage regimen. Althoughthese signs were still mild in nature, a decision was madeto stop treatment out of caution. These abnormalities
were not evident on testing 1 month later and the cat iscurrently in disease remission.
Necropsy findings
Four cats (CT56, CT62, CT72, CT75) were euthanized ordied within 2–5 days of enrollment and necropsies were
performed on all but cat CT75. A fifth cat (cat CT54) was
euthanized after 26 days of treatment. All five of thesecats presented with severe abdominal effusive disease.
Cats CT54 and CT56 had evidence upon necropsy ofwidespread pyogranulomatous vasculitis involving theabdominal viscera, central nervous system and eyes. Cat
CT56 also had compromise of the ileal wall in an area ofdense infiltrate and secondary bacterial sepsis. Cat CT72
had severe pyogranulomatous vasculitis restricted to theabdomen with moderate-to-severe peripheral edema andmineralization of the adrenal cortices. Cat CT62 ­sufferedfrom severe pyogranulomatous and fibrinosuppurative
peritonitis, which was complicated by an acute gastricperforation associated with plant material andintralesional bacteria indicative of sepsis. Cat CT75­
­presented with a chronic form of FIP characterized bysevere stunting of growth, a massive low protein/low
cell abdominal ­effusion, gallop rhythm indicative of
impaired cardiac function, and moderately severe peripheral edema. An echocardiogram showed bilateral atrial
enlargement but no indication of primary cardiac disease. The cat appeared to respond to GS-441524 and wasdischarged. The cat went into shock 2 days later and waseuthanized without necropsy.
FIPV was not detected by qRT-PCR in cats CT56, CT72
and CT75 at the time of necropsy, although pretreatment
ascites samples had tested positive. Ascites from cat CT54
had tested positive by qRT-PCR throughout treatment(Table 2) and tissues were still positive by immunohistochemistry at the time of necropsy.
Two additional cats were euthanized after successfully completing one or more rounds of treatment. Cat
CT57 was normal after one round of treatment but
relapsed with severe neurological signs 2 weeks later.
The cat failed to respond to retreatment and was euthanized. Lesions typical of FIP were found within thebrain and abdomen, but were negative for FIPV byimmunohistochemistry for nucleocapsid protein or for
7b RNA by qRT-PCR. Cat CT80 had been successfullyPedersen et al

Figure 8 Cross-section through the heart of cat CT80
showing extreme hypertrophy of the left ventricular walland septum and extreme narrowing of the chamber

treated for effusive abdominal FIP but developed
intense hindleg and lower back pain 4 weeks later andwas euthanized. The cat was found to have pronounced
thickening of the left ventricular wall and septum
causing severe constriction of the chamber (Figure 8).
The microscopic appearance of the left cardiac ventricular wall was typical of congenital feline hypertrophic
cardiomyopathy (HCM). No gross or microscopic
lesions of FIP were found in the abdomen, chest, eyes,brain or spine, and FIPV antigen and RNA were notdetected by qRT-PCR.

Discussion
GS-441524 is the second targeted antiviral drug after
GC376 to be evaluated for the treatment of cats with
FIP in the past two to three years.6,12,15 These two drugs
inhibited viral replication in two very different manners, either by terminating viral RNA transcription or
blocking viral polyprotein cleavage. Both processes
are well-established targets for several viral diseases
in people.1 A key question is how treatment with anucleoside analog compares with that of a viral protease inhibitor. The two drugs gave virtually identicalresults in tissue culture and experimental cat infection
studies.12,15 However, efficacy against naturally occurring FIP appeared greater with GS-441524 than GC376.
Six of 20 cats treated with GC376 remain in remission
to date (Pedersen NC, unpublished data, 2018) compared with 25/31 cats treated with GS-441524. Disease
relapses that did not respond to retreatment occurredin 14/20 cats with GC376 and only one cat treatedwith GS-441524.6 Eight of the 14 relapses associatedwith GC376 were neurological in nature,6 comparedwith 2/8 relapses with GS-441524. One of two neurological relapses in GS-441524-treated cats responded279
to retreatment at a higher dosage, whereas neurological relapses with GC376, even with increased dosage,
were no longer treatable.6 Both treatments caused similar injection site reactions. Both drugs appear to bequite safe, although GC376 interfered with the development of permanent teeth when given to younger
kittens.6
Although the results of field testing appear to favor
GS-441524, some of the difference may have been influenced by how the two drugs were administered. Efficacy
of GC376 may have improved if all 20 cats had beentreated without interruption for 12 weeks, rather thantreated for progressively longer periods starting at just 2
weeks.6 Five of the six cats cured with GC376 wereamong the seven cats that were treated continuously for
12 weeks, while only one of 13 cats treated one or moretimes for shorter periods of time was cured. These
shorter treatment times were essential for determining
the 12 week period used for all cats in the present study.
The GC376 field trial also involved fewer cats and wasconstrained by a limited supply of the drug, which madeit difficult to test other dosage regimens. Therefore,
GC376 should be further studied using a minimum of 12
weeks with a higher dosage and a larger number of cats
before making any final comparisons. It would also be
important at some time in the future to evaluate bothtypes of drugs in combination, as done for HIV/AIDS
and hepatitis C.
Early deaths must be anticipated in any trial of thistype, but how should they be considered in analyzing
efficacy? The five early deaths in this study were
included in the analysis of GS-441524 efficacy but
removed from consideration in the GC376 trial. If they
are included in the trial, it is important to determine
virus status at the time of death. Viral RNA was notdetected in the three necropsied cats that died after 2–5
days of GS-441524 treatment, suggesting that the drug
was effective but that the disease process was too far
advanced. This did not appear to be the case in a fourth
necropsied cat that survived for 26 days; viral RNA­levels did not decrease over the entire treatment period
and disease signs were unabated. Therefore, it is possible that this cat died because of failure to halt virus replication. Resistance to GS-5734 (Remdesivir), a prodrugof GS-441524, has been associated with amino acid mutations in RNA polymerase and proofreading exonucleasein tissue culture propagated coronaviruses.17 Whether asimilar type of resistance occurred in this cat remains to
be determined. Drug resistance was also seen in one cat
in the GC376 trial.6 Fortunately, none of the other cats inthe current trial showed signs of drug resistance at any
time. However, it is something that needs to be considered in future cats that fail to respond or respond poorly
to ­primary or secondary treatment.

280
The initial dosage of GS-441524 used in the present
study was determined from prior pharmacokinetic andexperimental infection studies with laboratory cats.12
These studies indicated that both 2.0 and 5.0 mg/kg SC
q24h for 14 days would be equally effective for field testing. Therefore, the 2.0 mg/kg dose was chosen for thefield trial, as it would cut drug usage by 60%. Although
this decision was validated in 18/26 cats, eight other catseither suffered disease relapses (two for a second time)or required longer treatment periods to get key bloodvalues back to normal. Therefore, a decision was madeto increase the dosage of GS-441524 from 2.0 mg/kg to
4.0 mg/kg SC q24h in cats that relapsed or required
extended treatment. The success of 4.0 mg/kg SC q24h
for at least 12 such cats, as well as one cat with neurological disease, led us to the conclusion that it was a more
effective dosage and should be the basis for future
treatment.
It was important to follow simple biological markers
of progress over the ⩾12 week treatment periods. PCV,
serum total protein, globulin and albumin levels, and the
A:G ratio were identified as useful markers. Based onthese parameters, it appeared that cats had not completely recovered after 6–10 weeks of treatment. Thisfinding validated the 12 week minimum treatment
period determined by an earlier GC376 field trial.6
Anemia of chronic disease (anemia of inflammation)affects 18–95% of people with acute and chronic infections and is normocytic/normochromic and not associated with iron deficiency.18,19 Plasma albumin levels
were also a good measure of disease activity, and low
albumin and low PCV are known to coincide in situations of chronic disease.19 Hyperglobulinemia in catswith FIP has been classified as infectious/inflammatory
and is caused by increases in all gamma globulin classes
and variable increases in alpha-2 globulins.20 The strong
tendency for cats with FIP to have high serum globulin
and low albumin levels makes the A:G ratio a particularly good indicator of disease activity.21
An expectation was that the pedigreed cats would not
respond as well to treatment because of a genetic weakness in their ability to respond immunologically to
FIPV22 and that younger cats with effusive FIP would be
most responsive.6 However, the pedigreed cats in thepresent trial responded equally well as the random-bredcats, and the breeds represented by the cats in the study
mainly mirrored the breeds’ current popularities. Oldercats, and cats with pure non-effusive FIP, also responded
as well to GS-441524 treatment as young cats and catswith effusive FIP. If a proportion of cats with ocular andneurological disease can also be successfully treatedwith GS-441524, no clinical manifestations of FIP should
be considered untreatable.
The safety profile of GS-441524 was impressive. No
systemic sign of toxicity based on CBC and serum

Journal of Feline Medicine and Surgery 21(4)chemistry values was observed over total treatment
periods from 12–30 weeks, with one possible exception.
One cat (CT53) had a mild rise in BUN and SDMA
8 weeks into a third round of treatment and prompting
a halt as a precautionary measure. Based on a previous
experience with GC376,6 there was concern about theeffect of GS-441524 on the development of permanentdentition. Three cats (CT52, CT74, CT77) in the present
study were 4 months of age or younger and still had
their juvenile dentition, and none showed any subsequent dental abnormalities. Injection site reactions wereobserved with GS-441524, but these were remarkably
low in number and were easily treated. It was not determined whether the drug, diluent or both were at fault.
The 1.5 pH of the diluent was well below the minimum
Food and Drug Administration (FDA) threshold of 4.5,
but drugs of this type are difficult to solubilize and stabilize at more physiologic pH. Nonetheless, more physiologic diluents should be evaluated.
One cat in the study (CT80) had confounding clinicalsigns. Although the cat presented with effusive abdominal FIP, it also had long-standing signs of vague hindlimb
lameness, lower back pain, periodic bouts of falling,
reluctance to jump to higher places, and unexplainable
and transient changes in behavior. These signs prompted
the cat to be treated for a long time after the abdominal
effusion disappeared. A decision was ultimately made todiscontinue treatment and see if more characteristic FIP
signs would recur. The cat was ultimately euthanized
and found to have congenital-type HCM and no residual
lesions of FIP or viral RNA in any tissues upon necropsy.
Dilated cardiomyopathy has been reported in 17.6% of
HIV-infected people on chronic antiretroviral therapy.23
It was concluded, however, that GS-441524 was not thecause of the heart disease in this cat. Heart disease in thiscat was hypertrophic rather than the dilative form seenin patients with HIV, and HCM is a common condition
among shelter cats.24

Conclusions
The results obtained from the 31 cats treated with
GS-441524 exceeded expectations and indicate that FIP,
regardless of signalment or disease form, is a treatabledisease using nucleoside analogs. The study design andtreatment parameters determined from this limited fieldtrial will be essential for future efforts in the commercialization of this or similar drugs against FIP.
Acknowledgements We wish to thank the staff of theCenter for Companion Animal Health for helping with drug
shipments (Lyra Pineda-Nelson and Nancy Bei) and data
presentation (Cynthia Echeverria). We are especially grate­
ful to the many owners and the 31 cats who participated inan emotional and taxing journey that succeeded beyond all­expectations. We are also grateful to the private veterinarians

Pedersen et al281

who assisted with periodic blood testing and were there for us
and their patients/owners when needed.

Supplementary material The following files are available:
Client consent form.
Figure S1: Mean liver enzyme levels (IU/l) in cats during treatment with GS-441524. No significant changes were observed
over the entire treatment periods.
Figure S2: Mean serum lipase and amylase levels (IU/l) in catsduring treatment with GS-441524. No significant changes wereobserved over the entire treatment periods.
Figure S3: Mean blood urea nitrogen and creatinine levels incats during treatment with GS-441524. No significant changes
were observed over the entire treatment periods.
Conflict of interest MP and EM are employees of Gilead
Sciences, Foster City, CA, USA, and hold stock interests in thecompany.

Funding Financial support for this study was obtained fromthe Center for Companion Animal Health, UC Davis, thePhilip Raskin Fund, Kansas City, and numerous donors to the
SOCK FIP organization as directed by Ms Carol Horace. The
GS-441524 used in this trial was provided by Gilead Sciences,
Foster City, CA.

ORCID iD Niels C Pedersen
0001-6269-929X

https://orcid.org/0000-

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