Niels C. Pedersen, Nicole Jacque 

November 3, 2021

Introduction 

Resistance to antiviral diseases has been well documented for diseases like HIV/AIDs and hepatitis C. In  some cases, this resistance is present in the infecting virus, but more frequently it is a result of  prolonged drug exposure. Resistance to both GC376 [1] and GS-441524 [2] have also been documented  in cats with naturally acquired FIP. Resistance develops from mutations in regions of the viral genome  containing the targets for the antiviral drug. For instance, several amino acid changes (N25S, A252S or  K260 N), were found in the protease (3CLpro) of an FIPV isolate from a cat with resistance to GC376 [3].  The N25S change in 3CLpro was found to confer a 1.68-fold increase in the 50% inhibitory concentration of GC376 in tissue cultures [3]. Resistance to GC376, although recognized in the initial field trials, has not  been described at the current time. GC376 is not as popular a treatment for FIP and has not been  recommended for cats with ocular or neurological FIP [1].  

Natural resistance to GS-441524 was seen in one of 31 cats treated for naturally occurring FIP [2]. One  cat among 31 in the original field trial of GS-441524 also appeared to be resistant, as viral RNA levels did  not decrease over the entire treatment period and disease signs were unabated. Although this virus was  not studied, resistance to GS-5734 (Remdesivir), a prodrug of GS-441524, has been created in tissue  culture with amino acid mutations in RNA polymerase and proofreading exonuclease [4]. 

Resistance to GS-441524 has been confirmed in a proportion of cats that have been treated for FIP with  GS-441524 over the last 3 years, especially among cats with neurological FIP [5]. Resistance to GS 441524 is usually partial and higher dosages will often cure the infection or significantly reduce disease  signs if treatment is maintained. Interestingly, resistance to GS-441524 is also being detected in Covid 19 patients being treated with Remdesivir [12]. An immunocompromised patient developed a protracted course of SARS-CoV-2 infection. Remdesivir therapy initially alleviated symptoms and greatly  reduced virus levels, but her illness recurred along with a great increase in viral replication. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase which  was not present in pre-treatment specimens and conferred a ∼6-fold increase in resistance. 

The history of Molnupiravir and its recent application to treatment of FIP has been described [6].  However, there are no current studies documenting natural or acquired resistance to Molnupiravir.  Molnupiravir has been shown to function as an RNA mutagen inducing multiple defects in the viral  genome [7], while Remdesivir/GS-441524 is a non-obligate RNA chain terminator, [8], suggesting that its  resistance profile will be different.  

Overcoming GS-441524 resistance 

Drug resistance can only be overcome in two manners: 1) by progressively increasing the antiviral  dosage to achieve drug levels in body fluids that exceed the level of resistance), or 2) by using another  antiviral drug that has a different resistance mechanism, either by itself or in combination. Until now, the first option has been the one chosen and has proven effective in many cases. However, GS-441524 resistance can be total or so high that increasing the dosage is no longer viable. In such cases, the  second option has been increasingly utilized. Currently available alternatives to GS-441524, although  still from the unapproved market, are GC376 and Molnupiravir.  

Antiviral drug treatment regimens for GS-441524 resistance GC376/GS-441524 

A combined GS/GC regimen has proved successful on cats treated with GS-441524 at doses as high as 40  mg/kg without cure due to GS-441524 resistance. It is preferable to intervene as soon as GS-441524  resistance is noted, which allows the cat to be cured sooner and with less money spent by the owner.  

Rainman is the current supplier of GC376, which is supplied in 4 ml vials at a concentration of 53 mg/ml. 

GS/GC dosage: The GS dosage (SC or PO equivalent) for combined antiviral drug treatment cats is the  same as that required to reasonably control disease signs. This is usually the last dosage used before  treatment was stopped and a relapse occurred. GC376 is added to this dosage of GS-441524 at a dosage  of 20 mg/kg SC q24h regardless of form of FIP. This is sufficient for most cats, including many neuro FIP  cats, but some will need higher dosages. If this fails to achieve remission of clinical signs or bloodwork is  concerning, the GC376 dosage is increased by 10 mg/kg increments to as high as 50 mg/kg SC q24h.  

Length of treatment: Eight weeks of combined GC/GS therapy is recommended, which is on top of  whatever previous GS mono therapy has been completed. Some cats have been cured with 6 weeks of  combined therapy, but relapses are more likely to occur than after 8 weeks. 

Side effects: Most cats experience no serious side effects. However, about one in five cats may experience nausea or discomfort at the beginning of treatment and occasionally longer. These side  effects do not seem to be dose-dependent and can be treated with anti-nausea medications, such as Cerenia, Ondansetron, or Famotidine. Ondansetron appeared to work better in some cats.  

Molnupiravir 

Molnupiravir has been reported to be effective as a sole treatment for cats with FIP by at least one  Chinese sellers of GS-441524 [9], but no reports on its use for cats with resistance to GS-441524.  However, it is unlikely that resistance to GS-441524 will extend to Molnupiravir. The fact that it has been  found to be effective as an oral medication also makes it attractive as a solo treatment, as many cats  with GS-441524 resistance have suffered from injections over very long periods.  

The field trial for Molnupiravir purportedly consisted of 286 cats with various forms of naturally  occurring FIP seen in pet clinics in US, UK, Italy, Germany, France, Japan, Romania, Turkey, and China. No  deaths occurred among 286 cats that participated in the trial, including seven cats with ocular (n=2) and  neurological (n=5) FIP. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258  after 8 weeks. All treated cats remained healthy 3-5 months later, a period during which relapses would  be expected in cats not successfully cured. This data provides compelling evidence for the safety and  efficacy of Molnupiravir for cats with various forms of FIP. However, it is hoped that this field trial will be written in manuscript form, submitted for peer review, and published. Nevertheless, it is now being sold  to owners of cats with FIP. At least one other major seller of GS-441524 is also interested in using  Molnupiravir for FIP, indicating a demand for additional antiviral drug treatments to cats with FIP.  

Molnupiravir dosage: A safe and effective dosage for Molnupiravir in cats with FIP has not been  established by well controlled and monitored field trials such as those conducted for GC376 [1]  and GS-441524 [2]. However, at least one seller out of China has provided some  pharmacokinetic and field-testing data on Molnuparivir in cats with naturally occurring FIP in  their advertising flier for a product called Hero-2081 [9]. This information does not clearly state  the amount of Molnupiravir in one of their “50 mg tablets” and the actual dosing interval (q12h  or q24h?). The dosage used in this study also appeared to be too high. Fortunately, an  estimated starting dosage for Molnupiravir in cats with FIP can be obtained from published in vitro cell culture studies of EIDD-1931 and EIDD-2801 [15] and laboratory and field studies of  GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 uM/ul against FIPV in cell  culture, while the EC50 of GS-441524 is around 1.0 uM/ul [18]. They both have similar oral  absorptions of around 40-50%, so an effective subcutaneous (SC) dosage for Molnupiravir  would be approximately one-half the recommended 4 mg/kg SC q24h starting dosage for GS 441524 [14], or 2 mg/kg SC q24h. The per-os (PO) dosage would be doubled to account for less  efficient oral absorption to a dosage of 4 mg/kg PO q24h. An estimated starting oral dosage for An effective dosage for Molnupiravir in cats with FIP can also be calculated from available data  on Covid-19 treatment. Patients being treated for Covid-19 are given 200 mg of Molnupiravir  PO q12h for 5 days. This dosage was obviously calculated from a pharmacokinetic study done  on people, and if an average person weighs 60-80 kg (70 kg), the effective inhibitory dosage is  ~3.0 mg/kg PO q12h. A cat has a basal metabolic rate 1.5 times a human, and assuming equal  oral absorption for both people and cats, the minimum cat dosage by this calculation would be  4.5 mg/kg PO q12h for non-ocular and non-neurological forms of FIP. If Molnupiravir crosses  the blood-to-eye and blood-to-brain barrier at equal efficiency to GS-441524 [3,18], the dosage  would be increased ~1.5 and ~2.0 times to allow for adequate penetration into aqueous humor  and cerebrospinal fluid for cats with ocular (~8 mg/kg PO, q12 h) or neurological FIP (~10 mg/kg PO, q12h), respectively. These dosages are comparable to those used in ferrets , where 7 mg/kg  q12h sustained sterilizing blood levels against influenza virus (1.86 uM) of drug over 24 h [10]. Dosages  in ferrets of 128 mg/kg PO q12h produced near toxic blood levels, while a 20 mg/kg PO q12h produced  only marginally higher blood levels [10].  

Molnupiravir/GC376 or Molnupiravir/GS-441524 

Combinations of Molnupiravir with GC376 or GS-441524 will find increasing use, not merely as  to synergize or add to their individual antiviral effects, but as a way to prevent drug resistance.  Drug cocktails have been highly effective in preventing drug resistance in patients with  HIV/AIDS [11]. However, there is insufficient evidence currently for the safety and efficacy of  combinations of Molnupiravir with either GC376 or GS-441524 as the initial treatment of FIP. 

References

[1] Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M,  Meadows JM, Chang KO. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired  feline infectious peritonitis. J Feline Med Surg. 2018; 20(4):378-392. 

[2] Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. efficacy and  safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline  infectious peritonitis. J Feline Med Surg. 2019; 21(4):271-281.  

[3] Perera KD, Rathnayake AD, Liu H, et al. Characterization of amino acid substitutions in feline  coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease  inhibitor. J. Vet Microbiol. 2019;237:108398. doi:10.1016/j.vetmic.2019.108398 

[4]. Agostini ML, Andres EL, Sims AC, et al. Coronavirus susceptibility to the antiviral remdesivir (GS 5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI:  10.1128/mBio.00221-18. 

[5] [28] Pedersen NC. 2021. The neurological form of FIP and GS-441524 treatment.  https://sockfip.org/the-neurological-form-of-fip-and-gs-441524-treatment/ 

[6] Pedersen NC. The long history of beta-d-n4-hyroxycytidine and its modern application to treatment  of covid019 in people and FIP in cats. https://sockfip.org/the-long-history-of-beta-d-n4-hydroxycytidine and-its-modern-application-to-treatment-of-covid-19-in-people-and-fip-in-cats/. 

[7] Agostini, M. L. et al. Small-molecule antiviral beta-d-N (4)-hydroxycytidine inhibits a  proofreading-intact coronavirus with a high genetic barrier to resistance. J. Virol. 2019; 93,  e01348. 

[8] Warren, T. K. et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus  in rhesus monkeys. Nature 2016; 531, 381–385. 

[9] [26] FIP Warriors CZ/SK – EIDD-2801 (Molnupiravir) 

EIDD-2801 (Molnupiravir)

[10] Toots M, Yoon JJ, Cox RM, Hart M, Sticher ZM, Makhsous N, Plesker R, Barrena AH, Reddy PG,  Mitchell DG, Shean RC, Bluemling GR, Kolykhalov AA, Greninger AL, Natchus MG, Painter GR, Plemper  RK. Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human  airway epithelia. Sci Transl Med. 2019;11(515):eaax5866.  

[11] Zdanowicz MM. The pharmacology of HIV drug resistance. Am J Pharm Educ. 2006;70(5):100.  doi:10.5688/aj7005100 

[12] Gandhi, S, Klein J, Robertson A, et al. De novo emergence of a remdesivir resistance  mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised  patient: A case report. medRxiv, 2021.11.08.21266069AID  

Case Reports

Rocky – DSH MN Neuro FIP  

A 9‐month‐old castrated male domestic short hair cat obtained as a kitten from a rescue group  was presented with a several‐week history of seizures with increasing frequency, ataxia, and  progressive paresis. Bloodwork was unremarkable. Treatment for FIP was initiated at 15 mg/kg  BID GS-441524 tapered to SID over approximately a week. The cat showed improvement within  24 hours of starting treatment, with seizures halted and increased mobility. Within 5 days of  treatment, the cat was again ambulatory. About 2 weeks from the beginning of treatment  however, the cat experienced loss of vision, decreased mobility, resumption of seizures, and  difficulty swallowing. Adjustments in levetiracetam and prednisolone dose were made as well as  changes in GS-441524 formulation followed by transient improvements in mobility and  swallowing, and decreased incidence of seizures, however overall, the cats condition declined.  The dose of GS-441524 was progressively raised in increments to as high as 25 mg/kg with little  to no improvement. At this point, the cat was switched to oral GS at 25 mg/kg (estimated to be  equivalent to approximately 12.5 mg/kg) and within 3 days the cat became ambulatory, vision  improved, and seizures ceased, along with increases in energy and appetite. The cat continued to  experience improvement for about 4 weeks on oral GS-441524, then plateaued for about 3 weeks  before a rapidly progressing paresis. Oral doses as high as [30 mg/kg SC equivalent] were tried  to no effect. The cat was then switched to injectable GS-441524 at a dose of 20 mg/kg, and the  cat was again ambulatory within 4 days with good appetite and energy. After 2 weeks GC376  was added to the dosing regimen at 20 mg/kg BID. The cat completed 6 weeks of combined GS 441524 and GC376 therapy, and then discontinued treatment. While the cat has some permanent  neurological deficits, the cat has been stable with good mobility, appetite, and activity for 9  months since cessation of antiviral therapy.  

Video of Rocky: https://www.youtube.com/watch?v=RXB_NnfcMOY  

Bucky – DSH MN Neuro/ocular FIP  

A 4‐month‐old castrated male domestic short hair cat obtained as a kitten from a rescue group  was presented with a 1-month history of lethargy and progressive history of ataxia, rear limb  paresis, pica, uveitis, anisocoria, and urinary and fecal incontinence. Bloodwork mostly  unremarkable except for a mild hyperglobulinemia. The A/G ratio was 0.6. The cat was treated at  10 mg/kg GS-441524 SC SID for 3 weeks. Activity, mentation, and uveitis improved within  72 hours of treatment. Slow improvement in mobility and ocular symptoms were seen over the  first 2 weeks but then plateaued. At 3 weeks, the GS-441524 dosage was increased to 15 mg/kg  GS-441524 SC SID due to continued neurological and ocular deficits. Additionally, enlargement  of the left eye due to glaucoma was noted at this time, and the eye continued to swell until it was  removed in the 8th week of treatment.  

Due to continued weakness/lack of coordination in his pelvic area and increasing lethargy, in  week 9 the GS-441524 dosage was increased to 20 mg/kg SC SID, [or equivalent oral dose] and  20 mg/kg SC BID GC376 was added to the regimen several days later. Dramatically increased  activity and willingness to jump onto elevated surfaces was seen within 48 hours of starting treatment with GS376. Combined GS-441524 and GC376 therapy was maintained for 8 weeks.  The cat has residual incontinence issues post treatment but is otherwise clinically normal 6  months post treatment.  

Boris – Maine Coon MI wet ocular FIP  

A 5‐month‐old intact male Maine Coon cat, obtained from a breeder, presented with lethargy,  inappetence, abdominal ascites, coughing, anemia and neutrophilia. No chemistry panel was  done at diagnosis. The cat was treated at 6 mg/kg GS-441524 SC SID for 8 weeks. Six weeks  into treatment, x ray revealed nodules in his lungs, and at 8 weeks hyperglobulinemia persisted.  The dosage of GS-441524 was then increased to 8 mg/kg SC SID for 4 weeks. Only minor  improvement in bloodwork and x-rays were noted, and the dosage of GS-441524 was increased  to 12 mg/kg SC SID for 4 weeks, followed by an increase to 17 mg/kg for 11 weeks, 25 mg/kg  for 4 weeks, and 30 mg/kg for 4 weeks. At 25 weeks into treatment, ultrasound noted pleural  irregularities on the left side, and x rays showed no improvement in lung nodules. Additionally, uveitis and retinal detachment were noted in the right eye. Lung aspirates were obtained,  showing inflammation consistent with FIP. At 33 weeks into treatment, 20 mg/kg SC BID  GC376 was added to the regimen, and the combined GS-441524 and GC376 therapy was  continued for 12 weeks. Increased activity was noted within days. Within 5 weeks, weight gain  accelerated, coughing decreased, and energy level increased. Bloodwork showed improvement in  the A/G ratio and chest radiographs showed reduction in nodules in the lungs. After 84 days of  combined antiviral therapy, the A/G ratio was 0.85 and the cat appeared clinically normal. The  cat is currently 3 months post treatment.