SUMMARY OF FIP TREATMENT USING GS-441524 (Dr. Pedersen)

Original article: SUMMARY OF GS-441524 TREATMENT FOR FIP
Niels C. Pedersen, DVM PhD, Professor Emeritus,
Pet Health Center, School of Veterinary Medicine, UC Davis

We use the same criteria to monitor treatment as described in clinical study published in JFMS (Journal of Feline Medicine and Surgery). Owners should monitor temperature, weight, activity, appetite, and clinical signs of the original disease at daily or weekly intervals. Blood tests - hematology and biochemistry (including serum protein values - total protein, albumin, globulin, A: G ratio) at the beginning of treatment and then every 4 weeks. It is always useful to update these values along with the weight in the form of a graph. The aim is to have a healthy, sensitive and active cat at the end of 12 weeks of treatment and with normal blood test values, especially in terms of hematocrit, total protein, globulin, albumin and A: G ratios. Significant weight gain is also a good sign, and some young or particularly emaciated cats can more than double their weight during treatment. This is, of course, an idealized treatment, and it should be appreciated that upward adjustments may be required if the response is slow or if complications such as ocular or neurological impairment occur during treatment.

Supportive (symptomatic) care may be required to stabilize cats that are critically ill at the time of diagnosis or during the first days of treatment with GS-441524 (GS). Abdominal effusion should not be aspirated unless it compresses the chest and interferes with respiration, as it is quickly replaced at the expense of the rest of the body. However, thoracic effusions are usually associated with varying degrees of dyspnoea and should be eliminated. Chest effusions return much more slowly. Symptomatic care also often includes fluids and electrolytes to suppress dehydration, antibiotics suspected of secondary bacterial infection and anti-inflammatory drugs (usually systemic corticosteroids), and rarely blood transfusions. Some cats with eye problems may also need topical medications to suppress severe inflammation and increased intraocular pressure (glaucoma).

Corticosteroids such as prednisolone should only be used during the first days of GS treatment and should be discontinued when there is a rapid improvement in health. Long-term use of corticosteroids with GS is strongly discouraged as it may mask the signs of improvement caused by GS, especially in cats with neurological FIP, has no therapeutic power and may interfere with the development of a protective immune response to FIP. It is possible that this immune response plays a major role in the final cure. If cats are on chronic steroid therapy, no dose reduction is required as there is no evidence that cats experience severe adrenal atrophy, which occurs in humans during long-term steroid therapy. Many owners, GS treatment consultants and veterinarians will use various promoted supplements to improve liver, kidney or immune system health, as well as vitamins such as B12. These substances do not have proven effectiveness and I consider them a waste of money.

Treatment with GS, which is the most common, can also be complicated by ulcers / lesions at the injection site. Treatment is difficult for both owners and cats because injections can be painful. In some cats, especially those with neurological impairments, there is a problem with the development of partial drug resistance, which requires an increase in dose. The response to treatment is usually within 24-72 hours and most cats return to normal or approach normal within 2-4 weeks, which is a good sign. We anticipate that the success rate of FIP treatment with GS-441424 is greater than 80%, given treatment failure due to misdiagnosis of FIP, inappropriate dosing, health complications, and drug resistance. Young cats are easier to treat and have a higher cure rate than cats older than 7 years. Cats with wet or dry FIP, with uncomplicated neurological or ocular symptoms, are easier to treat than cats with neurological FIP.

The starting dose for cats with wet or dry FIP without signs of ocular or neurological disease is 4-6 mg / kg daily for 12 weeks, with younger cats and wet FIP tending toward the lower limit and dry cases to the upper limit. Cats with ocular lesions and no neurological symptoms start with a dose of 8 mg / kg daily for 12 weeks. Cats with neurological symptoms start at a daily dose of 10 mg / kg for 12 weeks. If cats with wet or dry FIP initially show ocular or neurological symptoms, they switch to appropriate ocular or neurological doses. There is an oral form of GS available from at least two sources in China (Spark, Mutian), but I do not use it, so I do not know a comparable dosage. However, I do not recommend this if the injection dose rises above 10 mg / kg per day, as the effectiveness of oral absorption decreases at these high doses.

I recommend adjusting the dosage by weekly weight control. The weight gain of many of these cats can be huge, either because they are so skinny at first or they grow, or both. If weight loss occurs at the beginning of treatment, I remain at the original dose and do not reduce it. Failure to gain weight during treatment is considered a bad sign. We do not increase the dose unless there are serious reasons for this, such as worsening or improved blood test results, slow improvement, poor activity, restoration of the original clinical symptoms, or a change in the form of the disease, including ocular or neurological symptoms. This is where common sense comes in, because you don't want to get stuck on one blood level, which is not quite common, but does not affect the overall health of the cat. For example, globulin may still be a little high, but other important blood test values and health are very good. If there is a good reason to increase the dose, it should always be from +2 to +5 mg / kg per day and for at least 4 weeks. If these 4 weeks cause a prolongation of the 12-week duration of treatment, it is because of this dose adjustment. A positive response to any dose increase can be expected, and if you do not see an improvement, it means that the dose is still not high enough, drug resistance is developing, you have a bad GS brand, the cat does not have FIP, or there are other diseases that affect treatment.

One of the most difficult decisions is determining when to stop treatment. Although some cats, often younger with wet FIP, can be cured as early as 8 weeks, and possibly earlier, the usual duration of treatment is 12 weeks. Some cats may even require a dose adjustment and longer treatment periods. Critical blood levels such as hematocrit, total protein, albumin and globulin levels, and total white blood cell and absolute lymphocyte counts usually return to normal in treated cats after 8-10 weeks, when there is often an unexpected increase in activity levels. It is assumed, but there is no evidence yet, that after 8-10 weeks, the cat will develop its own immunity to infection. This is a situation that occurs in the treatment of hepatitis C in humans, which is also a chronic infection caused by the RNA virus, which often requires up to 12 or more weeks of antiviral treatment.

Unfortunately, there is no simple test to determine when a cure has taken place, and the fear of relapse often leads owners, treatment advisers and veterinarians to extend treatment beyond 84 days. Fear of relapses will also make people involved in the decision-making process too cautious about a single blood value that is slightly abnormal (eg, slightly high globulin or slightly low A: G ratio), or final ultrasound results suggesting suspected enlarged lymphatics. nodules, small amounts of fluid in the abdomen, or vague irregularities in organs such as the kidneys, spleen, pancreas, or intestines. It should be borne in mind that although most animals fall within the normal range of blood values, they are otherwise bell-shaped curves, and that there are a few exceptional patients who will have values at the edge of these curves. The ultrasound diagnosis must take into account the degree of pathology that may occur in the abdominal cavity affected by FIP, such as scars or some consequences in the form of organ changes in successfully treated cats. In situations where such questions arise, it is better to look more closely at the overall picture, and not just at one small part. The most important outcome of treatment is a return to normal health, which has two components - external health symptoms and internal health symptoms. External signs of health include a return to normal activity levels, an appetite, adequate weight gain or growth, and coat quality. The latter are often one of the best measures of health for a cat. Internal health symptoms are manifested by the return of certain critical values to normal based on periodic monitoring of complete blood counts and biochemistry. The most important values in the blood count are the hematocrit and the relative and absolute total number of white blood cells, neutrophils and lymphocytes. The most important values in biochemistry (or serum electrophoresis) are total protein, globulin, albumin and A: G ratio. Bilirubin is often elevated in cats by effusive FIP and may be useful in monitoring the severity and duration of inflammation. There are many other values in hematology and biochemistry panels, and it is not uncommon for some of them to be slightly higher or lower than normal, and it is better to ignore these values unless they are significantly elevated and associated with clinical symptoms - such as high urea and creatinine, which are also associated with increased water consumption, excessive urination, and abnormalities in urine analysis. The number of platelets in cats is notoriously low due to the trauma of blood collection and platelet aggregation, and should always be verified by a manual blood smear test. The final decision to discontinue or extend treatment when you encounter unclear doubts about different test procedures should always be based on external health manifestations more than on any single test result.

Different FIP groups have come up with different modifications of FIP treatment. Some groups will treat with an extremely high dose of GS from the beginning instead of increasing the dose only when indicated, or increase their GS dose in the last two weeks, or postpone treatment with a higher dose of GS in the hope of shortening the next two weeks. duration of treatment or reduce the likelihood of relapse. Some advocate the use of interferon omega or non-specific immunostimulants to further stimulate the immune system, and some use various other modifications. There is no evidence that modification of the extra high dose treatment will improve the cure rate. Similarly, interferon omega and non-specific immunostimulants have no demonstrated beneficial effects in FIP when administered as a single treatment or as adjuncts to GS. The practice of adding another antiviral drug, the viral protease inhibitor GC376, to the treatment of GS in cats that develop resistance to GS is also emerging, but this possibility still requires research. Finally, it is common for owners, treatment groups and veterinarians to add many supplements, tonics or injections (eg B12) to increase blood levels or to prevent liver or kidney disease. Such supplements are rarely needed in cats with pure FIP.

FIP relapses during the 12-week post-treatment observation period occur, and there is no simple blood test to predict whether a cure has occurred or is possible. Relapses usually involve infections that have entered the central nervous system (brain, spine, eyes) during treatment with wet or dry FIP, which has not been accompanied by neurological or ocular symptoms. The dose of GS-441524 used to treat these forms of FIP is often insufficient to effectively overcome the blood-brain or blood-eye barrier. The blood-brain barrier is more inaccessible than the blood-eye barrier, which explains why eye lesions are easier to treat than brain or spinal infections. Relapses that occur in the post-treatment period and that involve the eyes, brain or spine are usually treated for at least 8 weeks at an initial daily dose at least 5 mg / kg higher than the dose used during the primary treatment (eg 10, 12, 15 mg / kg daily). It is recommended that GS oral formulations not be used if the dose exceeds 10 mg / kg daily for injection, as intestinal absorption efficiency is reduced at high oral concentrations. Cats that cannot be cured of the infection at doses up to 15 mg / kg per day are likely to develop varying degrees of resistance to GS-441524. Partial resistance may allow the symptoms of the disease to be kept under control but not cured, while general resistance manifests itself in varying severity of clinical symptoms during treatment.

At the time of diagnosis, there may be resistance to GS-441524, but this is unusual. Rather, it occurs during treatment, and is often partial at first, leading to the need for higher dosing. In some cats, it may become complete. Resistance is a major problem in cats with neurological disease, especially those that have neurological symptoms or develop a brain infection during treatment, or during relapse after treatment has appeared to be successful. Many cats with partial drug resistance can be treated for signs of the disease, but relapse occurs as soon as treatment is stopped. The cats have been "treated" at the FIP for more than a year without healing, but eventually the resistance worsens or the owner runs out of money.

GS-441524 treatment shows no or minimal systemic side effects. It may cause mild kidney damage in some cats, but should not lead to kidney failure. Systemic vasculitis-type drug reactions have been observed in several cats and can be confused with injection site reactions. However, these drug reactions are in non-injectable areas and often go away on their own or respond well to short-term low-dose steroids. The main side effect of GS treatment is pain at the injection sites, which varies from cat to cat and according to the abilities of the person giving the injections (usually the owner). Injection site ulcers / lesions are a problem in some cats and usually occur when the injection site does not rotate (do not stay between the shoulders) and is not administered to the muscular and nervous layers under the skin. I recommend choosing places starting one inch behind the shoulder blades, down from the back to 1 to 2 inches in front of the tail and one third to half way down to the chest and abdomen. Many people use gabapentin before injections to relieve pain. The ulcers at the injection site are cleared of surrounding hair and gently cleaned 4 or more times a day with sterile cotton swabs soaked in dilute 1: 5 household hydrogen peroxide solution. They usually do not require any more complicated treatment and will heal in about 2 weeks.

We hope that the legal form GS-441524 will be available soon. The drug, called Remdesivir, is the greatest hope today, as Remdesivir breaks down into GS immediately when given intravenously to humans, mice, primates and cats. Remdesivir has received full US FDA approval, and similar approval is likely to follow in other countries. If so, it can be prescribed by any licensed human doctor and veterinarians. However, the use of Remdesivir in the United States is still limited to a specific subset of patients with Covid-19 and only under controlled conditions and with ongoing data collection. Until all restrictions are lifted, it will not be easily accessible for human use. I have no experience treating cats with Remdesivir instead of GS-441524. However, groups in Australia and some Asian countries are starting to use Remdesivir and are reporting the same results as GS-441524. The molar basis of Remdesivir is theoretically the same as GS-441524. GS-441524 free base has a molecular weight of 291.3 g / M, while Remdesivir has 602.6 g / M. Therefore, twice as much Remdesivir (602.6 / 291.3 = 2.07) would be needed to obtain 1 mg of GS-441524. The solvent for Remdesivir differs significantly from the solvent used for GS-441524 and is intended for IV use in humans. It is not known how diluted Remdesivir will behave when administered subcutaneously for 12 weeks or more. Mild signs of hepatotoxicity and nephrotoxicity have been observed with Remdesivir in humans. GS-441524 causes mild and non-progressive renal toxicity in cats, but without apparent hepatic toxicity. It is not clear whether the renal toxicity observed in humans receiving Remdesivir is due to its active ingredient (ie GS-441524) or to chemical agents designed to increase antiviral activity. Anivive is seeking GC376 approval for cats (and humans), but it will take another two or more years. GC376 is a viral protease inhibitor and acts differently from GS-441524, which inhibits early-stage viral RNA replication. Therefore, it is unlikely to have a significant synergistic viral inhibitory effect, but will be much more important in inhibiting drug resistance when used in combination therapy (such as combination antiviral therapy for HIV / AIDS).

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Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis

2/13/2019, Journal of Feline Medicine and Surgery; Translation 10.2.2021
Original article: Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis
Niels C PedersenMichel PerronMichael BannaschElizabeth MontgomeryEisuke MurakamiMolly Liepnieks, and Hongwei Liu

Abstract

Goals

The aim of this study was to determine the safety and efficacy of the nucleoside analog GS-441524 for cats suffering from various forms of naturally occurring feline infectious peritonitis (FIP).

Methods

Cats ranged in age from 3.4 to 73 months (mean 13.6 months); 26 cats had wet or mixed FIP, 5 dry form FIP. Cats with severe neurological and ocular FIP were not included in the study. The group started with GS-441524 at a dose of 2.0 mg / kg SC q24h for at least 12 weeks, which increased to 4.0 mg / kg SC q24h when indicated.

The results

Four of the 31 cats that had severe disease died or were euthanized within 2-5 days and the fifth cat after 26 days. The remaining 26 cats completed the planned 12 or more weeks of treatment. Eighteen of these 26 cats remain healthy at the time of publication (OnlineFirst, February 2019) after one round of treatment, while eight others suffered relapses within 3-84 days. Six of the relapses were non-neurological and two were neurological. Three of the eight rats were treated again with the same dose, while the five cats were increased from 2.0 to 4.0 mg / kg every 24 hours. Five cats treated with a second higher dose, including one with neurological disease, responded well and were healthy at the time of publication. However, one of the three cats re-treated with the original lower dose relapsed with neurological disease and was euthanized, while the two remaining cats responded favorably but a second relapse occurred. These two cats were successfully treated for the third time at a higher dose, which resulted in 25 long-term surviving cats. One of the 25 successfully treated cats was subsequently euthanized due to probable unrelated heart disease, while 24 remained healthy.

Conclusion and meaning

GS-441524 has been shown to be safe and effective in the treatment of FIP. The optimal dose was 4.0 mg / kg SC q24h for at least 12 weeks.

Keywords: Nucleoside analogue, GS-441524, feline infectious peritonitis, FIP, clinical study

Introduction

Drugs that inhibit viral replication have become a focal point in the treatment of human acute and chronic RNA and DNA infections. However, the development of interest in antiviral drugs for animal infections has been much slower. This is especially true for cats, which suffer from several chronic viral infections similar to humans. Infectious agents include feline leukemia and immunodeficiency virus (FeLV and FIV), feline herpesvirus (FHV), virulent systemic calicivirus, and coronavirus causing feline infectious peritonitis (FIPV). FeLV and FIV infections can be kept under control through testing, isolation and / or vaccination. The FHV-associated disease was the first feline viral infection to use an antiviral agent for treatment. Highly fatal systemic calicivirus affects only a small number of cats. FIPV infection is the best candidate for the development of antiviral drugs because vaccines are ineffective, the environment with many cats makes it very difficult to prevent and kills 0.3-1.4% cats worldwide.

The emergence of exotic diseases such as Ebola, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) in humans has prompted intensive research into drugs that inhibit RNA replication. One of the most promising antiviral drugs for nascent RNA viruses is the prodrug adenosine nucleoside monophosphate GS-5734 (Remdesivir; Gilead Sciences). GS-5734 demonstrated efficacy in experimental Ebola in rhesus monkeys and inhibited both epidemic and zoonotic coronaviruses in tissue cultures and mouse infectious models. These promising findings initiated research into GS-5734 and its original nucleoside GS-441524 against FIPV infection in cats. GS-441524 and GS-5734 have comparable EC50 (1.0 μM) and CC50 (> 100 μM) values against FIPV in feline cells. Therefore, it was decided to focus on the less chemically complex GS-441524 for further testing with laboratory cats. A pharmacokinetic study in two laboratory cats showed sustained and effective plasma levels of GS-441524 over 24 hours after a dose administered subcutaneously (SC) or intravenously (IV). These results were extended to 10 laboratory cats with experimentally induced abdominal effusion feline infectious peritonitis (FIP). This study demonstrated that GS-441524 is highly effective against experimental FIP, paving the way for this clinical study.

The aim of this study was to demonstrate the safety and efficacy of GS-441524 in the treatment of cats with naturally occurring FIP. Small molecule drugs, such as GS-441524, weigh <900 daltons and are approximately 1 nm in size and can easily penetrate cells and interact with key target molecules. Unlike previously published substances or drugs that inhibit FIPV by inhibiting cellular processes by viruses used for their replication, small molecules such as GS-441524 interfere directly with virus-encoded replication processes.

Materials and methods

Preparation of the drug

GS-441524 was provided by Gilead Sciences as a pure and highly stable powder, which was diluted to a concentration of 10 or 15 mg / ml in 5% ethanol, 30% propylene glycol, 45% PEG 400, 20% water (pH 1.5 with HCl). The solution was placed in sterile 50 ml glass vials in which it was shaken until dissolved and then subjected to a sonic water bath for 5 to 20 minutes until clear. The diluted drug was refrigerated and used within 3-4 weeks.

Study concept

This study was conducted in accordance with Protocols 19336 and 19863, approved by the Institutional Committee on the Care and Use of Animals and the Clinical Trials Evaluation Committee of the Veterinary Teaching Hospital of the University of California, Davis. The institutional rules exclude the use of sick cats from shelters or similar establishments, due to the requirement of legal ownership / adoption and treatment under specific conditions with the consent of the owner (supplementary material). The study did not include a control group because there is no effective comparative treatment. The placebo group was not included either, as in vitro and in vivo preparatory studies indicated that GS-441524 would be safer and more effective than any treatment.

Case selection and diagnosis confirmation

Cats with FIP were received from owners or their veterinarians who were looking for current treatment options or access to a previous study of antiviral drugs. The initial diagnosis of FIP was based primarily on characteristic signaling, clinical history and symptoms of the disease, results of routine laboratory tests, and examination of abdominal or thoracic effusions. A more definitive diagnosis based on RT-PCR or immunohistochemistry was desirable, but not a prerequisite for inclusion. Cats with overt ocular or neurological disease were excluded due to doubts about the ability of antiviral drugs, including GS-441524, to cross the blood-brain or blood-eye barrier.

31 cats and their owners were admitted to the study (Table 1). Owners or representatives of 26 cats came for initial treatment directly to UC Davis and five owners and their cats (CT59, CT73, CT76, CT78, CT80) were treated by a local veterinarian. The cats present at UC Davis were re-evaluated, their FIP diagnosis was re-confirmed on the basis of signaling, clinical history, physical examination, results of previous laboratory tests and complete blood count (CBC), serum protein and effusion analyzes. Thoracic or abdominal effusion in cats with wet FIP was confirmed positive for FIPV 7b RNA by RT-PCR. Cats with signs of non-fusion FIP were further tested by abdominal and thoracic ultrasonography for primary lesions. The eye disease was confirmed by the VMTH Ophthalmology Service, UC Davis. The neurological condition in cases with possible symptoms of central nervous system disease was evaluated by the VMTH neurological service.

Table 1
List of 31 cats included in the test, including laboratory designation, cat name, breed, clinical form of infectious peritonitis (FIP) and date of diagnosis

IDNameDate of birthTribeGenderOriginDate of diagnosisFIP form
CT52Moon9.1.2017SavannahFBreeder24.4.2017Abdominal effusion
CT53Ice Bear2.8.2016DLHMCRescue team5.5.2017Abdominal effusion
CT54Charolett11.7.2016SiberianFBreeder15.4.2017Abdominal effusion
CT55Dempsey26.6.2016DSHMCRescue team15.4.2017Abdominal effusion
CT56Mudsa1.7.2016DSHMCShelter12.5.2017Abdominal effusion
CT57Boone31.10.2016DSHFSRescue team8.5.2017Abdominal effusion
CT58Justyna17.4.2016RagdollFBreeder25.5.2017Abdominal effusion
CT59Bubba11.4.2011DLHMCWandering10.4.2017Abdominal non-fusion
CT60Joey25.7.2016DSHMCRescue team20.5.2017Abdominal effusion
CT61Hudson1.7.2016DSHMCRescue team29.5.2017Thoracic effusion
CT62Luca10.3.2016DSHMCRescue team30.5.2017Abdominal effusion
CT63Bao Bao6.11.2016DSHMCRescue team3.6.2017Abdominal effusion
CT64Cedrick27.6.2016DSHMCRescue team22.5.2017Abdominal non-fusion
CT65Mona14.3.2016Exotic SH / PersianFBreeder11.6.2017Thoracic effusion
CT66Squeekers7.6.2016DSHFSShelter14.6.2017Abdominal effusion
CT67Double2.3.2016RagdollFSBreeder20.6.2017Abdominal effusion
CT68Tuckerman8.5.2016Maine CoonMCRescue team22.6.2017Abdominal effusion
CT69Danny16.6.2015SnowshoeMCShelter22.6.2017Thoracic effusion
CT70Tolstoy1.8.2014DSHMCRescue team25.6.2017Abdominal effusion
CT71Amadeus29.6.2016DSHMCWandering20.6.2017Thoracic effusion
CT72Bella25.2.2017British SHFBreeder20.6.2017Abdominal effusion
CT73Siersha8.8.2015DSHFSShelter21.6.2017Abdominal non-fusion
CT74Maive4.3.2017SiberianFSBreeder7.7.2017Abdominal effusion
CT75Lucy31.3.2017DSHFRescue team10.7.2017Abdominal effusion
CT76Pie20.7.2016Exotic SHMBreeder28.6.2017Abdominal effusion
CT77Mila15.3.2017SiberianFSBreeder3.7.2017Abdominal effusion
CT78Polly1.3.2016DSHMCRescue team22.7.2017Abdominal non-fusion
CT79Oona21.9.2016HimalayanFBreeder18.7.2017Chest non-fusion
CT80Fezzik17.10.2016DLHMCWandering25.7.2017Abdominal effusion
CT81Jewelkat8.9.2016PersianFSBreeder1.8.2017Thoracic effusion
CT82Tiko8.4.2016DSHMCRescue team6.8.2017Abdominal effusion
F = uncastrated female; FS = castrated female; M = male uncastrated; MC = castrated male; DLH = domestic longhair; DSH = domestic shorthair; SH = short-haired;

Treatment regimen

Based on previous tissue culture experiments and pharmacokinetic studies in laboratory cats, the initial dosing regimen for GS-441524 was set at 2.0 mg / kg SC q24h. Based on experience with the 3CL protease inhibitor GC376 against naturally occurring FIP, a minimum treatment period of 12 weeks was established. Treatment was extended by one or more weeks in cats that still had abnormal serum protein levels. In later stages of the study, the dose was increased from 2.0 to 4.0 mg / kg in cases where treatment had to be prolonged or when the disease had relapsed. Every 4 weeks, a new dose of 1 or 3 ml Luer lock syringes with 1 inch Luer 22G (0.7x25mm) Luer needles was sent to the owners. The syringes were stored in a refrigerator and warmed to room temperature before administration. Injections were given along the spine from 2 cm behind the shoulder blades to half of the lumbar region and halfway down to the adjacent chest and hips.

Monitoring during the initial treatment period

At the time of entry into testing, the cats had stopped all treatment that was not necessary - antibiotics, corticosteroids, interferons, pentoxifylline, non-steroidal anti-inflammatory drugs, or painkillers. During their stay at UC Davis, cats were monitored every 12 hours for temperature, appetite, activity, urination and defecation. Blood was collected at 1-3 day intervals to evaluate hematocrit, total protein, bilirubin, white blood cell count and differential white blood cell count.

Ascites samples were taken initially and then taken at one or more daily intervals for as long as possible and tested for FIPV 7b RNA transcript levels by quantitative (q) RT-PCR (IDEXX molecular diagnostics). Formalin-fixed tissue sections from five dissected cats were subjected to immunohistochemistry for FIPV nucleocapsid protein.

Monitoring of initial and long-term response to treatment

Cats were released for home treatment when a significant favorable response to treatment was noted, usually within 3-5 days. During this period, owners were instructed on the proper administration of subcutaneous injections and were encouraged to continue daily records of body temperature, activity, appetite, defecation and urination, and weekly body weight measurements. CBC and serum chemical panel were performed at monthly intervals by local veterinarians or during VMTH visits. Any abnormal symptoms or behaviors should be recorded and reported immediately. Reasonable euthanasia was usually performed by the owner's veterinarian or, if possible, at UC Davis. The bodies were immediately cooled and sealed in plastic bags, and sent within 2 or fewer days in ice-cooled containers to UC Davis by express mail. Autopsies were performed by one of the authors (ML) at the Anatomic Pathology Service, School of Veterinary Medicine, UC Davis. The owner's request for the final disposition of the body was respected.

The results

Disease signaling and presentation

The study included 31 cats aged 3.4 to 73 months (mean 13.6 months) (Table 1). Eighteen cats were domestic short-haired and long-haired, 13 cats represented representatives of 10 different breeds (Table 1). Domestic cats were adopted from cat rescue organizations (n = 13), shelters (n = 2) or stray cats from the area (n = 3). The study included 14 females (7 neutered; 7 neutered) and 17 males (1 neutered; 16 neutered).

Twenty-six of the 31 cats had wet FIP (6 thoracic, 20 abdominal). Five cats had non-fusion FIP; four of them (CT59, CT64, CT73, CT78) with disease located in the abdomen (mesenteric and ileo / cecal / colic lymph nodes) and one (CT79) in the chest (lungs, hilar lymph nodes) (Table 1). Four additional cats showed signs of earlier dry FIP, which turned into an effusion form (CT57, CT65, CT67, CT71) (Table 1). Gross symptoms of eye disease corresponding to FIP were confirmed by ophthalmoscopic examination in three of the 31 cats (CT56, CT65, CT71). Two cats (CT71, CT80) reluctantly or were unable to jump to elevated sites at all, indicating neurological impairment.

Treatment results

Four cats were euthanized (CT62, CT72, CT75) or died (CT56) during the first 2-5 days due to serious illness and other complications, and the fifth cat was euthanized (CT54) after 26 days due to lack of response to treatment (Figure 1). Treatment was uninterrupted, with the exception of three cats who were given a two-week rest period at week 4 (cat CT80) or week 8 (cat CT53, CT71) due to injection problems and skin reactions (Figure 1). After the second relapse, the CT53 cat was treated for 8 and not 12 weeks due to an increase in blood urea and an increase in serum levels of symmetric dimethylarginine (SDMA).

Figure 1.
Treatment time scale and clinical outcome of 31 cats enrolled in clinical study GS-441524. The treatment period is indicated by a solid line (dose 2 mg / kg) or a dashed line (dose 4 mg / kg). Asterisks indicate a relapse point. The end date of treatment for cats that have achieved permanent clinical remission is indicated in parentheses. The time point and cause of death are marked with a cross

The clinical response of the 26 cats that completed at least 12 weeks of treatment was dramatic. The fever usually resolved within 12-36 hours (Figure 2), along with a significant increase in appetite, activity levels, and weight gain on a daily basis. Abdominal effusions quickly disappeared within 1-2 weeks, starting at about 10-14. the day after starting treatment. Cats with thoracic effusions, usually showing shortness of breath, were sucked out by practical private veterinarians before the pleural effusions were aspirated before arriving at UC Davis. Residual shortness of breath and thoracic effusion responded quickly to treatment and were no longer visible at all after 7 days. Jaundice resolved slowly over 2–4 weeks, with a decrease in hyperbilirubinemia. Signs of eye disease began to disappear within 24-48 hours and ceased to be apparent on the outside even for ophthalmoscopic examination within 7-14 days. Enlarged mesenteric and ileo / cecal / colic lymph nodes began to shrink during treatment. According to the owners' estimates, all 26 cats looked normal or almost normal on the outside after 2 weeks of treatment. After 2 weeks of treatment, the emphasis was on monitoring several blood test parameters. Key values included hematocrit, total white blood cell count, absolute lymphocyte count, total serum protein, serum globulin, serum albumin, and albumin: globulin ratio (A: G).

Figure 2.
Mean (solid line) and 1 SD (standard deviation) (dashed) body temperatures during the first 5 days of GS-441524 treatment. The normal temperature range for cats is 37.7-39.1 ° C (100-102.5 ° F). Temperatures dropped to the normal range within 12-36 hours of treatment

Eighteen of the 26 cats that received at least 12 weeks of uninterrupted primary treatment required no further treatment. However, eight other cats suffered disease relapses within 3–84 days (mean 23 days) (Figure 1). This group included three cats that temporarily discontinued initial treatment (CT53, CT71, CT80), and five cats (CT53, CT57, CT60, CT68, CT73) that required extended primary treatment (Figure 1). The disease relapses in 2/8 cats (CT57, CT71) were apparently neurological in nature with high fever and strong posterior ataxia and incoordination, while the disease relapses in the remaining six cats consisted mainly of fever, anorexia and decreased activity. Only one cat (CT60) had an obvious abdominal discharge during the relapse. One cat (CT57) was euthanized 2 weeks after relapse with neurological symptoms that did not respond to the second round of treatment.

In eight cats, it was decided to increase the dose of GS-441524 from 2.0 to 4.0 mg / kg, either due to prolongation of treatment (CT77, CT80) or due to one (CT60, CT68, CT71, CT73) or two relapses ( CT53, CT63), or because the relapse had a neurological form (CT71). All eight cats responded positively to the booster regimen.

A total of 25/26 cats treated for 12 weeks or more achieved permanent remission of FIP, although one subsequently died of an unrelated heart problem (see "Autopsy Findings"). The longest surviving cats at the time of publication (OnlineFirst, February 2019) stopped treatment in August 2017 and the shortest in May 2018, all after the end of the observation period, in which relapse could still occur (ie 84 days after the end of treatment). The 24 surviving cats will be carefully monitored for recurrence of symptoms and will be regularly tested for total protein, globulin, albumin and A: G ratios during the first year. Less intensive monitoring will be performed for the rest of the cats' lives. Owners were advised to avoid unnecessary strain on cats during the first 3 months, even though four cats (CT52, CT58, CT65, CT79) and one cat (CT76) underwent trouble-free castration.

Indicators of favorable response to treatment

The simplest long-term measure of treatment effectiveness was body weight. Weight gain of 20–120% occurred during and after treatment, even in cats that were 1 year or older at the time of diagnosis. Significant growth also appeared in younger cats, as their owners independently noted. These significant post-treatment growth accelerations suggested that FIP was subclinical in many cats for some time before diagnosis and affected their growth. CBC (hematology) and chemical profile (biochemistry) have also been shown to monitor the later effects of treatment and to observe possible drug toxicities.

CBCs (Hematology)

Cats showed an increased white blood cell count, which fell to normal during the first 2 weeks of treatment (Figure 3a). Lymphopenia recorded at the time of admission disappeared during the first week of treatment (Figure 3b). Mild to moderate anemia was observed on admission, resulting in hematocrit (PCV) (Figure 4). Hematocrit did not return to normal until 6-8 weeks of treatment. Absolute total white blood cell and lymphocyte counts were the only significant values during the first week of treatment, while PCV provided a more accurate picture of the course of treatment during the first 8 weeks.

Figure 3.
(a) Mean standard deviation white blood cell count in 26 cats that completed a primary treatment regimen for 12 weeks or more. (b) Mean absolute lymphocyte count in blood with standard deviation in 26 cats that had completed at least 12 weeks of treatment
Figure 4.
Hematocrit (PCV) with a standard deviation for 26 cats that have completed at least 12 weeks of treatment. The dotted line indicates the growth trend of PCV over time

Serum protein changes

Cats with FIP often showed higher than normal total serum protein levels, high serum globulin levels, low serum albumin levels, and low A: G ratios (Figures 5-7). Serum protein abnormalities progressively improved and reached normal values after 8-10 weeks of treatment (Figures 5-7). Total protein levels were the least informative, indicating a low R2 (0.1883) trend line (Figure 5). However, 3 weeks after the start of treatment, there was a dramatic and transient increase in total protein levels (Figure 5). This phenomenon was related to an increase in serum globulins (Figure 6a) at a time of rapid regression of abdominal effusions.

Figure 5.
Mean serum total protein levels and standard deviation for 26 cats that have completed at least 12 weeks of treatment
Figure 6.
(a) Mean serum globulin levels and standard deviation for 26 cats that have completed at least 12 weeks of treatment. (b) Mean serum albumin levels and standard deviation for 26 cats that have completed at least 12 weeks of treatment
Figure 7.
Mean albumin: globulin (A / G) ratios and standard deviation for 26 cats that have completed at least 12 weeks of treatment

Plasma globulin levels increased during the first 3 weeks of treatment, peaked and then slowly decreased to a maximum reference value of 4.5 g / dl or less by week 9 (Figure 6a). Although globulin levels appear to reflect treatment status over time, a low R2 (0.3621) indicated that this was a less reliable indicator of treatment progress.

Serum albumin levels of 26 cats treated for at least 12 weeks were usually low (⩽3.2 g / dl) at the time of treatment (Figure 6b). Albumin levels then increased slowly and reached normal levels after 8 weeks. The trend line for this increase in albumin was high R2 (0.79), making serum albumin levels as well as PCV a good indicator of treatment progress. As expected, the A: G ratio showed an equally strong trend line over time and around the 8th week of treatment the level exceeded 0.70 (Figure 7).

Decreased viral RNA levels in ascitic fluid cells in association with treatment

During the first 2-9 days of antiviral treatment, sequential ascites samples were taken from eight cats and tested for viral RNA levels by qRT-PCR (Table 2). The most reliable source of FIPV RNA was whole effluents or their cell fractions. In 7/8 cats, viral RNA levels dropped within 2-5 days, often to undetectable levels. One cat (CT54) did not show a significant decrease in viral RNA levels within 9 days.

Table 2
Levels of feline infectious peritonitis 7b RNA transcription in whole ascites or ascitic fluid cell fraction during initial treatment GS-441524

Sample IDTreatment daysSample typeCopies of viral RNA / ml
CT520Ascites9.44 × 104
3AscitesUndetectable
CT540Ascites8.49 × 105
2Ascites6.97 × 104
4Ascites2.44 × 103
7Ascites2.07 × 103
9Ascites6.46 × 104
CT620Ascites5.96 × 103
2Ascites1.53 × 103
8AscitesUndetectable
CT740Cells6.51 × 106
2Cells3.39 × 105
CT750Cells9.08 × 106
3Cells4.75 × 105
4Cells2.50 × 105
CT770Ascites5.47 × 104
2Ascites3.93 × 103
CT800Ascites4.10 × 103
2AscitesUndetectable
CT820Ascites1.13 × 104
5AscitesUndetectable

Side effects observed during and after treatment

Limited injection site reactions. Two types of injection site reactions have been observed and it has not been established whether they were due to the drug, the diluent or both. Immediate responses to pain were manifested by vocalization, occasional growling, and postural changes lasting 30-60 seconds. These initial reactions eased over time as owners became more skilled at injecting and cats gradually adapted to this routine. Sixteen of the 26 cats treated experienced injection site reactions (Table 3). Reactions were most common during the first 4 weeks and progressed to open wounds in only 7/16 cats. Ulcerations healed within 2 weeks by trimming the surrounding hair and gently cleaning the wound with a cotton swab soaked in one part household hydrogen peroxide and two parts water twice a day. Only three cats had noticeable scars at the injection sites.

Table 3
Injection site reactions in 16 of 26 cats treated with GS-441524 for 12 weeks or more

Cat IDSuperficial lesionsOpen woundsScars
CT53310
CT58100
CT60002
CT61500
CT63220
CT64101
CT65911
CT66320
CT68400
CT71510
CT73710
CT74310
CT761000
CT78700
CT79200
CT82200
Most lesions were superficial and included the epidermis and did not require any treatment, while some progressed to open wounds that healed within 2 weeks of topical treatment. Some reactions left small permanent scars

Systemic drug reactions. GS-441524 treatment was remarkably safe for a total of 12-30 weeks. No long-term abnormalities were observed in CBC values (Figures 3 and 4). Liver and kidney function tests and amylase / lipase levels remained normal during and after treatment (Supplemental Figures S1 - S3). The only exception was the CT53 cat, which had a progressive increase in blood urea (BUN) to 35 mg / dl (reference interval [RI] 16–37 /g / dl) and a sudden increase in SDMA (20 µg / dl) (RI 0- 14 /g / dl) after 8 weeks in the third round of treatment in a 4 mg / kg booster regimen. Although these symptoms were still mild in nature, it was decided to discontinue treatment. These abnormalities were no longer present when tested 1 month later and the cat is currently in remission.

Autopsy findings

Four cats (CT56, CT62, CT72, CT75) were euthanized or died within 2-5 days of study entry, and necropsies were performed on all but the CT75 cat. The fifth cat (CT54 cat) was euthanized after 26 days of treatment. All five of these cats had severe abdominal effusion disease. At CT54 and CT56, necropsy revealed evidence of extensive pyogranulomatous vasculitis involving the abdominal viscera, central nervous system, and eyes. In the CT56 cat, the ileal wall was also compromised in the area of ​​dense infiltrate and secondary bacterial sepsis. The CT72 cat had severe abdominal pyogranulomatous vasculitis with moderate to severe peripheral edema and adrenal cortex mineralization. The CT62 cat suffered from severe pyogranulomatous and fibrinosuppurative peritonitis, which was complicated by acute gastric perforation associated with plant material and intralesional bacteria suggestive of sepsis. The CT75 cat exhibited a chronic form of FIP characterized by severe growth retardation, massively low protein / low cell effusion, accelerated cardiac function suggestive of impaired cardiac function, and moderate peripheral edema. The echocardiogram showed bilateral atrial enlargement, but no sign of primary heart disease. The cat appeared to respond to GS-441524 and was released. The cat fell into shock 2 days later and was euthanized without autopsy.

At the time of necropsy of CT56, CT72 and CT75 cats, no FIPV virus was detected using qRT-PCR, although pre-treatment ascites samples were positive. Cat ascites CT54 showed a positive qRT-PCR result throughout treatment (Table 2) and the tissues were still immunohistochemically positive at the time of necropsy.

After successfully completing one or more rounds of treatment, two more cats were euthanized. The CT57 cat was normal after one round of treatment, but relapsed with severe neurological symptoms 2 weeks later. The cat did not respond to re-treatment and was killed. Lesions typical of FIP were found in the brain and abdomen, but were negative for FIPV determined by immunohistochemistry for nucleocapsid protein or for 7b RNA by qRT-PCR. The CT80 cat was successfully treated for effusive abdominal FIP, but 4 weeks later she developed severe hind leg and lower back pain. The cat was found to have a marked thickening of the left ventricular wall and septum, which caused severe ventricular narrowing (Figure 8). The microscopic appearance of the left ventricular wall was typical of congenital feline hypertrophic cardiomyopathy (HCM). No gross or microscopic FIP lesions were detected in the abdomen, chest, eyes, brain or spine, and neither FIPV nor FIPV RNA was detected by qRT-PCR.

Figure 8.
CT80 cat's heart section showing extreme left ventricular and septal wall hypertrophy and extreme ventricular narrowing

Discussion

GS-441524 is the second targeted antiviral drug tested for the treatment of FIP in the last 2-3 years after GC376. The two drugs inhibit viral replication in two very different ways, either by terminating viral RNA transcription or by blocking the cleavage of the viral polyprotein. Both processes are a well-known target for the treatment of some human viral diseases. A key issue is to compare nucleoside analogue treatment with viral protease inhibitor therapy. The two drugs gave virtually identical results in tissue culture studies and experimentally infected cats. However, the efficacy against naturally occurring FIP appeared to be higher with GS-441524 than with GC376. Six of the 20 cats treated with GC376 remain in remission to this day (Pedersen NC, unpublished data, 2018) compared to 25/31 cats treated with GS-441524. Diseases that did not respond to re-treatment occurred in 14/20 cats with GC376 but only in one cat treated with GS-441524. 8 of the 14 GC376-associated relapses were neurological, compared to 2/8 GS-441524 relapses. One of the two neurological relapses in cats treated with GS-441524 responded to re-treatment with a higher dose, whereas neurological relapses with GC376, even at an increased dose, were no longer treatable. Both treatments caused similar injection site reactions. Both drugs appear to be relatively safe, although GC376 interfered with the development of permanent teeth when given to younger kittens.

Although the results of the clinical study appear to favor GS-441524, some differences may have been affected by the way the two drugs were administered. The effectiveness of GC376 could be improved if all 20 cats were treated without interruption for 12 weeks, instead of being treated progressively over longer periods starting at only 2 weeks. Five of the six cats treated with GC376 were among the seven cats that were treated continuously for 12 weeks, while only one of the 13 cats treated once or more was treated for a shorter period of time. These shorter durations of treatment were necessary to determine the 12-week period used for all cats in this study. In addition, the GC376 clinical trial included fewer cats and was limited by limited drug delivery, making it difficult to test other dosing regimens. Therefore, GC376 should be further studied prior to any final comparison using a minimum of 12 weeks with a higher dose and more cats. It would also be important to evaluate both types of drugs in combination at some point in the future, as is the case with HIV / AIDS and hepatitis C.

Premature deaths should be considered in any study of this type, but how should they be considered in an efficacy analysis? Five premature deaths in this study were included in the GS-441524 efficacy analysis, but were excluded in study GC376. It is important to determine the status of the virus at the time of death to include deaths in the study. Viral RNA was not detected in three necropsed cats that died after 2-5 days of treatment with GS-441524, indicating that the drug was effective but the disease was at an advanced stage. This was not the case for the fourth dissected cat, which survived 26 days; viral RNA levels did not decrease throughout the treatment period and the symptoms of the disease did not improve. Therefore, it is possible that this cat died as a result of an unsuccessful cessation of virus replication. Resistance to GS-5734 (Remdesivir), a prodrug of GS-441524, has been associated with amino acid mutations in RNA polymerase and corrective exonuclease in tissue culture propagated coronaviruses. Whether this cat has developed similar resistance remains to be determined. Drug resistance was also observed in one cat in the GC376 test. Fortunately, none of the other cats in the current study showed signs of drug resistance. However, for future cats that do not respond at all or do not respond well to primary or secondary treatment, this option should be considered.

The initial dose of GS-441524 used in the present study was determined based on previous pharmacokinetic and experimental infectious studies with laboratory cats. These studies indicated that 2.0 and 5.0 mg / kg SC q24h for 14 days would be equally effective in the clinical study. Therefore, a dose of 2.0 mg / kg was chosen for clinical trials, as this would reduce drug consumption by 60%. Although this decision was confirmed in 18/26 cats, eight other cats either suffered relapses (two even twenty) or required a longer duration of treatment to get key blood levels back to normal. Therefore, a decision was made to increase the dose of GS-441524 from 2.0 mg / kg to 4.0 mg kg SC q24h in cats that relapsed or required prolonged treatment. The success rate of 4.0 mg / kg SC q24h in at least 12 such cats, as well as in one cat with a neurological disease, led us to the conclusion that this is a more effective dosage and should become the basis for future treatment.

It was important to monitor simple biological indicators of progress over ⩾ 12 weeks of treatment. HCT (PCV) levels, serum total protein, globulin and albumin levels, and the A: G ratio were identified as useful markers. Based on these parameters, it was shown that the cats had not yet fully recovered after 6-10 weeks of treatment. This finding confirmed the minimum 12-week duration of treatment determined in a previous GC376 clinical trial. Chronic anemia (inflammation anemia) affects 18-95% people with acute and chronic infections and is normocyte / normochromic and unrelated to iron deficiency. Plasma albumin levels were also a good indicator of disease activity, and low albumin and low HCT (PCV) are known to correspond to the incidence of chronic disease. Hyperglobulinemia in cats with FIP has been classified as infectious / inflammatory and is caused by an increase in all classes of gamma globulins and variable increases in the alpha-2 globulin fraction. The marked tendency of cats with FIP to high serum globulin and low albumin levels makes the A: G ratio a particularly good indicator of disease activity.

Purebred cats were not expected to respond as well to treatment due to their genetically impaired ability to respond immunologically to FIPV, and younger cats with wet FIP were expected to respond best to treatment. In this study, however, purebred cats eventually responded as well as regular cats, and the breeds represented by the cats in the study reflected the most popular current breeds. Older cats and cats with pure non-fusion FIP responded to GS-441524 as well as young cats and cats with wet FIP. Assuming that some cats with ocular and neurological diseases can also be treated with GS-441524, it can be said that no more manifestations of FIP may be considered incurable.

The safety profile of GS-441524 was impressive. Based on CBC and serum chemistry, no significant systemic signs of toxicity were observed during the total treatment periods of 12 to 30 weeks, with one possible exception. One cat (CT53) had a slight increase in BUN and SDMA in the 8th week of the third round of treatment and forced treatment to be stopped as a precautionary measure. Based on previous experience with GC376, there have been concerns about the effect of GS-441524 on the development of permanent teeth. Three cats (CT52, CT74, CT77) in this study were 4 months of age or younger and still had juvenile teeth and none showed any subsequent dental abnormalities. Injection site reactions were observed with GS-441524, but their number was remarkably low and easy to treat. It has not been established whether the drug, diluent or both are to blame. Diluent pH 1.5 was well below the FDA (Food and Drug Administration) minimum threshold of 4.5, but drugs of this type are difficult to dissolve and stabilize at a more physiologically acceptable pH. Nevertheless, more physiological diluents should be evaluated.

One cat in the study (CT80) had disturbing clinical signs. Although the cat showed effusive abdominal FIP, it also had long-term symptoms of vague hind limb curvature, low back pain, regular falling episodes, reluctance to jump to higher ground, and unexplained and transient behavioral changes. These symptoms led to the treatment of the cat long after the abdominal effusion disappeared. Finally, a decision was made to discontinue treatment and see if the characteristic symptoms of FIP returned. The cat was eventually euthanized and necropsied that it had a congenital HCM type and no residual FIP or viral RNA lesions in any tissues. Dilated cardiomyopathy has been reported in 17.6% HIV-infected people on chronic antiretroviral therapy. However, it was concluded that GS441524 was not the cause of the heart disease in this cat. Heart disease in this cat was hypertrophic in contrast to the dilatation form observed in HIV patients, and in addition, HCM is relatively common in shelter cats.

Conclusions

The results obtained from 31 cats treated with GS-441524 exceeded all expectations and suggest that FIP, regardless of signaling or disease form, is a treatable disease using nucleoside analogs. The study design and treatment parameters resulting from this limited clinical trial will be important for further efforts in the commercialization of this or similar anti-FIP drugs.

Additional material

Owner consent form

Figure S1. Mean liver enzyme levels (IU / L) in cats during treatment with GS-441524. No significant changes were observed throughout the treatment periods.
Figure S2. Mean serum lipase and amylase levels (IU / L) in cats during treatment with GS-441524. No significant changes were observed throughout the treatment periods.
Figure S3. Mean BUN and creatinine levels in cats during treatment with GS-441524. No significant changes were observed throughout the treatment periods.

Thanks

We thank the staff of the Center for Pet Health for their help in transporting medicines (Lyra Pineda-Nelson and Nancy Bei) and for presenting the data (Cynthia Echeverria). We are especially grateful to the many owners and 31 cats who took part in an emotional and demanding journey that exceeded all expectations. We are also grateful to the practical private veterinarians who helped with the regular blood tests and were there for us and their patients / owners when needed.

Notes

Received: December 28, 2018

Additional material: The following files are available: Client consent form.

Figure S1: Mean liver enzyme levels (IU / l) in cats during GS-441524 treatment. No significant changes were observed throughout the treatment periods.

Figure S2: Mean serum lipase and amylase levels (IU / l) in cats during GS-441524 treatment. No significant changes were observed throughout the treatment periods.

Figure S3: Mean urea and creatinine blood levels in cats during GS-441524 treatment. No significant changes were observed throughout the treatment periods.

Conflict of Interest: MP and EM are employees of Gilead Sciences, Foster City, CA, USA and have stakes in the company.

Funding: Financial support for this study was provided by the UC Davis Center for Animal Health, the Philip Raskin Fund in Kansas City, and numerous SOCK FIP donors as directed by Carol Horace. GS-441524 used in this experiment was provided by Gilead Sciences, Foster City, CA.

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Read "Efficacy and safety of nucleoside analogue GS-441524 in the treatment of cats with naturally occurring feline infectious peritonitis"

A mouse study showed that GS-441524 suppresses SARS-CoV-2

2.11.2020

Original article on news-medical.net

In animal studies, Chinese researchers found that a metabolite of the antiviral drug Remdesivir called GS-441524 could help inhibit the SARS-CoV-2 virus, which causes COVID-19.

The virus has infected more than 46.85 million people worldwide and already has more than 1.2 million lives killed. To date, there is no effective antiviral drug available to treat this infection, and there are no vaccines available to block the infection.

Remdesivir, a broad-spectrum antiviral drug developed by the biopharmaceutical company Gilead Sciences, has demonstrated initial success against this viral infection. This medicine is approved or authorized for emergency use in the treatment of COVID - 19 in 50 countries.

A new study entitled "Remdesivir Metabolite GS-441524 Efficient Inhibits SARS-CoV-2 Infection in Mouse Model" was released online in advance on the server bioRxiv before going through the review process.

Proposed metabolic pathway of remdesivir and chemical structures of metabolites

Basic facts

The COVID-19 pandemic is currently raging without an effective antiviral drug. In most cases, SARS CoV-2 only leads to mild respiratory symptoms, but in some individuals it can lead to acute severe respiratory syndrome (ARDS) and be fatal.

Remdesivir is currently the only COVID-19 treatment approved by the US Food and Drug Administration (FDA). Remdesivir is an inhibitor of RNA-dependent RNA polymerase (RdRP). The recent publication of the results of the World Health Organization's (WHO) Solidarity Trial questioned the effectiveness of Remdesivir against SARS CoV-2. The results of the study questioned its effectiveness in shortening hospital stays and reducing the risk of death in patients with COVID-19.

Pharmacokinetics of Remdesivir

Studies have shown that remdesivir is metabolised after intravenous administration to active metabolites called GS-704277, GS-441524 (original nucleoside) and GS-443902. The most important and important metabolite is GS-441524. GS-441524 has a long half-life of about 27 hours, while Remdesivir in its unaltered form has a half-life in the body of about 1 hour. GS-441524 also has greater bioavailability compared to unchanged Remdesivir. The authors write that the implications of these differences in the pharmacokinetics of unchanged remdesivir and its major metabolite are not clearly understood.

Efficacy of remdesivir and its metabolite

Laboratory studies have shown that GS-441524 is less active against Ebola virus, hepatitis C virus and respiratory syncytial virus (RSV) compared to unchanged Remdesivirus. GS441524 is converted to monophosphate in the body. This is the most important step in activating the drug. Remdesivir, in its unchanged form, bypasses this step and also has a better ability to penetrate the cell. However, studies have shown that GS441524 has potent activity against human coronaviruses (CoVs), including SARS CoVs 1 and 2, Middle East coronaviruses (MERS-CoVs), etc. It is also effective in inhibiting feline coronavirus infectious peritonitis (FIP) (FCoV). GS-441524 has shown success in the treatment of FIP 96%, the researchers explain.

Purpose of the study

The purpose of the study was to compare the efficacy of GS-441524 and Remdesivir against SARS-CoV-2 in vitro.

The efficacy of Remdesivir and GS-441524 was observed against SARS CoV-2 and mouse hepatitis virus (MHV) in mouse models. Drug pharmacokinetic parameters were also tested in animal models and cell cultures. Vero E6 cells (African green monkey kidney cells), calu-3 (human lung adenocarcinoma cell) and caco-2 cells (colorectal adenocarcinoma) were used for this study. They also used genetically modified or AAV-hACE2 transduced mice and mice with mouse hepatitis virus (MHV).

The overall results showed that the original nucleotide of Remdesivir, GS-441524, was able to inhibit SARS-CoV-2 replication in Vero E6 and other cells. The results of the study were:

  • GS-441524 was found to have good plasma distribution in animal bodies after administration
  • GS-441524 had a long half-life in the bodies of mice - almost 4.8 hours
  • GS-441524 was found to have better plasma stability compared to Remdesivir.
  • GS-441524 has been found to be effective in reducing viral load in affected organs in MHV transduced mice. Organ toxicity also decreased with drug use.
  • The authors stated that these results were "consistent with previously published studies that the relative efficacy of GS-441524 and remdesivir depends on the cell type."
  • GS-441524 did not cause toxicity to the treated cells, suggesting "its good safety profile," the researchers wrote.
  • "GS-441524 treatment was able to significantly suppress virus replication in the lungs by more than 99.9 percent," the researchers wrote.

Conclusions and implications

The study researchers concluded that GS-441524 was the major and major metabolite of remdesivir in plasma. The effects of remdesivir on COVID-19 may be due in part to this metabolite. The team wrote that their study showed that GS-441524 could be "a promising and inexpensive drug candidate in the treatment of COVID-19 and future CoV diseases."

* Important note

BioRxiv publishes preliminary scientific reports that have not been peer-reviewed and should therefore not be considered validated as relevant to clinical practice / health-related behavior, nor should they be treated as official information. Read "A mouse study showed that GS-441524 suppresses SARS-CoV-2"

Dr. Pedersen on the relationship between Remdesivir and GS441524

Original article: SOCKFIP

Dear veterinarians, cat owners and the public,

I am still asked about the relationship between GS-441524 and the very promising treatment for Covid-19 - Remdesivir. GS-441524 is a biologically active ingredient in Remdesivir and has been commonly used worldwide for the safe and effective treatment of feline infectious peritonitis (FIP) for over 18 months. FIP is a common and fatal coronary heart disease in cats. GS-441424 and Remdesivir are almost identical drugs. Remdesivir is a form of GS-441424 that Gilead Sciences has decided to use in humans to treat COVID-19, and clinical trials are currently underway in China, the United States, and several other countries. Remdesivir is called product. The prodrug is transformed by infected cells into the active ingredient, which in this case is GS-441524 with the addition of one phosphate group (i.e. GS-5734). Gilead researchers slightly modified GS-5734 to protect the added phosphate group and allow absorption into the cells. This form of GS-441524 is known as Remdesivir. Upon entering the cell, its enzymes remove protection to give GS-5734. GS-5734 is further activated in the cells by the addition of two additional phosphates to form the triphosphate form GS-441524. This is a molecule that inhibits the production of viral RNA. We decided to use GS-441524 to treat FIP because it had the same antiviral properties as Remdesivir and at that time Gilead Sciences did not anticipate its use in humans. GS-441524 is also much cheaper than Remdesivir. Therefore, there was no apparent conflict between the use of one form for cats and another form for humans. However, Gilead believed that our research on cats would affect the approval of Remdesivir for use in human medicine and refused to allow GS-441524 for the treatment of animals. This refusal, coupled with the desperate need for FIP treatment worldwide, led to the Chinese black market with GS-441524. FIP is a major problem for domestic cats in China, and Chinese cat owners have demanded FIP treatment even more desperately than owners in other countries. The first papers describing the treatment of FIP with GS-441524 were published in 2018 and 2019, and thousands of cats have since been treated. Despite this experience, the medical community, including researchers, was apparently unaware of the use of GS-441524 to treat FIP and its relationship to Remdesivir. Veterinarians have considerable but underappreciated experience with coronaviruses, coronavirus diseases and vaccines in pigs, calves and poultry. Ferrets also suffer from a serious FIP-like disease caused by their own coronavirus species. What happens to GS-441524 for cats if Remdesivir is shown to be safe and effective in treating Covid-19? GS-441524 is the first critical step in the production of Remdesivir and it is logical to assume that it will lead to the existence of competition in its use for cats and humans. On the positive side, the worldwide approval of Remdesivir may also help to change the opposition to the granting of rights to use GS-441524 in animals. If Remdesivir were approved for human use (although not approved by GS-441524 itself), it could also become legally available for use in veterinary medicine. However, the safety and efficacy of Remdesivir for the treatment of FIP have not been established.

-Niels C. Pedersen, DVM, PhD, School of Veterinary Medicine, UC Davis .. Read “Dr. Pedersen on the relationship between Remdesivir and GS441524 ”

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