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Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis

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Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned cats with various forms of feline infectious peritonitis (FIP). Methods Twenty cats from 3.3–82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study. Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However, disease signs recurred 1–7 weeks after primary treatment and relapses and new cases were ultimately treated for a minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1–7 weeks of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged 3.3–4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment and at the time of writing for 5–14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12 weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat with only mesenteric lymph node involvement that went into remission after three relapses that required progressively longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption of permanent teeth in cats treated before 16–18 weeks of age. Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has opened the door to targeted antiviral drug therapy.
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https://doi.org/10.1177/1098612X17729626
Journal of Feline Medicine and Surgery
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Introduction
Drugs that directly inhibit virus replication have become
mainstays in the treatment of chronic viral infections
such as HIV/AIDS,1 hepatitis C virus (HCV),2 hepatitis B
virus, herpesvirus and acute infections such as influ-
enza. RNA viruses such as HIV-1 and HCV possess ideal
targets for virus inhibition such as RNA-dependent RNA
polymerase and protease. Proteases are a particularly
good target because they are involved in virus matura-
tion (HIV) or production of functional viral proteins
(HCV). Protease inhibitors are also used in combination
with inhibitors of reverse transcription for HIV/AIDS
for lifelong therapy, and combinations of different
Efficacy of a 3C-like protease
inhibitor in treating various forms of
acquired feline infectious peritonitis
Niels C Pedersen1, Yunjeong Kim2, Hongwei Liu1,
Anushka C Galasiti Kankanamalage3, Chrissy Eckstrand4,
William C Groutas3, Michael Bannasch1, Juliana M Meadows5
and Kyeong-Ok Chang2
Abstract
Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned
cats with various forms of feline infectious peritonitis (FIP).
Methods Twenty cats from 3.3–82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field
trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered
subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study.
Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However,
disease signs recurred 1–7 weeks after primary treatment and relapses and new cases were ultimately treated for a
minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1–7 weeks
of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats
had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged
3.3–4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment
and at the time of writing for 5–14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12
weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat
with only mesenteric lymph node involvement that went into remission after three relapses that required progressively
longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection
and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption
of permanent teeth in cats treated before 16–18 weeks of age.
Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has
opened the door to targeted antiviral drug therapy.
Accepted: 3 August 2017
1 Center for Companion Animal Health, School of Veterinary
Medicine, University of California, One Shields Avenue, Davis,
CA, USA
2 Department of Diagnostic Medicine and Pathobiology, College of
Veterinary Medicine, Kansas State University, Manhattan, KS, USA
3 Department of Chemistry, Wichita State University, Wichita, KS,
USA
4
Department of Veterinary Microbiology and Pathology,
Washington State University, Pullman, WA, USA
5
Department of Veterinary Medicine and Epidemiology, School of
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,
One Shields Avenue, Davis, CA 95616, USA
Email: ncpedersen@ucdavis.edu
729626JFM0010.1177/1098612X17729626Journal of Feline Medicine and SurgeryPedersen et al
research-article2017
Original Article
2 Journal of Feline Medicine and Surgery
protease inhibitors have been highly effective in curing
HCV infection in people.2 Therefore, it is not surprising
that viral protease should also be an attractive target for
research on RNA virus infections of animals. Kim et al
synthesized peptidyl compounds that target 3C-like pro-
teases (3CLpro) and evaluated them for their efficacy
against feline coronavirus (FCoV) and feline calicivirus,
as well as important human RNA viruses that encode
3CLpro or related 3C protease.3–6 They identified a series
of compounds that showed potent inhibitory activity
against various coronaviruses, including FCoV, with a
wide margin of safety. The in vivo efficacy of their
3CLpro inhibitors was evaluated in mice infected with
murine hepatitis virus A59, a murine coronavirus, and
found to cause significant reductions in virus titers and
pathologic lesions.5
There are currently no commercially available antivi-
ral drugs for coronavirus infections in people or animals,
and the studies of Kim et al showed that, as a proof of
principal, inhibition of 3CLpro can lead to suppression
of coronavirus replication in vivo.4,5 They suggested that
some of their 3CLpro inhibitors may be used as thera-
peutic agents against these important viruses in domes-
tic and wild cats. This was demonstrated to be the case in
a subsequent study using experimental feline infectious
peritonitis (FIP) virus (FIPV) infection in laboratory
cats.6 Although experimental FIPV infection is highly
fatal once the infection reaches a definable stage, 14–20
days of GC376 treatment caused rapid disease remission
in six cats that has lasted over 12 months at the time of
writing in the remainder.
Materials and methods
Official protocols
This study was conducted under protocol 18731
approved by the Institutional Animal Care and Use
Committee and the Clinical Trial Review Board of the
Veterinary Medical Teaching Hospital Clinical Trials
Committee, University of California, Davis. This proto-
col detailed the conditions of the testing of a novel pro-
tease inhibitor, GC376, in client-owned cats. Each owner
was required to read and consent to the conditions of the
trial (supplementary material).
Organization of clinical trial
The present study was designed to evaluate the 3CLpro
inhibitor GC376 in a group of cats with naturally occur-
ring FIP. The study did not incorporate a placebo group,
because, as noted by Miller and Brody,7 ‘the leading ethi-
cal position on placebo-controlled clinical trials is that
whenever proven effective treatment exists for a given
condition, it is unethical to test a new treatment for that
condition against placebo’. GC376 was proven to be
highly effective in curing cats of experimentally induced
FIP prior to the study and an effective treatment was
presumably available. A natural history of disease group
substituted for placebo controls.8 None of the 20 treated
cats demonstrated lasting favorable responses to treat-
ments they received prior to GC376 therapy.
Institutional rules precluded the use of cats obtained
from shelters or similar facilities for research even of this
type, thus requiring that all cats be legally owned/
adopted and treated with strict client consent (supple-
mentary material). Cats with clinically apparent neuro-
logic disease were also not included. Twenty cats from
various regions of the USA, of varying age and with dif-
ferent presenting forms of FIP were ultimately enrolled in
the trial. This relatively small group of cats allowed valu-
able insights into trial design, owner interaction and com-
pliance, monitoring safety and efficacy, establishing a
minimal dosage regimen, evaluating disease relapses
during or following treatment, and determining the clini-
cal forms of FIP most amenable to treatment. This infor-
mation will, hopefully, assist in the additional testing
required for licensing and eventual commercialization of
GC376 and for conducting similar trials of future antiviral
drugs for FIPV and other chronic viral infections of cats.
Description of treatment cohort
Twenty cats and their owners were ultimately included
in the trial and pertinent information on each cat is
given in Table 1 and on the entire trial group in Figure 1.
Cats presented to the study with varying degrees of pre-
testing by their primary-care veterinarians. This testing
usually included a complete blood count (CBC) with
total plasma protein, globulin (G), albumin (A), A:G;
serum chemistry profile and analysis of effusions,
including total protein, actual or estimated cell count,
and type of inflammatory cells. Additional testing had
been performed on a small proportion of cats and
included FIPV antibody titers, abdominal or thoracic
ultrasound, biopsies of diseased tissue and quantitative
real-time PCR (qRT-PCR) on effusions.
Cats with clinical signs indicative of neurologic
involvement were excluded from the trial based on ear-
lier unpublished experimental studies with GC376. One
cat that was a long-term survivor of an earlier pharma-
cokinetic and efficacy trial of GC376 had a recurrence of
FIP manifested by neurologic signs 6 months after what
appeared to be successful treatment of acute infection.6
This cat failed to respond to a repeat course of GC376,
which prompted a study of the ability of the drug to pen-
etrate into the brain. GC376 levels in the brains of labora-
tory cats were only 3% of plasma drug concentrations.
Disease conrmation
The diagnosis of FIP was confirmed at the time of entry
into the study based on signalment, clinical history,
examination of prior laboratory test results, physical
examination and repeat of basic blood and effusion
Pedersen et al 3
analyses. Manual palpation of the abdomen was usually
sufficient to identify ascites, enlarged mesenteric lymph
nodes, enlargement of the cecum and associated ileo-
cecal-colic lymph nodes, masses in the kidneys and
colonic infiltration. Manual palpation was augmented,
when necessary, by ultrasonography. Eyes were exam-
ined initially with directed light for any abnormalities in
the retina, precipitates in anterior chamber or on the
back of the cornea, and aqueous flare. The presence of
ocular disease was confirmed when in question by com-
plete ophthalmoscopic examination conducted by the
ophthalmology service of the Veterinary Medical
Teaching Hospital (VMTH), UC Davis. The presence of
FIPV was further confirmed by qRT-PCR,6 either from
abdominal or thoracic effusions taken at the time of
admission or at the time of necropsy. Sequencing of the
FIPV protease gene was carried out on cats that relapsed
while on therapy, to determine whether potential muta-
tion conferring drug resistance had occurred.5,6
The diagnosis of dry-to-wet FIP in three cats (CT03,
CT10 and CT14) was based on diffuse enlargement of the
colon and histories of loose stools, blood and mucus in the
stool, straining to defecate and small-caliber stools prior to
occurrence of abdominal effusions. Colonic FIP has been
described as a specific variant form of non- effusive FIP.9
Dry-to-wet FIP was also suspected in cats CT01, CT02 and
CT12, owing to a stunting of growth that preceded the
appearance of abdominal effusions by many weeks.
Treatment regimen
GC376 was synthesized in a highly pure form and formu-
lated at a concentration of 53 mg/ml in 10% ethanol and
90% polyethylene glycol 400, as described previously.6
GC376 was administered subcutaneously (SC) at a
Table 1 Signalment, environmental origin, major presenting clinical signs and principal lesions found at necropsy
following treatment with GC376 protease inhibitor
ID/name Age
(months)
Weight
(kg)
Sex Breed Origin Presenting signs Clinical status/necropsy lesions
Gross Histologic
CT01 (Echo) 5.6 1.64 FS DSH KR Peritonitis, stunted B, Int
CT02 (Cate) 6 2.67 FS DLH KR Peritonitis, stunted B, E, Int, L, MLN
CT03 (Pancake) 7.86 3.18 MC Him Cattery Dry (Col) to wet +Int, L, MLN, S, Om, P
CT04 (Kratos) 82 4.8 MC DSH KR Dry (MLN) Remission
CT05 (Scooter) 10 4.25 MC DSH KR Dry (E, MLN, K) B, E, L, K, MLN
CT07 (Mac) 6.6 2.6 MC DSH KR Dry (Col) +E, Int, L, MLN, S, K, A,
Lu
CT08 (Phoebe) 4.2 2.18 FS DSH KR Dry (E) B, E, K, MLN, S
CT09 (Sammy) 10.5 2.89 MC DSH KR Dry (MLN, K) ?* B*
CT10 (Bandit) 17.9 4.06 MC Him Cattery Dry (Col) to wet +B, E, Int, L, MLN, K,
Om, P, Lu
CT12 (Daisy) 7.5 2.5 FS DSH KR Peritonitis, stunted B, Int, L, S
CT13 (Leo) 7.4 1.97 MC Sphynx Cattery Dry (E, K) +B, E, Int, L, MLN, S, K
CT14 (Muffin) 8 2.94 FS DSH KR Dry (Col) to wet +E, Int, L, MLN, K, Om, P
CT15 (Flora) 4.3 2.39 F DSH FC Peritonitis Remission
CT16 (Bean) 4 1.4 FS DSH KR Peritonitis, stunted +B, E, Int, L, MLN, S,
Om, P
CT17 (Peanut) 4.4 2.3 M DSH KR Peritonitis Remission
CT18 (Smokey) 4 1.84 MC DSH KR Peritonitis Remission
CT20 (Cloud) 3.3 1.55 M RM Cattery Pleuritis (MLN) Remission
CT21 (Phoebe) 4.8 1.92 F DSH KR Peritonitis Remission/relapse/
retreat
CT22 (Pepper) 3.3 1.6 F Siberian Cattery Peritonitis +B, E, Om, MLN, Lu, Dia
CT23 (Oakely) 3.9 3.1 FS DSH KR Peritonitis Remission
Mean 10.28 2.59
SD 17.22 0.94
FS = female spayed; F = entire female; DSH = domestic shorthair; KR = kitten rescue; B = brain; Int = intestine; DLH = domestic longhair; E
= eye; L = liver; MLN = mesenteric lymph nodes; Him = Himalayan; Col = colon; S = spleen; Om = omentum; P = peritoneum; MC = male
castrated; M = entire male; K = kidney; A = adrenal gland; Lu = lung; FC = feral colony; RM = Ragmuffin; Dia = diaphragm
*No necropsy performed but terminal neurologic signs
Severe cerebral edema, no typical inflammatory lesions noted
4 Journal of Feline Medicine and Surgery
dosage of 15 mg/kg q12h SC, unless stated otherwise.
The effective dosage for cats with experimentally induced
FIP was 10 mg/kg/ q12h SC, but the dosage was raised
to 15 mg/kg after the first cat (CT01) failed to respond to
a lower dose of 10 mg/kg suggested by earlier pharma-
cokinetic studies.6 This was a clinical decision based on
this one cat’s response to treatment.
Monitoring response to treatment Cats with FIP based on
pretesting and initial evaluation at the time of presenta-
tion to UC Davis were hospitalized for at least 5 days
and immediately started on treatment. They were closely
evaluated at least twice daily for rectal temperature,
pulse, respiration, appetite and activity. Clumping litter
was used, allowing for daily evaluation of stool volume
and consistency, and urination. Whole blood was col-
lected in EDTA or heparin by venepuncture prior to
starting treatment, at 2 day intervals while hospitalized,
at the time of discharge and at 2 week intervals for the
first month and at monthly or greater intervals thereaf-
ter. Routine blood testing at each time point included at
a minimum a hematocrit, total plasma protein, icterus
index, total white blood cell count, differential white
blood cell percentages, and absolute neutrophil, lym-
phocyte, and monocyte and eosinophil counts. Blood
serum chemistry values were taken periodically to check
for potential drug toxicities. Samples of abdominal effu-
sions were obtained by paracentesis every other day if
obtainable, which was usually for the first 3–7 days. Cats
presenting with dyspnea were examined by thoracic
ultrasonography and a fluid sample obtained by ultra-
sound-guided paracentesis. Effusions were examined
for presence of fibrinous precipitates, an admixture of
neutrophils and small/large mononuclear cells, inten-
sity of yellow discoloration, viscosity by threading and
total protein content. Cell pellets from peritoneal or tho-
racic effusions were also examined by qRT-PCR for viral
RNA levels as previously described.6
Cats were discharged to their owners when a positive
response to treatment was noted, usually within 5 days.
The owner(s) were instructed either by the trial veteri-
narian or primary-care veterinarian on how to adminis-
ter the medication twice a day by subcutaneous injection.
Injection sites were varied to include the topline from the
nape to the mid-back and on the sides of the chest and
flanks. Care was taken to avoid depositing drug into the
dermis or sequentially at the same subcutaneous site.
Owners were encouraged to keep daily logs on rectal
temperature, activity, appetite, defecation and urination,
and weekly to bi-weekly body weights. Periodic blood
samples for CBC and serum chemistry values were
obtained by the owners’ personal veterinarians and sent
Figure 1 Demographics of cats enrolled in the trial. (a–c) Pie charts summarizing the percentage of patients: (a) by age in
months, (b) breed or (c) origin. (d) A bar graph showing the feline infectious peritonitis (FIP) forms of the enrolled patients.
M = months; DSH = domestic shorthair; DLH = domestic longhair; MD = Maryland; OH = Ohio; Tx = Texas; FL = Florida;
IL = Illinois; CT = Connecticut; CA = California
Pedersen et al 5
to commercial veterinary diagnostic laboratories. Any
abnormal signs or behaviors were to be noted and
promptly reported. Euthanasia, when required, was con-
ducted either at UC Davis or by the primary-care veteri-
narian. Bodies of cats euthanized by primary-care
veterinarians were immediately refrigerated and sent on
ice packs by overnight express mail to UC Davis for nec-
ropsy. The owners’ request for care and final disposition
of the body was honored.
Results
Determining the duration of treatment
The first five cats in the trial were treated initially for 2
weeks (CT01, CT02, CT03, CT04 and CT05). A rapid
improvement in health was observed in all cats and treat-
ment was stopped. Despite the favorable initial response,
disease signs recurred 1 (CT01, CT05), 2 (CT03, CT04) or
7 (CT02) weeks after the 2 week treatments ended (Figure
2). The cats were then retreated, the rationale being to
progressively extend primary and secondary treatment
periods for as long as their FIP remained responsive to
GC376 (see CT04, CT22, Figure 2). New cats that entered
the trial were next treated for 3 (CT07) or 4 weeks (CT08,
CT16). Cats CT08 and CT16 responded initially, but their
disease signs reappeared while on treatment. Cat CT08
developed neurologic disease, whereas cat CT16 had a
recurrence of abdominal lesions (Table 1). The primary
and secondary treatment periods were then extended to
9 weeks (CT07, CT09, CT10, CT14) (Figure 2). Cat CT09
developed neurologic signs during the 9 week primary
treatment and was eventually euthanized when disease
signs became severe. Cat CT07 developed neurologic dis-
ease 6 weeks after starting the second treatment. At that
point, all new cats admitted to the trial, and earlier cats
such as CT10, were treated or retreated for at least 12
weeks. The benefit of 12 weeks of treatment was most
apparent in cat CT04 that had been treated three other
times for shorter periods and then eventually relapsed
(Figure 2). Treatment was stopped in cats that had no
clinical or laboratory disease signs after 12 weeks of
either primary or secondary treatments. It was deter-
mined that a minimum treatment period should be
around 12 weeks. Cat CT21 was treated for 17 weeks
because of delayed improvement in total protein and
white blood cell counts (Figure 2). This cat suffered a
relapse of pleural FIP 13 weeks later and is at the time of
writing receiving more treatment.
Response to initial treatment and favorable
response indicators
A dramatic and progressive improvement in health was
observed in 19/20 cats during the first 1–4 weeks of
Figure 2 The time scale for treatment and clinical outcome of 20 cats entered into a field trial of GC376 protease inhibitor.
Periods during which cats were treated are identified by solid lines. The date of the last day of treatment for the six cats that
achieved a sustained clinical remission is indicated. Cat 21 was still on treatment at the time of writing. The remaining 13
cats succumbed to non-neurologic (FIP) or neurologic FIP (neuro-FIP) after being off primary or secondary treatments for
0–7 weeks
6 Journal of Feline Medicine and Surgery
treatment. The exception was cat CT16, which responded
with a decrease in rectal temperature during the first
4 days of treatment. However, the fever returned and
health continued to deteriorate over the next 23 days and
the cat was euthanized. The fever (>102.5°F) in the other
19 cats disappeared within 24–48 h, associated with a par-
allel improvement in appetite, activity, growth and weight
gain. Abdominal effusions were usually non-detectable
within 2 weeks. The residual thoracic effusion remaining
after initial therapeutic drainage was largely gone after 3
days in cat CT20. Renal masses in cats CT02 and CT13 also
decreased rapidly in size and were non-palpable after 2
weeks. Enlarged mesenteric lymph nodes were somewhat
slower to return to normal size. Palpable thickening of the
colon and associated ileo-cecal-colic masses were the
slowest to resolve and colonic signs persisted in cat CT03
in the face of treatment and a return to otherwise normal
health. Jaundice, a frequent finding in younger cats with
effusive FIP, slowly resolved over 2 weeks or more, in par-
allel with decreasing hyperbilirubinemia. Signs of ocular
disease began to clear within 48 h and were gone by 1
week, regardless of initial severity (Figure 3).
Weight gain was a simple and accurate measure of
growth and improvement of health. The value of monitor-
ing this parameter was typified by CT04, the oldest cat in
the trial (Figure 4a). Cat CT04 presented with significant
weight loss of 30%. It gained weight after each round of
treatment, began to lose weight shortly before each disease
relapse, and regained and added to its weight after each
treatment. It regained all its lost weight after 9.3 months off
and on treatment (4.8 kg to 7.19 kg). All kittens in sustained
remission steadily gained weight during and after antivi-
ral treatment, indicating normal growth continued with
antiviral treatment (Figure 4b). One long-term survivor
was spayed (CT15) and two castrated (CT17, 20) without
complications during their disease remissions.
Lymphopenia was a common presenting clinical fea-
ture of cats with wet FIP (Figure 5), and tended to be
directly correlated with the severity of abdominal
inflammation, as indicated by the viscosity, presence of
fibrin tags, protein content, cell count and degree of yel-
low coloring of the effusion. Lymphopenia improved
with treatment in all the cats with wet FIP except CT16,
but it was not helpful in predicting disease relapses that
occurred thereafter (Figure 5a). Lymphopenia was not as
evident in cats with dry FIP and not as helpful as other
parameters in assessing treatment response (Figure 5b).
Total plasma protein levels as an indirect measure of
globulin concentration were frequently elevated at pres-
entation, but the values were highly variable over the
first 4 weeks and often increased transiently during the
resorption of effusions. Cats that ultimately failed treat-
ment tended to have higher total plasma protein concen-
trations at the onset of treatment and tended to maintain
higher levels during treatment than cats that success-
fully achieved sustained remission (Figure 6).
Decrease in viral RNA levels in cells from ascitic
uid associated with treatment
Sequential ascites samples were collected from some of
these same cats over the first 6–25 days of antiviral treat-
ment and tested for levels of viral RNA by qRT-PCR.
FIPV levels are frequently low or negative in blood from
cats with FIP and are highest in cells from effusions.10
Therefore, cells from ascites or pleural effusions were
the most reliable source of FIPV RNA. Cats CT15, CT16
and CT17 had 955, 1699 and 2937 times higher levels of
viral RNA, respectively, than CT02, which had the low-
est virus load in effusion prior to treatment (Figure 7).
Viral RNA levels decreased by up to 1,567,463-fold
within 2 weeks compared with pretreatment values,
except for cat CT16 (Figure 8), which had the second
highest viral RNA levels prior to antiviral treatment
among the 12 cats with effusion samples available for
testing (Figure 7). The failure of viral RNA levels of
CT16 to rapidly decrease, along with severe lymphope-
nia, may explain why it did not respond to treatment.
CT10 also had a somewhat slower drop in virus levels
and relapsed twice after antiviral treatment. It is note-
worthy that viral RNA levels in ascites cells from cats
CT15, CT17 and CT18 decreased the fastest and were
also among the five cats that went into a sustained dis-
ease remission. Whether this was a property of the indi-
vidual FIPV isolates or the form and severity of the
host’s disease was not determined.
Treatment failure due to recurrence of abdominal
FIP or occurrence of neurologic disease
Thirteen of 20 cats in the trial ultimately succumbed to
recurrence of disease. One cat (CT16) failed to show sig-
nificant improvement and was euthanized 3 weeks after
Figure 3 Appearance of the eyes of cat CT08 before starting
treatment (a) and 1 week later (b). This cat developed severe
neurologic signs 3 weeks after starting treatment
Pedersen et al 7
starting a 4 week treatment regimen (Figures 2 and 8),
whereas the other 12 experienced variable periods of
disease remission following primary or secondary
treatment lasting from 3–17 weeks (average 7.8 weeks)
(Figure 2). All but one (CT09) of these 13 cats was
necropsied (Table 1). Eight of these cats were euthanized
because of severe neurologic signs and five because of
recurrences of abdominal disease (Figure 2). Three of
the cats that succumbed to neurologic disease (CT05,
CT08, CT13) presented because of ocular FIP or ocular
involvement was detected secondarily on examination
(Table 1). The earliest signs of neurologic disease
included fever that persisted in the face of continued
treatment, listlessness, sporadic muscle twitches of ears
and muscles, abnormal swallowing movements, com-
pulsive stretching of the limbs and loss of normal men-
tation indicated by brief staring episodes or withdrawal.
These signs persisted in the face of treatment for days
or weeks, but ultimately progressed to incoordination
and tonic/clonic seizures. The appearance and rapid
progression of neurologic signs was more apt to occur
after discontinuation of treatment than during treat-
ment (Figure 2).
Five cats (CT03, CT07, CT10, CT14 and CT16) had
recurrence of typical intra-abdominal lesions in the
absence of neurologic signs during or after treatment
(Table 1). Four of them presented with ileocecal masses
(CT03, CT07 and CT14) or an enlarged colonic lymph
node (CT10) that decreased in size (CT03, CT10 and
CT14) or became no longer palpable (CT07) following
primary treatment. However, CT03 continued to suffer
from severe constipation, straining and toothpaste-like
stool. The severity of the colonic obstruction necessitated
a colon resection, which relieved the clinical signs but did
not prevent eventual recurrence of abdominal disease.
All three cats that presented with severe ileo-cecal-colic
Figure 4 Antiviral treatment and body weight changes. (a) Cat CT04, a 6.8-year-old castrated male that presented with dry
feline infectious peritonitis (FIP), received four rounds of antiviral treatment of increasing duration and as indicated by the
dotted boxes. It lost weight preceding each relapse and gained increasingly more weight after subsequent treatment. (b)
Weight gains of four kittens of 3.5–4.4 months of age during and following antiviral treatment are depicted by dots. A dotted
box indicates the duration of antiviral treatment (12 weeks)
Figure 5 Mean and SD of absolute lymphocyte counts
for treated patients with (a) wet or (b) dry feline infectious
peritonitis (FIP). (a) Twelve cats (open circle) that presented
with the abdominal or thoracic effusion and followed for
up to 12 weeks. A thirteenth cat (CT16, closed circle) with
abdominal effusion responded poorly to treatment. (b) Seven
cats that presented with the dry or dry-to-wet forms of FIP and
followed for up to 6 weeks of treatment
8 Journal of Feline Medicine and Surgery
infiltrates still had evidence of this form of FIP at nec-
ropsy and immunohistochemistry demonstrated FIPV
antigen in macrophages within the granulomatous
inflammation (Figure 9).
Attempts to treat neurologic disease by increasing
drug dose and treatment duration
An attempt was made to alleviate neurologic signs by
increasing the dose of GC376, thus increasing blood
levels and the amount of drug that passed across the
blood–brain barrier. Cat CT01 presented with effusive
FIP and was initially treated with GC376 (10 mg/kg
q12h SC for 9 days). The cat responded well, but fever
returned on day 9 and the dosage was increased to 15
mg/kg q12h for 5 days. The fever disappeared and treat-
ment was stopped at day 14. Fever returned 3 days later
along with vague neurologic signs consisting of muscle
twitching, abnormal limb stretching and abnormal
Figure 6 Mean and SD of total plasma protein levels among 20 cats over a 12 week period. Thirteen cats suffered fatal relapses
at various weeks during treatment (W) and seven cats went into a sustained remission after 12 weeks of treatment (W-SV)
Figure 7 Relative baseline feline infectious peritonitis virus
(FIPV) RNA levels in effusion samples from the enrolled
patients prior to antiviral treatment. Quantitative real-time PCR
was performed on the pretreatment effusion samples of the
enrolled patients. The relative baseline viral RNA levels as
fold differences compared with the pretreatment viral level
of CT02, the cat with the lowest levels of RNA. The level of
RNA transcripts was calculated for each patient using the ∆Ct
method with a beta-actin reference gene
Figure 8 Reduction in feline infectious peritonitis virus RNA
from sequential effusion samples during GC376 treatment
of cats CT10, CT12, CT15, CT16, CT17, CT18 and CT23.
Each point indicates the fold reduction of viral RNA level over
that measured prior to treatment (day 0). Virus RNA levels
were determined using quantitative real-time PCR by the ∆Ct
method and a beta-actin reference gene
Pedersen et al 9
swallowing motions. The cat was immediately put back
on treatment at a dosage of 15 mg/kg q12h SC and its
condition improved but shortly worsened with return of
fever and the same vague neurologic signs with mild
incoordination. The dosage was then increased to 50
mg/kg q12h SC for 14 days and its condition improved
to near normal. Treatment was stopped but neurologic
signs immediately returned. The cat was then treated for
four additional days at 50 mg/kg q12h SC, during which
time the neurologic signs once again improved. However,
a decision was made to stop all treatment after that time.
The cat’s condition remained stable for 1 week and then
the cat developed extreme incoordination, dementia and
tonic/clonic seizures. Euthanasia was performed and a
necropsy demonstrated lesions only in the brain.
Cat CT12 responded well to treatment at a dosage of
15 mg/kg q12h SC; the rectal temperature returned to
normal within 48 h and the abdominal effusion disap-
peared within 2 weeks. The cat appeared normal after
the second week of treatment but then developed a per-
sistent fever of 102.5–104°F. The owners felt that the cat
was otherwise normal in activity and appetite so the
treatment was continued at the same dosage. However,
the fever persisted, subtle behavior signs were noted and
the cat failed to grow as expected. The cat continued
treatment for 15 more weeks during which time the drug
dosage was transiently decreased twice (ie, to 10 mg/kg
q12h and 15 mg/kg q24h) for several days, but the fever
increased and activity decreased each time and the 15
mg/kg q12h dosage was reinstituted. The cat continued
to show signs of variable fever and vague behavioral
signs, but the owners were optimistic about the cat’s
appetite and level of activity. The cat’s treatment was
then stopped as further use of the drug for this purpose
could not be justified. The cat’s condition remained
unchanged with persistent fever, reclusive behavior and
failure to grow for another 5 weeks. Severe neurologic
signs consisting of incoordination, dementia and sei-
zures appeared at week 22 and the cat was euthanized.
Gross and microscopic lesions were limited to the brain.
Testing for the emergence of viral resistance
The emergence of a drug-resistant virus was considered
in cat CT03, which relapsed with abdominal lesions after
an initial favorable response to granulomatous colitis
and dry-to-wet abdominal FIP. Granulomatous lesions
were still present in the abdomen at the time of necropsy
and no gross or microscopic lesions were found in the
brain (Table 1). Therefore, disease recurrence was not
related to neurologic disease and persistent FIPV anti-
gen was identified in macrophages in granulomatous
lesions. A sequence comparison was performed between
the 3CLpro of the pretreatment effusions and omentum
collected at necropsy 95 days later. However, no amino-
acid substitution was found in 3CLpro, indicating that
the emergence of a drug-resistant virus was not the cause
of the cat’s recurrent disease.
The 3CLpro sequences of pretreatment viral RNA
obtained 25 days (CT16), 139 days (CT02), 149 days
(CT12) and 231 days (CT10) later at the time of necropsy
were also compared. No differences were observed in
3CLpro over this time. Sequences also remained
unchanged in CT02, CT16 and CT12 from the times of
presentation to necropsy. The viral 3CLpro from lung and
spleen of cat CT10, which relapsed twice over 8 months
and retreated, had an Asp-to-Ser substitution at position
25 and a Lys-to-Asp substitution at position 260 com-
pared with virus from pre-treatment abdominal fluid.
The exact effects of these mutations on protease function
are currently being investigated. It is reported that genetic
evolution of viral protein quasi-species arises over time
in patients chronically infected with an RNA virus (HCV)
and may lead to sporadic amino acid changes.11
Occurrence of sustained clinical remissions
Seven of the 20 cats in the GC376 treatment trial, all of
which received at least 12 weeks of continuous treatment,
were categorized as potential treatment successes based
on more than 12 weeks of disease remission following ces-
sation of treatment (Figure 2). Six of these kittens pre-
sented with acute effusive disease of the abdomen (CT15,
CT17, CT18, CT21, CT23) or chest (CT20) at 3.3–4.4 months
of age and were treated continuously for 12 or 17 (cat
CT21) weeks (Table 1, Figure 2). A seventh cat (CT04), a
6.8-year-old random-bred castrated male presenting with
dry FIP limited to a mesenteric lymph node, also achieved
Figure 9 Section from the greatly thickened wall of resected
colon from cat CT03. Immunoperoxidase (brown color)
staining for feline infectious peritonitis virus antigen is seen
in macrophages around the periphery of a granulomatous
lesion. Virus persistence in the colon occurred in the
presence of treatment and regression of other signs of
disease (eg, effusive peritonitis)
10 Journal of Feline Medicine and Surgery
a long-term remission, but only after four rounds of treat-
ment of increasing duration (Table 1, Figure 2).
Six of these long-term survivors had abnormalities in
their CBC, hematocrit and total proteins at the onset of
treatment, but had completely normal blood values at
the time treatment was stopped. However, cat CT21 still
had elevated plasma protein levels and an increased
white cell count after 12 weeks and was continued on
treatment for another 5 weeks. Plasma protein and white
cell counts were improved after 5 additional weeks of
treatment but still not within normal parameters.
Thirteen weeks after stopping treatment the cat devel-
oped a typical FIP effusion in the chest with fever. The
chest fluid was drained to improve breathing and the cat
started on a second round of GC376 and and at the time
of writing was afebrile, active and eating normally after
8 weeks of treatment. Treatment will last for 12 weeks if
no signs of disease reappear.
Side effects observed during and after treatment
Two side effects of consequence were observed during
and after treatment with GC376. The drug often caused
stinging on injection. Subcutaneous swellings occurred
when too many injections were given in the same site but
rapidly resolved. A deep localized ulceration occurred
between the shoulder blades in one cat (CT12) at about
week 14 of an 18 week treatment period. However, no
evidence of dermal FIP was observed at necropsy and it
was most likely a response to continuous injections into
the same site. A survey of the seven long-term survivors
demonstrated a palpable focal subcutaneous thickening.
Four pea-sized nodules between the shoulder blades in
one cat were calcified on radiographs. These nodules
along with some surrounding fibrous tissue were surgi-
cally removed. Three other long-term survivors have 1–3
small focal areas of permanent hair loss in injection sites
that are obscured by the surrounding coat (Figure 10).
The owners and their veterinarians were asked to peri-
odically inspect these lesions for any changes in charac-
ter and to check for any new ones.
The most consequential side effect associated with
long-term treatment involved juvenile dentition. The
normal formation, growth and eruption of permanent
teeth were delayed in all four kittens that were treated
starting at 3.3–4.4 months of age. The canines, incisors,
fourth premolars and molars were the least affected,
whereas second and third premolars were the most
affected (Figure 11). The adult teeth appeared smaller
than normal and this coupled with delayed eruption led
to either retention of deciduous canines, failure of decid-
uous teeth to be shed or partial eruption of abnormal
permanent teeth lingual to retained deciduous teeth. No
other anatomic or physiologic defects have been
observed in any of the long-term survivors or noted on
those cats that were necropsied.
Necropsy ndings
The bodies of 12/13 cats that ultimately failed treatment
were submitted for necropsy including gross and histo-
logic examination and immunohistochemistry on dis-
eased tissues for FIPV antigen. Tissues collected and
examined included representative sections of all major
Figure 10 A focal area of permanent hair loss caused by
inadvertent deposition of GC376 in the epidermis of cat CT21.
These areas were usually covered by hair and not outwardly
visible
Figure 11 The adult dentition of cat CT17, which was treated
for 12 weeks with GC376 starting at 4.4 months of age.
There is retention of the upper left deciduous canine. The
upper second and third premolars appear to be deciduous.
Small permanent third premolars have partially erupted
lingual to the deciduous third upper premolars. The gingiva
surrounding the retained canine and premolars is inflamed.
The adult canines also appear smaller than normal. The
permanent right canine and fourth upper premolars appear
to have erupted normally
Pedersen et al 11
abdominal and thoracic organs, brain and eye. Gross
examination identified three distinct presentations. Five
cats had no gross evidence of active FIP (CT01, CT02,
CT05, CT08, C12), three had lesions consistent with non-
effusive FIP (CT07, CT10, CT13) and four had effusive
peritonitis with multiorgan involvement (CT03, CT14,
CT16, CT22). Histology of the five cats lacking gross evi-
dence of disease demonstrated mostly mild mononuclear
infiltrates, usually perivascular inflammation in the eye,
liver, intestinal wall and kidney. The three cats with non-
effusive FIP had mild-to-severe inflammation in many
organs with the most severe lesions occurring in the eye,
mesenteric lymph nodes, kidney and lung. The three cats
with effusive FIP had severe pyogranulomatous
inflammation in multiple abdominal organs including
the omentum, peritoneum, intestinal wall, mesenteric
lymph nodes, liver and spleen.
Severe inflammation stereotypic of cerebral FIP was
present in the brains of all but one (CT07) of the eight
cats that presented to necropsy with no gross evidence of
FIP or with non-effusive FIP. The one cat without charac-
teristic brain lesions had severe cerebral edema. In con-
trast, typical FIP lesions were absent in the brains of all
three cats that presented to necropsy with effusive FIP.
Stereotypic FIP lesions of the brain were characterized
by moderate-to-severe chronic meningoencephalitis and
ventriculitis associated with periventricular parenchy-
mal necrosis (Figure 12a). The fourth ventricle was most
severely affected and meningitis was most commonly
observed ventral to the cerebellum and brainstem. Thick
perivascular cuffs associated with vasculitis were fre-
quently observed. FIP antigen was demonstrated by
immunoperoxidase staining in the brain of 6/7 cases
with stereotypic cerebral FIP (Figure 12b). Tissues from
the 11 cats that were necropsied were tested for the pres-
ence of FIPV RNA by qRT-PCR. All of them tested posi-
tive thus establishing the persistence of virus in cats
failing treatment.
Discussion
Success with GC376 treatment against experimental
FIPV infection prompted us to investigate whether
GC376 could be equally efficacious against naturally
occurring FIP.6 There are significant differences between
experimental effusive abdominal FIP and the naturally
occurring disease. Experimental disease bypasses the
critical early stage that starts as kittens with exposure to
an innocuous feline enteric coronavirus (FECV).12
Naturally occurring FIP results from specific mutants
that arise following FECV infection and FIP occurs in the
presence of FECV immunity.13,14 Experimental FIP, in
contrast, is induced in coronavirus naïve cats by an intra-
peritoneal injection of a large dose of laboratory cat-
passaged and purified FIPV. Naturally occurring disease
is often subclinical for many weeks or months before
outward signs of disease are observed, whereas experi-
mental disease signs appear within 2–4 weeks and rap-
idly progresses. Naturally occurring FIP presents in a
variety of clinical forms, whereas the experimental infec-
tion is almost always effusive and abdominal. FIP in
nature is also affected by a milieu of disease enhancing
cofactors, while experimental disease occurs in cats that
are free of such extraneous influences.15 Differences may
explain why a small proportion of cats naturally exposed
to FIPVs develop disease, whereas 80–100% of experi-
mentally infected cats die.15,16 Our predictions proved
correct and naturally occurring FIP was much more dif-
ficult to treat than the experimental disease. It must be
stressed, however, that this trial would not have been
Figure 12 Photomicrographs of a lesion in the brain of cat
CT08. This cat developed severe neurologic disease while
on initial GC376 treatment. (a) The fourth ventricle contains
proteinaceous fluid admixed with numerous neutrophils and
macrophages that multifocally extend into the surrounding
rarefied neuropil. Large cuffs of lymphocytes and plasma
cells surround blood vessels (*) (hematoxylin stain, ×20
magnification). (b) Multiple cells resembling peritoneal
macrophages (outlined by the small rectange in box in
Figure 4a) demonstrate positive immunoreactivity for feline
infectious peritonitis antigen (hematoxylin counterstain, ×600
magnification)
12 Journal of Feline Medicine and Surgery
approved without the information obtained from phar-
macokinetic, acute and chronic toxicity, and efficacy
studies conducted on laboratory cats.
This was the first attempt to use a targeted antiviral
drug against a systemic and highly fatal disease of vet-
erinary importance. Although no specific antiviral drugs
are yet available for coronavirus infections in people or
animals, antiviral drugs for other viral infections of peo-
ple, such as HCV and HIV-1, have been developed for
treatment and the use of these drugs provided a sound
base for their application to animal diseases such as FIP.
HCV mainly infects liver cells, causing persistent viral
infection in a majority of people. However, only about
20–30% of them develop liver diseases in 20–30 years.
HCV infection can be cleared with non-specific antiviral
treatment (interferon and ribavirin) over 6–12 months in
about half of patients, and recent introduction of direct-
acting antiviral drugs of 3–6 months, duration consider-
ably increased the cure rate to more than 90%.2 HIV
infection in people leads to a prolonged asymptomatic
state and eventually to advanced HIV disease. HIV-1
infects T cells and macrophges and survives in a latent
state. More than 30 antiretroviral drugs, most of them
used in combinations of two or more drugs, have been
successfully used to reduce viral load to undetectable
levels in the blood of HIV/AIDS patients. However, the
virus rebounds on discontinuation of antiviral treat-
ment, necessitating life-long antiviral treatment.
Dissemination of virus to the brain, which is mainly
mediated by virus-infected macrophages, and subse-
quent development of neurologic disease occurs in more
than 50% of HIV infections.17 Therefore, neurologic
impairment still remains an important problem even in
this antiviral treatment era. These precedents of antiviral
treatment of HCV and HIV-1 infections show that treat-
ment outcome (viral clearance vs viral persistence),
treatment duration (finite vs continous) and the presence
of neurologic sequelae are greatly influenced by viral
pathogenesis.
The present study was limited to 20 cats with FIP, rep-
resenting a spectrum of ages and disease forms. Although
the number of treated cats was small, a surprising amount
of information was gleaned, such as how long to treat,
potential side effects, how to identify the clinical form of
FIP most likely to respond to the therapy, and potential
indicators for treatment failures and successes. The field
study was based on the experiences gained from phar-
macokinetic and efficacy studies undertaken in labora-
tory cats. The initial treatment period was set to be 2
weeks based on experimental studies, but it was ulti-
mately extended to 12 weeks and longer based on experi-
ences obtained as the trial progressed. This final treatment
period was closer to the 3–6 months used to treat HCV
infection in people with direct-acting antiviral drugs.2
Difficulties in treating neurologic disease was also
anticipated from experimental studies. Side effects were
acceptable and included stinging upon injection and der-
mal and subcutaneous inflammation when too much
drug was given in the same spots. This was also seen first
in laboratory cats. A more serious side effect, which was
not seen beforehand in laboratory cats, was limited to kit-
tens and involved retarded development of adult teeth
and retention or delayed loss of deciduous teeth.
GC376 treatment was successful in inducing a signifi-
cant remission of disease signs and regression of lesions
in 19/20 cats. This result confirms our findings of rapid
reversal of clinical signs in laboratory cats with experi-
mental FIP treated with GC376,6 and extended our
knowledge of the drug’s effects on a broad spectrum of
forms of naturally occurring FIP. Cats came from various
parts of the USA and even Peru, thus confirming that
geographically diverse field strains of FIPV were equally
susceptible to this inhibitor. Marked reductions in viral
RNA transcripts occurred in effusions within days of
treatment and were associated with rapid improvement
of health. However, disease remission was sustained for
3 months and longer in only 7/20 of these cats. Failure to
achieve long-term disease remission was ultimately
associated with the occurrence of neurologic disease in
the absence of gross abdominal lesions or a recurrence/
persistence of gross abdominal lesions in the presence of
histologic lesions in the brain and/or eyes. These find-
ings indicate that FIPV has a greater propensity to spread
from the body cavities to the brain than previously
assumed, especially if given enough time. This spread
most likely involves infected macrophages that enter the
brain through small blood vessels in the meninges and
ependyma.18,19
Cats that developed neurologic disease did so either
while on treatment (CT05, CT08, CT22) or 2 (CT01, CT02,
CT09), 3 (CT13) or 6 (CT10) weeks after treatment was
stopped. The most likely explanation for this delay, as
well as some therapeutic benefit of higher dosages, was
that some GC376 was still able to penetrate into the
brain. GC376 levels in cerebrospinal fluid were only 3%
of plasma in the brain at 2 h after a subcutaneous injec-
tion at a dosage of 10 mg/kg (unpublished data).
Although the relative brain drug concentrations in these
cats were low, they were still 21.4-fold higher than the
levels required to inhibit virus replication in tissue cul-
ture. Given this finding, it was assumed that higher dos-
ages would allow more drug into the brain. This
assumption was bolstered by experiences with two cats
that manifested neurologic signs. Increasing the dosage
of GC376 to 50 mg/kg q12h in one cat (CT01) resulted in
a noticeable improvement but did not eliminate the signs
of brain disease. Extending treatment for a period of
almost 3 months at a dosage of 15 mg/kg q12h appeared
to delay the progression of neurologic signs in a second
cat (CT12), whereas attempts to reduce the total daily
Pedersen et al 13
dosage in this cat to 10 mg/kg q12h or 15 mg/kg q24h
caused neurologic signs to worsen. This suggested that
dosages of 15 mg/kg q12h or above allowed enough
GC376 to cross the blood–brain barrier to retard but not
eliminate neurologic signs.
The high incidence of central nervous system (CNS)
diseases in this study was greater than previously
reported and unexpected given that cats with signs of
brain or spinal cord involvement were excluded from
the trial.20 CNS disease was much more likely to occur in
older cats with dry or dry-to-wet disease than in young
cats with wet FIP. This indicates that FIPV can enter the
brain in many cats, if given enough time. Infection of the
CNS appears to involve peritoneal-type macrophages,
as FIPV-infected cells in the brains of cats with neuro-
logic FIP more closely resemble peritoneal rather than
resident brain macrophages.18,19 This should not be sur-
prising, as macrophages migrate to various tissues,
including the brain, to carry out immune surveillance
and are also targets for a range of infectious agents, such
as FIPV and HIV-1. Infected macrophages play a major
role in viral dissemination to the brain in patients with
HIV and detection of virus in the brain can occur within
weeks of infection.21,22 However, neurologic impairment
usually occurs at a later stage. Anti-HIV drugs also
reduce the frequency of severe neurologic impairment,23
as observed with FIPV and GC376 in the present study.
There are also alternative explanations. It is possible that
extra-CNS involvement may inhibit the development of
brain disease and vice versa. It is common to see CNS
disease occur in the absence of visceral disease and vice
versa.13,20 Suppressing viral replication in the non-
neuronal tissues may also enhance positive selection for
mutants that are more neurotropic or neurovirulent.
However, proof for the latter would require considerable
studies using laboratory cats.
There were certain forms of FIP that appeared to
influence treatment success. The behavior of GC376
treatment in treating ocular FIP was paradoxical as it
responded extremely well to GC376. Even though ocular
lesions responded to treatment, all of the three cats with
eye involvement ultimately succumbed to brain disease,
thus supporting the close anatomic relationship of the
eye and the CNS. Chronic ileocecal and colon involve-
ment and stunted growth in older cats also carried a bad
prognosis in this study. A number of these cats appeared
to develop abdominal effusions only as a terminal mani-
festation of their disease. Host factors also associated
with reduced response to antiviral treatment in other
viral infections, such as HCV, include age, sex, liver cir-
rhosis or fibrosis, race or body weight.24
Emergence of resistance is a major concern for any
antiviral drug, but FIPV is rarely transmitted from cat to
cat,13,14 and drug resistance, if it does occur, would only
be a problem for individual treated cats and not the
entire population. Although viral resistance to GC376
was not observed in up to 20 passages in vitro,6 suggest-
ing that resistance is not easily acquired, long-term and
repetitive treatment in vivo may be a stronger selection
factor. However, viral resistance did not appear to be
responsible for relapses of abdominal diseases in five
treated cats. These cats had granulomatous-like masses,
often in the colon and ileo-cecal-colic lymph nodes,
which may have provided a protected place for viruses
to persist. Protection of pathogens within granulomas is
a well-documented phenomenon for mycobacteria and
applies to other pathogens such as viruses.24 The status
of liver disease (cirrhosis) in HCV infection also
increases the risk of relapse and necessitates longer
treatment, indicating that viruses may also be protected
from drugs when in certain protected sites.2 The forma-
tion of ‘protective granulomas’ involves a large number
of chemokines and cytokines and upregulation of
chemokine receptors, addressins, selectins and integ-
rins.25 Persistence of pathogens in such protected sites
may require a higher dose of drug and a longer treat-
ment period.
Treatment failures may also result from an inability of
the host to mount a protective immune response during
the period when virus replication is being suppressed.
Such a failure has been observed for HCV infection of
people.2 T-cell-mediated immunity plays an important
role in protective immunity, which occurs for about 20%
in acute infection of HCV,26 and an equal or greater pro-
portion of FIPV infection.15–17 The possible synergism of
T-cell-mediated viral clearance and antiviral drug ther-
apy in cats with FIP remains to be investigated. There
may also be merit in combining antiviral drugs and stim-
ulants of T-cell immunity for FIP treatment, such as the
combination of interferons and ribavirin for the treat-
ment of HCV infection.2
The sustained remission in 6/7 cats that were treated
for 12 weeks or longer was somewhat predictable. These
cats were 3.3–4.4 months of age when presented for acute
signs of either abdominal (C15, C17, C18, CT21, CT23) or
thoracic effusive FIP (CT20). This made them younger
than all but three (CT8, CT16, CT21 ) other cats in the trial
and more closely resembling the 16-week-old laboratory
cats with acute-onset effusive FIP that responded well to
GC376.6 The disease, being more acute, may allow less
time for virus to spread to the brain or eyes. The acute-
ness of their disease may have also allowed less time for
the infection to permanently compromise any protective
immune response. The seventh cat, CT04, was an extreme
to these six younger cats. CT04 was the oldest cat in the
study at 6.8 years that presented with substantial weight
loss (30%) and disease limited to mesenteric lymph
nodes. CT04 suffered disease relapses requiring reinstitu-
tion of treatment, but all relapses were identical to the
presenting condition and did not involve the CNS. Cats
14 Journal of Feline Medicine and Surgery
with this form of FIP have been known to undergo spon-
taneous remissions, indicating that there is a tipping
point between immunity and disease.13,27,28 Cats CT04
and CT21 demonstrated the wisdom of re-instituting
treatment when relapses occurred, providing that those
relapses do not involve the eyes or nervous system and
are still drug responsive.
Determining a minimal period of treatment was
based on progressively increasing treatment times based
on favorable response to treatment. The expectation
based on experimental studies was that 2 weeks of treat-
ment might be sufficient; hence, that was used as a start-
ing point. However, this study indicated that a minimum
treatment period was closer to 12 weeks, which was sur-
prisingly close to the usual 12 week period required to
treat humans with HCV using protease inhibitors.2
However, the treatment period for HCV can vary from
8–24 weeks in different people. Cat CT21 was outwardly
healthy, active and growing after 12 weeks of treatment,
but total proteins and white blood cell counts had still
not returned to normal as they had in the other six cats.
Nonetheless, a decision was made to stop treatment after
17 weeks because of the long period of outwardly nor-
mal health. Whether treating for a longer period of time
would have prevented a disease relapse 13 weeks after
stopping treatment is open to conjecture, but it does
raise the point of how long treatment is required in some
cats. It also raises the question of how long remission
must be sustained to declare the disease cured rather
than in a sustained remission. The longest disease-free
period has been over 11 months at the time of writing,
with five other cats free of infection signs for 5–9 months.
Based on clinical and histologic evidence of neurologic
disease at the time of fatal relapses, it would seem that
the virus will eventually reach the brain and that this
may be the most important limiting factor in antiviral
drug treatment of FIP.
Although only one-third of cats were long-term survi-
vors, the 20 cats in this trial provide a basis for future
studies with GC376 and other antiviral drugs that will
follow. Not all cats will be treatable, but this should not
stop the effort. Normal health was returned to almost all
treated cats in this limited study, albeit only for several
weeks or months. It is important to recognize the univer-
sality of viral pathogens and to embrace the pioneering
drug development that has and is being clinically
applied to human diseases such as HIV/AIDS, hepatitis
C, Middle East respiratory syndrome, severe acute res-
piratory virus, Ebola and influenza.
Conclusions
Inhibition of the 3CLpro of FIPV with GC376 was effec-
tive under the conditions of this study in decreasing virus
replication and causing remission of disease signs in cats
with naturally occurring FIP outside of the CNS. However,
sustained remissions in this study were more apt to occur
in kittens <18 weeks of age presenting with acute onset
wet FIP or in cats with dry FIP limited to a mesenteric
lymph node and less likely to occur in cats greater than 18
weeks of age presenting with dry, dry-to-wet or ocular
disease. Failure to achieve sustained remission was asso-
ciated with either a high incidence of neurologic disease
during or after treatment is stopped or to recurrence of
abdominal lesions. Antiviral treatment seemed to slow
the progression of neurologic disease but did not reverse
it at the dosage employed in this trial. The reason for
recurrence of extra-neurologic disease in the face of treat-
ment was not determined but was not associated with
mutations in the protease binding region.
Supplementary material Owner informed consent form.
Conict of interest YK, KOC and WCG have patent claims
on the protease inhibitors in the manuscript. The other authors
declare no potential conflicts of interest with respect to the
research, authorship, and/or publication of this article.
Funding Principal support for this study was made possible
by a grant from the Morris Animal Foundation, Denver, CO,
USA. Additional funds for technical support and animal care
were provided by the Philip Raskin Fund, Kansas City, SOCK
FIP, National Institutes of Health grant R01AI109039 and the
Center for Companion Animal Health, University of California,
Davis, CA, USA.
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... Many treatments, including prednisolone, feline interferon omega (rFeIFN omega) [46], polyprenyl immunostimulant [47], meloxicam [48], and most recently specific antivirals [49][50][51][52][53] have been used. Those treatments involved in the present study are detailed in Tables 1 and 2. There are three possible outcomes following the diagnosis of FIP and treatment: death due to FIP-related causes; total recovery; or remission, defined as an intermediate stage between cure and death and carrying the specter of relapse. ...
... In their report of their seminal study of successful treatment of FIP with an antiviral drug, Pedersen et al. wrote: "It raises the question of how long remission must be sustained to declare the disease cured, rather than in a sustained remission." [49] The purpose of this paper was to attempt to answer that question, but as opposed to duration of survival, we found that a return of elevated AGP to normal levels is a rapid and definitive marker for recovery from FIP. ...
... The state of remission from FIP in our series usually culminated in death, mainly by euthanasia due to a prolonged cachexic state, or sometimes acutely, presenting as acute haemolytic anaemia, collapse, or the onset of neurological signs. Neurological relapses were a problem in previous studies [49][50][51]. While parameters and a scoring system for predicting imminent death have been well documented [34], no such system has been previously defined to differentiate recovery from remission. ...
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Feline infectious peritonitis (FIP) is a systemic immune-mediated inflammatory perivasculitis that occurs in a minority of cats infected with feline coronavirus (FCoV). Various therapies have been employed to treat this condition, which was previously usually fatal, though no parameters for differentiating FIP recovery from remission have been defined to enable clinicians to decide when it is safe to discontinue treatment. This retrospective observational study shows that a consistent reduction of the acute phase protein alpha-1 acid glycoprotein (AGP) to within normal limits (WNL, i.e., 500 μg/mL or below), as opposed to duration of survival, distinguishes recovery from remission. Forty-two cats were diagnosed with FIP: 75% (12/16) of effusive and 54% (14/26) of non-effusive FIP cases recovered. Presenting with the effusive or non-effusive form did not affect whether or not a cat fully recovered (p = 0.2). AGP consistently reduced to WNL in 26 recovered cats but remained elevated in 16 cats in remission, dipping to normal once in two of the latter. Anaemia was present in 77% (23/30) of the cats and resolved more quickly than AGP in six recovered cats. The presence of anaemia did not affect the cat’s chances of recovery (p = 0.1). Lymphopenia was observed in 43% (16/37) of the cats and reversed in nine recovered cats but did not reverse in seven lymphopenic cats in the remission group. Fewer recovered cats (9/24: 37%) than remission cats (7/13: 54%) were lymphopenic, but the difference was not statistically different (p = 0.5). Hyperglobulinaemia was slower than AGP to return to WNL in the recovered cats. FCoV antibody titre was high in all 42 cats at the outset. It decreased significantly in 7 recovered cats but too slowly to be a useful parameter to determine discontinuation of antiviral treatments. Conclusion: a sustained return to normal levels of AGP was the most rapid and consistent indicator for differentiating recovery from remission following treatment for FIP. This study provides a useful model for differentiating recovery from chronic coronavirus disease using acute phase protein monitoring.
... Both lichen compounds show a behavior that is not new in the scenario of viral protease inhibitors. For instance, Boceprevir, a serine protease inhibitor used to treat HCV since 2011 covalently binds the NS3 protease [18], whereas GC376 is a covalent inhibitor of cysteine proteases of picornaviruses, noroviruses, and coronaviruses [40,41]. ...
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The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CL pro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CL pro. The compounds are slow-binding inactivators of 3CL pro with a Ki of 3.95 μM and 3.77 μM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki at about 3 × 10 −5 s −1 μM −1. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 μM, which is well below the Ki values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CL pro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors.
... The highly conserved catalytic mechanism of coronaviral M pro makes it an attractive therapeutic target for antiviral drug design. In fact, the di-peptidyl bisulfite adduct, GC376, has been successfully applied to treat feline infectious peritonitis in cats [12,13]. Encouraged by this success, GC376 has been widely tested for its in vitro and in vivo efficacy against various coronaviruses, including TGEV (transmissible gastroenteritis virus) [14], FIPV [12], PEDV [15], SARS-CoV [16], MERS-CoV [17], and SARS-CoV-2 [16,18]. ...
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Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (Mpro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV Mpro and can thermodynamically stabilize its folding. The structure of CCoV Mpro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV Mpro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV Mpro and other animal CoV Mpros with SARS-CoV-2 Mpro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral Mpros, SARS-CoV-2 Mpro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46–49 in animal coronaviral Mpros has been replaced by a stable α-helix in SARS-CoV-2 Mpro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV Mpros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV Mpros among different species.
... Paxlovid™ consists of a combination of nirmatrelvir (PF-07321332) and ritonavir. Nirmatrelvir belongs to a group of compounds previously shown to specifically inhibit the coronavirus protease 3CL and successfully tested against SARS and Feline coronaviruses [102,103]. It cannot be considered a true repurposed drug since it was initially synthesized with the purpose to be tested against SARS-CoV-2. ...
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In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) was first identified in the province of Wuhan, China. Since then, there have been over 400 million confirmed cases and 5.8 million deaths by COVID-19 reported worldwide. The urgent need for therapies against SARS-CoV-2 led researchers to use drug repurposing approaches. This strategy allows the reduction in risks, time, and costs associated with drug development. In many cases, a repurposed drug can enter directly to preclinical testing and clinical trials, thus accelerating the whole drug discovery process. In this work, we will give a general overview of the main developments in COVID-19 treatment, focusing on the contribution of the drug repurposing paradigm to find effective drugs against this disease. Finally, we will present our findings using a new drug repurposing strategy that identified 11 compounds that may be potentially effective against COVID-19. To our knowledge, seven of these drugs have never been tested against SARS-CoV-2 and are potential candidates for in vitro and in vivo studies to evaluate their effectiveness in COVID-19 treatment.
... Compared with GC376 (EC 50 = 51 nM) in the same assay, four of the analogs exhibited substantially improved antiviral potency: EB46, EB54, EB55, and NK01-63, with EC 50 values of 14 nM, 5 nM, 11 nM, and 6 nM, respectively ( Supplementary Fig. 17b, Table 1). The improved antiviral-EC 50 values in human Huh-7 ACE2 cells for these analogs are not only in line with their nanomolar biochemical-IC 50 For therapeutic consideration, we measured the cellular toxicity of all GC376 analogs in Huh-7 ACE2 cells ( Supplementary Fig. 17c). As no compound exhibited significant toxicity at the tested concentrations up to 50 µM, which is approximately 10,000-fold more than the antiviral-EC 50 values of the most potent GC376 analogs in the same cells against SARS-CoV-2, the therapeutic potentials of optimized GC376 analogs appeared promising and selective. ...
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The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses. Small molecule drugs promise to remain a valuable tool in controlling the ongoing COVID-19 pandemic. Here the authors describe optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for potential treatment of COVID-19.
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Hepatitis C virus (HCV) infection may lead to significant liver injury, and viral, environmental, host, immunologic and genetic factors may contribute to the differences in the disease expression and treatment response. In the early 2000s, dual therapy using a combination of pegylated interferon plus ribavirin (PR) became the standard of care for HCV treatment. In this PR era, predictive factors of therapy response related to virus and host have been identified. In 2010/2011, therapeutic regimens for HCV genotype 1 patients were modified, and the addition of NS3/4a protease inhibitors (boceprevir or telaprevir) to dual therapy increased the effectiveness and chances of sustained virologic response (SVR). Nevertheless, the first-generation triple therapy is associated with many adverse events, some of which are serious and associated with death, particularly in cirrhotic patients. This led to the need to identify viral and host predictive factors that might influence the SVR rate to triple therapy and avoid unnecessary exposure to these drugs. Over the past four years, hepatitis C treatment has been rapidly changing with the development of new therapies and other developments. Currently, with the more recent generations of pangenotipic antiviral therapies, there have been higher sustained virologic rates, and prognostic factors may not have the same importance and strength as before. Nonetheless, some variables may still be consistent with the low rates of non-response with regimens that include sofosbuvir, daclatasvir and ledipasvir. In this manuscript, we review the predictive factors of therapy response across the different treatment regimens over the last decade including the new antiviral drugs.
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Unlabelled: Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. Importance: Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats, and virulent, systemic feline calicivirus (vs-FCV) causes a highly fatal disease in cats for which no preventive or therapeutic measure is available. The genomes of these distinct viruses, which belong to different virus families, encode a structurally and functionally conserved 3C-like protease (3CLpro) which is a potential target for broad-spectrum antiviral drug development. However, no studies have previously reported a structural platform for the design of antiviral drugs with activities against these viruses or on the efficacy of 3CLpro inhibitors against coronavirus infection in experimental animals. In this study, we explored the structure-activity relationships of the derivatives of 3CLpro inhibitors and identified inhibitors with potent dual activities against these viruses. In addition, the efficacy of the 3CLpro inhibitors was demonstrated in mice infected with a murine coronavirus. Overall, our study provides the first insight into a structural platform for anti-FIPV and anti-FCV drug development.
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This review paper summarizes the epidemiology of hepatitis C virus (HCV) and chronic HCV infection, including HCV virology and treatment regimens. Specifically we focus on the evolution of past, current and future HCV treatment options, the reasons for treatment failure and the impact of resistance-associated variants on treatment success. This article is protected by copyright. All rights reserved.
Article
Objectives: The aim of the study was to evaluate central nervous system (CNS) lesions in non-effusive and effusive cases of feline infectious peritonitis (FIP) regarding aspects related to astrocytic and microglial reactions. Methods: Five necropsied cats that were naturally infected with FIP virus, confirmed by reverse transcriptase PCR and immunohistochemistry, with different intensities of CNS lesions, were studied. Brain and cerebellum were evaluated by light microscopy and immunohistochemistry for glial fibrillary acidic protein (GFAP) and vimentin to assess astrocytic morphology, and lectin histochemistry for Ricinus communis agglutinin-I (RCA-I) to detect microglia was performed to evaluate the glial response in the CNS of cats with FIP. Results: An important astrocytic response in many areas of the CNS of all cats, including the periventricular areas of lateral ventricles and fourth ventricle, the molecular layer of the cerebellum and cerebral cortex, was visualized. This astrocytic reactivity was associated with areas of granulomatous or pyogranulomatous vasculitis/perivasculitis in most cases, and it was characterized by multifocal to coalescing astrocytosis and astrogliosis with an increase in the expression of intermediate filaments, such as GFAP. However, astrocytes exhibited strong vimentin expression in neuroparenchyma with severe inflammatory and necrotic changes, but GFAP expression was mild or absent in these cases. A microglial response was present only in severe lesions, and RCA-I expression was detected primarily in gitter cells and resting microglia. Conclusions and relevance: The present study indicates a strong astrocytic response, including the presence of many less differentiated vimentin-positive astrocytes and gitter cells positive for RCA-1 in severe lesions in the CNS of cats with FIP.