Thirty‐two cats with effusive or non‐effusive feline infectious peritonitis treated with a combination of remdesivir and GS‐441524

Abstract Background GS‐441524 has been successfully used to treat feline infectious peritonitis (FIP) in cats. However, the use of its prodrug, remdesivir, in combination with a PO GS‐441524 containing product for the treatment of FIP has not yet been described. Objectives Describe treatment protocols, response to treatment and outcomes in cats with FIP treated with a combination of PO GS‐441524 and injectable remdesivir. Animals Thirty‐two client‐owned cats diagnosed with effusive or non‐effusive FIP including those with ocular and neurological involvement. Methods Cats diagnosed with FIP at a single university hospital between August 2021 and July 2022 were included. Variables were recorded from time of diagnosis, and subsequent follow‐up information was obtained from the records of referring veterinarians. All surviving cats were observed for the entire 12‐week treatment period. Results Cats received treatment with different combinations of IV remdesivir, SC remdesivir, and PO GS‐441524 at a median (range) dosage of 15 (10‐20) mg/kg. Clinical response to treatment was observed in 28 of 32 cats (87.5%) in a median (range) of 2 (1‐5) days. Twenty‐six of 32 cats (81.3%) were alive and in clinical and biochemical remission at the end of the 12‐week treatment period. Six of 32 cats (18.8%) died or were euthanized during treatment with 4 of the 6 cats (66%) dying within 3 days of starting treatment. Conclusions We describe the effective use of injectable remdesivir and PO GS‐441524 for the treatment of FIP in cats. Success occurred using different treatment protocols and with different presentations of FIP including cats with ocular and neurological involvement.

[5] GS-441524 is a nucleoside analog which prevents viral RNA replication by causing premature termination of viral RNA synthesis. 4mdesivir is monophosphate prodrug of GS-441524 which improves cellular penetration of the parent nucleoside. 5[8][9] Injectable GS-441524 has been successfully used for the treatment of effusive and non-effusive FIP in 25 of 31 cats treated with daily SC injections at a dosage of 2 to 4 mg/kg. 7Adverse effects were minimal and mostly attributable to injection site reactions.Disease relapse occurred in 8 of 26 cats within the first 3 to 84 days of treatment and was associated with lower treatment doses or treatment interruptions. 7Higher dosages of 5 to 10 mg/kg also have been effective in treating 3 of 4 cats with neurological FIP. 8 A PO multi-component drug (Xraphconn) containing GS-441524 was used to successfully treat 18 cats with FIP with minimal adverse effects. 9However, neither of these drug formulations are licensed, nor legally accessible, for veterinary use in the United Kingdom (UK).
1][12] As a result, access to injectable remdesivir for FIP treatment through a UK based compounding pharmaceuticals company became possible in August 2021 via the veterinary licensing cascade.This was followed by access to a PO formulation of GS-441524 3 months later.However, no studies have yet evaluated the combined use of injectable remdesivir and PO GS-441524 for the treatment of FIP in cats.9]13,14 Little therefore is known about optimal treatment doses for different disease presentations, treatment length and adverse effects with injectable remdesivir and PO GS-441524.
Ours is a retrospective, observational case series describing treatment protocols, adverse effects, treatment response including clinical and biochemical changes, outcomes and survival rates in 32 cats with FIP treated with a combination of PO GS-441524, injectable remdesivir or both.In our study, cats are described as achieving remission from FIP if they had resolution of all clinical signs and biochemical abnormalities by the end of the observed 12-week treatment period.
We aimed to describe treatment protocols and outcomes in cases of both effusive and non-effusive FIP as well as cats that presented with ocular and neurological involvement.

| MATERIALS AND METHODS
Ours was a retrospective study performed at the Queen Mother Hospital for Animals, Royal Veterinary College (London, UK) between August 2021 and July 2022.Ethical approval was granted by the institution's Ethical Review Board (URN SR2021-0208).Cats were included if a diagnosis of FIP was made by a board-certified small animal internal medicine specialist or a resident working under their supervision and the cat received treatment with either remdesivir or GS-441524.A minimum follow-up period of 12 weeks was required for all cats included, unless they were euthanized or died before the end of the follow-up period.A treatment period of 12 weeks was used for all surviving cats based on previous studies. 2,7,13All follow-up data was acquired from subsequent visits or from records of referring veterinarians.  3 | RESULTS

| Clinical presentation
Characteristics of 32 cats diagnosed with FIP are summarized in Figure 1 and Table S1.

| Presenting signs
The most common presenting signs for all 32 cats were lethargy

| Effusive and non-effusive disease
Twenty-five cats presented with effusive FIP.Four cats had pleural effusion, 16 cats had peritoneal effusion and 5 cats had bi-cavitary effusions.Seven cats presented with non-effusive FIP.

| Ocular signs
Six cats had evidence of ocular involvement.Four cats had chorioretinitis, 1 cat had left anterior uveitis with green iridial discoloration, 1 cat had bilateral anterior uveitis with keratoprecipitates, and 1 cat had bilateral uveitis and chorioretinitis.

| Neurological signs
Five cats presented with neurological signs.All 5 cats had marked obtundation, 2 cats had proprioceptive ataxia, 1 cat had cerebellar ataxia, and 2 cats had vestibular ataxia.

| Diagnostic testing
Hematologic and biochemical findings are summarized in Table 1 and Table S1.Diagnostic imaging, cytologic, histopathologic, and immunocytochemistry findings are summarized in Table 2. Five of 16 cats (31%) that had non-invasive blood pressure (NIBP) measured at presentation were hypotensive (Doppler NIBP <80 mmHg).

| Co-morbidities
One cat developed congestive heart failure while hospitalized and echocardiography was suggestive of hypertrophic cardiomyopathy phenotype.One cat developed phlebitis and necrosis of the right cephalic skin at a previous IV catheter site and required multiple general anesthetic administrations for surgical debridement and primary closure.One cat underwent sedation for nasogastric feeding tube placement and developed suspected aspiration pneumonia.
One cat that received IV meloxicam off-license for 3 days before presentation developed worsening acute kidney injury within 48 h of presentation.One cat (Ragdoll with effusive FIP) showed a slow clinical response to treatment with first signs of improvement after 5 days.This cat subsequently developed suspected aspiration pneumonia after placement of a nasogastric tube and was discharged 12 days into treatment.The cat suffered from a seizure 24 h after discharge with suspected progressive neurological FIP and was subsequently euthanized.
Excluding the 4 cats that were euthanized within 3 days of starting treatment, 26 of 28 cats (92%) were still alive at the end of the 12-week treatment period.

| Serum albumin: globulin ratios
Median serum albumin: globulin ratios continued to increase for all cats at 4, 8, and 12 weeks into treatment (Figure 3).A significant increase in albumin: globulin ratios occurred for cats with albumin: globulin ratios performed at both time points from week 0 to week 4 (P < .01),from week 4 to week 8 (P < .01)and from week 8 to week 12 (P = .01).

| DISCUSSION
Our case series demonstrates the effective use of injectable remdesivir and PO GS-441524 for the treatment of naturally-occurring FIP.When cats survived the initial 3 days of treatment, outcome was considered excellent, with 26 of 28 cats (92.8%) still alive and in clinical remission at the end of the 12-week treatment period.This outcome is consistent with previous success rates reported with SC GS-441524 treatment. 2,7,8Our study also supports client-reported survey results on a PO GS-441524-like compound for the successful treatment of FIP. 16rein, we describe treatment protocols for FIP using a combination of IV remdesivir, SC remdesivir and PO GS-441524.No stud- ies have yet assessed optimal dosing of remdesivir, the prodrug of GS-441524, for the treatment of FIP in cats.4,17 Optimal dosing of PO GS-441524 also has not yet been established although dosages of 5 to 10 mg/kg for 12 weeks have been used successfully. 9,16Doses equivalent to the doses used for IV remdesivir were used in our study because of the poor oral bioavailability of GS-441524 identified in other species despite its molecular weight being roughly half that of remdesivir. 18ditional pharmacokinetic studies are needed however to determine the optimal dosing of both remdesivir and PO GS-441524 for cats with FIP.
Because of the small number of cats receiving each treatment protocol and the retrospective nature of our study, comparisons cannot be made among treatment protocols and conclusions cannot be drawn on associated treatment success.However, effective treatment did occur using all treatment combinations.Treatment protocols were decided on a case-by-case basis and were influenced by several factors.A PO formulation of GS-441524 was not available for the period of August to November 2021 and, as a result, all cats were transitioned from IV to SC remdesivir.However, once available, cats were subsequently transitioned onto an equivalent dose of PO GS-441524 after variable durations of SC treatment.Subcutaneous injections were associated with a larger financial burden, difficulties in administration, poor patient tolerance and adverse effects such as pain on injection.As a result, SC injections in many patients consisted only of a short course or were negated altogether.In contrast, PO GS-44152 did not appear to be associated with clinically relevant complications and was well tolerated as has been reported previously. 9These findings also are consistent with previously reported owner perceptions that administration by injection was a major disadvantage in SC GS-441524 treatment. 16Poor compliance with treatment previously has been associated with an increased risk of relapse and must be considered when designing treatment protocols. 7The success of combining IV remdesivir and PO GS-441524 for the treatment of FIP in our study, in light of the disadvantages of SC remdesivir injection, therefore may lead to preferential use of PO GS-441524 over SC remdesivir injections in the future.
Treatment success also occurred in our study with different presentations of FIP including cats with neurological and ocular involvement.Cases of neurological and ocular FIP require higher doses of injectable GS-441524 because of poor drug access across the bloodocular and blood-brain barriers.without neurological signs developed neurological signs after discharge >1 week into treatment and subsequently were euthanized.These findings suggest that neurological disease developed and progressed despite treatment in these cats.This phenomenon has been reported previously, but the cause of treatment failure is unknown. 7It is possible that sub-therapeutic concentrations of GS-441524 within the cerebrospinal fluid and the development of viral resistance may play contributory roles. 2,7,13Additional pharmacokinetic studies therefore are required to determine optimal remdesivir and GS-441524 dosing in cases with ocular and neurological involvement.
In our study 6 of 32 cats did not survive the treatment period.
Four of these cats (67%) were hypoglycemic on presentation, which may reflect disease severity and the presence of severe inflammatory response syndrome or sepsis.Unfortunately, necropsy examination and blood cultures were not available for all cats.All 4 cats died within 72 h of starting treatment.Mortality in our study however also was influenced by other contributing factors including financial constraints resulting in cats not being allowed sufficient time to respond to treatment.True rates of clinical response therefore may be higher than our case series suggests.Additional investigations are required to identify whether cats that do not respond are refractory to treatment because of the presence or development of viral resistance or because of severe and irreversible inflammatory changes. 7,13 our study, 3 of 5 Ragdolls were euthanized because of their disease.Three of the 4 Ragdolls that survived more than 24 h demonstrated a delayed response to treatment and relapsed while receiving treatment, raising concerns for a poorer response in this breed.Only 1 Ragdoll in our study survived to 12 weeks without evidence for clinical relapse, however, there were too few Ragdolls in our study to determine if this breed is associated with poorer outcome, because this finding has not been reported in previous studies using GS-441524.Future studies investigating breed-specific F I G U R E 3 Box and whisker plot depicting albumin: globulin ratios of 25 cats with feline infectious peritonitis at 4, 8, and 12 weeks into treatment with remdesivir and/or GS441524.
differences in response to treatment in a larger cohort of cats are warranted to investigate this association further.
Follow-up hematology and serum biochemistry results were available for 25 of 26 cats, but only 9 cats had blood tests performed at every time point, which likely was a consequence of financial constraints of the owners.In our study, albumin: globulin ratios continued to improve significantly at every time point.This observation supports previous findings that the albumin: globulin ratio may be a useful treatment monitoring marker. 7Furthermore, complete resolution of hyperbilirubinemia, hyperglobulinemia, and hypoalbuminemia only occurred 8 to 12 weeks into treatment in some cats.These findings, along with similar findings in previous studies, therefore may support a minimum treatment period of 12 weeks. 7Additional studies are needed to identify the most effective treatment duration for the different presentations of FIP using different treatment protocols.
In our study, 32% of the cats had more than a 2-fold increase in ALT activity above the limit of the RI at 4, 8, or 12 weeks into treatment.This finding has been reported previously in cats treated with GS-441524. 9In humans with COVID-19, minor serum aminotransferase increases occur in patients treated with remdesivir without other evidence of hepatic injury, and resolve after cessation of treatment. 19,20The cause of hepatopathy with remdesivir and GS-441524 treatment is not understood, but may be the result of direct toxicity because of inhibition of mitochondrial RNA polymerase. 19,20 our study follow-up requirement was only the 12-week treatment period.As a result, the frequency of relapse of clinical signs and the duration of sustained remission after finishing 12 weeks of treatment cannot be established.Additional studies are required to establish longterm follow-up and outcome.
In conclusion, our case series demonstrates the effective use of injectable remdesivir and PO GS-441524 for the treatment of effusive and non-effusive FIP in cats.Success occurred using different treatment protocols, and with different presentations of FIP, including cats with ocular and neurological involvement.Additional prospective studies are required to identify the most effective treatment protocols for the different presentations of FIP, and to provide longterm followup information after the end of treatment.

ACKNOWLEDGMENT
No funding was received for this study.

CONFLICT OF INTEREST DECLARATION
United Kingdom based Specials Company BOVA contributed financially to the treatment of 1 cat in this study.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Approved by the Royal Veterinary College University, URN SR2021-0208.

2. 1 |
Data collection 2.1.1 | Signalment and presenting signs The following information was collected and recorded for all cats: weight, age, breed, sex and neuter status, body condition score, presenting signs and days from first clinical signs to diagnosis.Clinical examination findings were recorded including ocular examination by a board-certified ophthalmologist or resident under their direct supervision and neurological findings by a board-certified neurologist or resident under their direct supervision.All cats were classified as having effusive or non-effusive FIP based on the presence or absence of pleural or peritoneal effusion or both on point-of-care ultrasound examination or other imaging modalities.

3. 4 . 1 |
Intravenous remdesivir treatmentThirty of 32 cats commenced treatment with IV remdesivir.Median (range) time from admission to starting treatment was 1 (0-9) days and duration of IV remdesivir treatment was 5 (1-10) days.For all cats, remdesivir was diluted 50 : 50 with saline and given as a constant rate infusion over 1 h.Overall starting dosage was 15 (10-20) mg/kg.Nine cats (mainly those with stable effusive FIP) were given a starting dosage of 10 mg/kg once daily, 15 cats 15 mg/kg and 6 cats 20 mg/kg.Higher doses were used for cats with neurological FIP, those with ocular involvement or those with unstable effusive FIP, including those with hypotension, hypoglycemia, and severe pleural effusion.3.4.2 | Subcutaneous remdesivir treatmentEleven cats received SC remdesivir treatment after initial IV remdesivir treatment.Two cats (1 effusive FIP and 1 non-effusive, neurological FIP) received only SC remdesivir treatment after IV treatment and completed the 84-day treatment course.One cat received 2 doses of SC remdesivir before euthanasia.Eight cats received SC injections of remdesivir for a median (range) of 14 (2-68) days before transitioning onto an equivalent dose of PO GS-441524 to complete the 84-day treatment course.Median (range) dosage of SC remdesivir was 10 (10-15) mg/kg.3.4.3| Oral GS-441524 treatment Two cats never received IV remdesivir and were started on PO GS-441524 from initial presentation.One cat was markedly azotemic on presentation and was euthanized 48 h after beginning treatment because of worsening acute kidney injury.The second cat completed an 84-day course of PO GS-441524.Sixteen cats transitioned immediately from IV remdesivir to an equivalent dose of PO GS-441524 after 5 (5-10) days and completed the entire 84-day course.The dosage of PO GS-441524 was 15 (10-20) mg/kg.
Table showing diagnostic findings in 32 cats with feline infectious peritonitis.No clinically relevant adverse effects were observed with IV remdesivir or from PO GS-441524 treatment.Seven cats reacted adversely to SC injections with immediate pain reactions.All 7 of these cats received gabapentin before SC injection and 10 cats received topical vived to 12 weeks developed pyrexia and anorexia 2 days after finishing the initial treatment course.Clinical investigations were suspicious of a relapse, and the cat was started on a second 84-day course of GS-441524 at a dosage of 20 mg/kg (previously 10 mg/kg).The cat was in clinical remission after finishing the second 84-day treatment Two cats were euthanized after discharge at 12 and 13 days into treatment.One cat (Ragdoll with non-effusive FIP) showed an initial response to 15 mg/kg IV and SC remdesivir treatment and was discharged after 7 days.This cat developed thoracolumbar myelopathy within 24 h of returning home.The cat was restarted on IV remdesivir at 20 mg/kg for 5 days with no clinical improvement and was euthanized 12 days into treatment.Necropsy examination confirmed the clinical suspicion of progressive FIP within the central nervous system.
Table showing biochemical abnormalities present in cats with feline infectious peritonitis at time points 0, 4, 8, and 12 weeks into treatment with a combination of injectable remdesivir and oral GS-441524.