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"

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

Original article: Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis
Published: 13.9.2017, SAGE - Journal of Feline Medicine and Surgery

Niels C Pedersen,1 Yunjeong Kim,2 Hongwei Liu,1 Anushka C Galasiti Kankanamalage,3 Chrissy Eckstrand,4 William C Groutas,3 Michael Bannasch,1 Juliana M Meadows,5 and Kyeong-Ok Chang2

Abstract

The goal

The safety and efficacy of the 3C-Like protease inhibitor GC376 was tested in a group of client-owned cats with various forms of infectious peritonitis (FIP).

Methods

Twenty-four cats aged 3.3 to 82 months (mean 10.4 months) with various forms of FIP were enrolled in the clinical trial. Fourteen cats had wet or mixed FIP and six cats had dry FIP. GC376 was administered subcutaneously every 12 hours at a dose of 15 mg / kg. Cats with neurological symptoms were excluded from the study.

The results

Nineteen of the 20 GC376-treated cats recovered within 2 weeks of starting treatment. However, symptoms of the disease returned after 1-7 weeks of primary treatment, and relapses and new cases were finally treated for at least 12 weeks. Relapses that stopped responding to treatment occurred in 13 of these 19 cats within 1-7 weeks of initial or repeated treatment. Severe neurological disease occurred in 8/13 cats that failed treatment, and abdominal lesions recurred in five cats. At the time of writing, seven cats were in remission. Five kittens aged 3.3–4.4 months with wet FIP were treated for 12 weeks and were in remission for 5–14 months (mean 11.2 months) after treatment and at the time of writing. The sixth kitten was in remission for 10 weeks after 12 weeks of treatment, but relapsed and responded well to the second round of GC376 treatment. The seventh was a 6.8-year-old cat with mesenteric lymph node involvement, who managed to achieve remission after three relapses, which required successively longer repeated treatments for 10 months. Treatment side effects included injection burning and occasional foci of subcutaneous fibrosis and hair loss. In cats treated before 16.-18. week, there was a slow development and abnormal growth of permanent teeth.

Conclusions and significance

GC376 has been shown to be promising in the treatment of cats with specific forms of FIP, opening the door to targeted antiviral therapy.

Introduction

Drugs that directly inhibit viral replication have become key supports in the treatment of chronic viral infections such as HIV / AIDS, hepatitis C virus (HCV), hepatitis B virus, herpesvirus and acute infections such as influenza. RNA viruses, such as HIV-1 and HCV, contain ideal targets for inhibiting viruses, such as RNA-dependent RNA polymerase and protease. Proteases are a particularly good target because they are involved in virus maturation (HIV) or in the production of functional viral proteins (HCV). Protease inhibitors are also used in combination with reverse transcription inhibitors in the lifelong treatment of HIV / AIDS. Combinations of different protease inhibitors are highly effective in treating HCV infection in humans. Therefore, it is not surprising that viral protease should also be an attractive target for research into animal infections caused by RNA virus. Kim et al. synthesized peptidyl compounds that target 3C-like (3CLpro) proteases and evaluated their efficacy against feline coronavirus (FCoV) and feline calicivirus, as well as important human RNA viruses that encode 3CLpro or a related 3C protease. They identified a series of compounds that showed strong inhibitory activity against various coronaviruses, including FCoV, with a high safety margin. The efficacy of their 3CLpro inhibitors has been tested in mice infected with hepatitis A59 virus, murine coronavirus, and has been found to cause significant reductions in virus titers and pathological lesions.

There are currently no commercially available antiviral drugs for coronavirus infections in humans or animals, and studies by Kim et al. demonstrated that inhibition of 3CLpro could lead to suppression of coronavirus replication in vivo. They have shown that some of their 3CLpro inhibitors are useful as therapeutic agents against these important viruses in domestic and wild cats. This was confirmed by a study using experimental infection with feline infectious peritonitis virus in laboratory cats. Although experimental FIPV infection is highly fatal, once the infection has reached a definable stage, 14-20 days of GC376 treatment resulted in rapid remission of the disease in six cats, which lasted more than 12 months at the time of publication.

Materials and methods

Official protocols

This study was conducted in accordance with Protocol 18731 approved by the Institutional Committee on the Care and Use of Animals and the Clinical Trials Evaluation Committee of the Veterinary Medical Teaching Hospital at the University of California, Davis. This protocol details the testing conditions for the new protease inhibitor GC376 in client-owned cats. Each owner was required to read and agree to the study conditions.

Organization of a clinical trial

The subject of the study was the evaluation of the 3CLpro GC376 inhibitor in a group of cats with naturally occurring FIP. The study did not include the placebo group because, as Miller and Brody noted, "the main ethical principle in placebo-controlled clinical trials is that if there is a proven effective treatment for a given condition, testing against placebo is unethical." GC376 has already been shown to be highly effective in the treatment of cats with experimentally induced FIP prior to this study, suggesting an existing effective treatment. The placebo control was replaced by a group with a naturally occurring disease. None of the 20 treated cats showed persistent beneficial responses to the treatment they received prior to GC376 treatment.

The institutional rules precluded the use of cats obtained from shelters or similar research facilities of this type, and required that all cats be legally owned / adopted and treated with the express consent of the owner. Cats with clinically obvious neurological disease were excluded. The study eventually included 20 cats from different areas of the United States, of different ages, and with various forms of FIP. This relatively small group of cats provided valuable insights into the design of the trial, interaction and compliance with the owner, safety and efficacy monitoring, determination of the minimum dosing regimen, evaluation of disease relapses during or after treatment, and determination of clinical forms of FIP that are most appropriate for treatment. This information will hopefully assist in further testing needed for the licensing and possible commercialization of GC376 and for performing similar tests on future antiviral drugs for FIPV and other chronic feline viral infections.

Test group description

Twenty cats and their owners were included in the experiment, and the relevant information for each cat is shown in Table 1 and for the entire experimental group in Figure 1. The cats were enrolled in the study with varying degrees of preliminary testing by primary care veterinarians. This testing usually involved a complete blood count (CBC) with total plasma protein, globulin (G), albumin (A), A: G; serum chemical profile and effusion analysis, including total protein, actual or estimated cell numbers and inflammatory cell type. A small proportion of the cats underwent additional testing, which included FIPV antibody titers, abdominal or thoracic ultrasound, affected tissue biopsies, and real-time quantitative PCR (qRT-PCR) from the effusions.

Table 1
Basic data, origin, main clinical signs and main findings at autopsy after treatment with protease inhibitor GC376

ID / NameAge (months)Weight
(kg)
GenderTribeOriginSymptomsConditionAutopsy
CT01 (Echo)5.61.64FSDSHKRPeritonitis, stunted-B, Int
CT02 (Cate)62.67FSDLHKRPeritonitis, stunted-B, E, Int, L, MLN
CT03 (Pancake)7.863.18MCHimCTDry (Col) to moist+Int, L, MLN, S, Om, P
CT04 (Kratos)824.8MCDSHKRDry (MLN)Remission
CT05 (Scooter)104.25MCDSHKRDry (E, MLN, K)-B, E, L, K, MLN
CT07 (Mac)6.62.6MCDSHKRDry (Col)+E, Int, L, MLN, S, K, A, Lu
CT08 (Phoebe)4.22.18FSDSHKRDry (E)-B, E, K, MLN, S
CT09 (Sammy)10.52.89MCDSHKRDry (MLN, K)?*B*
CT10 (Bandit)17.94.06MCHimCTDry (Col) to moist+B, E, Int, L, MLN, K, Om, P, Lu
CT12 (Daisy)7.52.5FSDSHKRPeritonitis, stunted-B, Int, L, S
CT13 (Leo)7.41.97MCSphynxCTDry (E, K)+B, E, Int, L, MLN, S, K
CT14 (Muffin)82.94FSDSHKRDry (Col) to moist+E, Int, L, MLN, K, Om, P
CT15 (Flora)4.32.39FDSHFCPeritonitisRemission
CT16 (Bean)41.4FSDSHKRPeritonitis, stunted+B, E, Int, L, MLN, S, Om, P
CT17 (Peanut)4.42.3MDSHKRPeritonitisRemission
CT18 (Smokey)41.84MCDSHKRPeritonitisRemission
CT20 (Cloud)3.31.55MRMCTPleuritis (MLN)Remission
CT21 (Phoebe)4.81.92FDSHKRPeritonitisRemission / relapse / re-treatment
CT22 (Pepper)3.31.6FSiberianCTPeritonitis+B, E, Om, MLN, Lu, Dia
CT23 (Oakely)3.93.1FSDSHKRPeritonitisRemission
Average10.282.59
SD17.220.94
FS = castrated female; F = uncastrated female; DSH = domestic shorthair; KR = rescued kitten; B = brain; Int = intestine; DLH = domestic longhair; E = eye; L = liver; MLN = mesenteric lymph nodes; Him = Himalayan; Col = colon; S = spleen; Om = omentum; P = peritoneum; MC = castrated male; M = uncastrated male; K = kidney; A = adrenal gland; Lu = lungs; FC = cat colony; RM = ragmuffin; Dia = diaphragm, SD = Standard deviation; CT = Kennel

* No autopsy was performed, but terminal neurological symptoms were present
† Severe cerebral edema, no typical inflammatory lesions have been reported

Figure 1.
Demographics of study cats. (a-c) Pie charts summarizing the percentage of patients: (a) age in months, (b) breed, or (c) origin. (d) Bar graph showing forms of feline infectious peritonitis (FIP) enrolled patients.
M = months; DSH = domestic shorthair; DLH = domestic longhair; MD = Maryland; OH = Ohio; Tx = Texas; FL = Florida; IL = Illinois; CT = Connecticut; CA = California

Cats with clinical signs of neurological impairment were excluded from the study based on previous unpublished experimental studies with GC376. One cat that survived a previous study of the pharmacokinetics and efficacy of GC376 had a recurrence of FIP with neurological symptoms 6 months after appearing to be a successful treatment for acute infection.6 This cat did not respond to a repeated GC376 penetration study that prompted a penetration study. to the brain. GC376 levels in laboratory cat brain represented only 3% plasma drug concentrations.

Confirmation of the disease

The diagnosis of FIP was confirmed at study entry based on baseline data, clinical history, examination of previous laboratory tests, physical examination, and repetition of baseline blood and effusion tests. Manual abdominal palpation was usually sufficient to identify ascites, enlarged mesenteric lymph nodes, appendix enlargement and associated ileocecal-colic lymph nodes, renal tumors, and colonic infiltration. Manual palpation was supplemented with ultrasound if necessary. The eyes were initially examined with direct light for any abnormalities in the retina, for clots in the anterior chamber or on the back of the cornea, and flashes in the ventricular water. The presence of ocular disease was confirmed by a complete ophthalmoscopic examination performed by the Ophthalmological Service of the Veterinary Medical University Hospital (VMTH), UC Davis. The presence of FIPV was further confirmed by qRT-PCR, either from abdominal or thoracic effusions collected at the time of admission or at the time of necropsy. Sequencing of the FIPV protease gene was performed on cats that relapsed during treatment to determine if there was a potential mutation causing drug resistance.

The diagnosis of dry-wet (mixed) FIP in three cats (CT03, CT10 and CT14) was based on diffuse colon enlargement and a history of loose stools, blood and mucus in the stools, defecation strains and small caliber stools before abdominal effusions. Colon FIP has been described as a specific variant form of non-fusive FIP. Mixed FIP was also suspected in cats CT01, CT02 and CT12 due to growth arrest, which preceded the occurrence of abdominal effusions by many weeks.

Treatment regimen

GC376 was synthesized in highly pure form and prepared at a concentration of 53 mg / ml in 10% ethanol and 90% polyethylene glycol 400 as described above. GC376 was administered subcutaneously (SC) at a dose of 15 mg / kg every 12 hours of SC, unless otherwise stated. The effective dose for cats with experimentally induced FIP was 10 mg / kg / q12 h SC, but the dose was increased to 15 mg / kg after the first cat (CT01) did not respond to the lower 10 mg / kg dose determined by previous pharmacokinetic studies. It was a clinical decision based on this cat's response to treatment.

Monitoring response to treatment
Based on preliminary testing and initial evaluation at the time of presentation at UC Davis, cats with FIP were hospitalized for at least 5 days and started treatment immediately. They were examined in detail for rectal temperature, pulse, respiration, appetite and activity at least twice a day. A clumping litter was used to allow daily evaluation of stool volume and consistency and urination. Whole blood was collected into EDTA or heparin by venipuncture before treatment, at two-day intervals during hospitalization, at discharge time, and at two-week intervals during the first month and at monthly or longer intervals thereafter. Routine blood tests at each time point included minimal hematocrit, total plasma protein, icteric index, total white blood cell count, differential white blood cell count, and absolute neutrophil, lymphocyte, and monocyte and eosinophil counts. To check the potential toxicity of the medicines, blood serum chemistry values were recorded regularly. Abdominal effusion samples were obtained by paracentesis every other day if they could be obtained, which was usually the first 3-7 days. Cats with shortness of breath were examined by thoracic ultrasonography and a fluid sample was obtained by ultrasound-guided paracentesis. The effluents were examined for the presence of fibrin clots, neutrophil and small / large mononuclear cell admixtures, yellowing intensity, fiber viscosity, and total protein content. Cell pellets from peritoneal or thoracic effusions were also examined by qRT-PCR for viral RNA levels as previously described.

Cats were issued to their owners when a positive response to treatment was noted, usually within 5 days. The owner (s) were instructed by either the chief veterinarian or the primary care veterinarian how to administer the drug twice daily by subcutaneous injection. The injection sites were varied to include the upper line from the nape of the neck to the middle of the back and to the sides of the chest and hips. Care was taken to avoid storing the drug in the dermis or gradually in the same subcutaneous site. Owners were encouraged to maintain daily records of rectal temperature, activity, appetite, defecation and urination, and weekly to biweekly body weights. Periodic blood samples for CBC and serum chemistry were collected by the owners' personal veterinarians and sent to commercial veterinary diagnostic laboratories. Any abnormal symptoms or behavior should be noted and reported immediately. Euthanasia was performed at either UC Davis or a primary care veterinarian, as appropriate. The bodies of the cats killed by the primary care veterinarians were immediately cooled and sent in ice packs by express mail to UC Davis for autopsy. The owners' requirements for treatment and final disposition of the body were respected.

The results

Determination of treatment duration

The first five cats in the study were initially treated for 2 weeks (CT01, CT02, CT03, CT04 and CT05). A rapid improvement in health was observed in all cats and treatment was discontinued. Despite a favorable initial response, symptoms of the disease were repeated 1 (CT01, CT05), 2 (CT03, CT04) or 7 (CT02) weeks after the end of the 2-week treatment (Figure 2). The cats were then treated again, due to a gradual prolongation of the primary and secondary treatment time until their FIP remained sensitive to GC376 (see CT04, CT22, Figure 2). New cats that entered the study were further treated for 3 (CT07) or 4 weeks (CT08, CT16). Cats CT08 and CT16 initially responded, but their symptoms reappeared during treatment. Cat CT08 developed neurological disease, while cat CT16 had recurrent abdominal lesions (Table 1). Primary and secondary treatment times were then extended to 9 weeks (CT07, CT09, CT10, CT14) (Figure 2). Cat CT09 developed neurological symptoms during the 9-week basic treatment and was eventually sacrificed when the symptoms of the disease became severe. CT07 developed neurological disease 6 weeks after the start of the second treatment. From this point, all new cats were accepted into the study and older cats, such as CT10, were treated or re-treated for at least 12 weeks. The benefit of 12 weeks of treatment was most evident in the CT04 cat, which had previously been treated three times with a shorter treatment period followed by relapse (Figure 2). Treatment was discontinued in cats that had no clinical or laboratory signs of disease after 12 weeks of primary or secondary treatment. It was found that the minimum treatment period should be around 12 weeks. Cat CT21 was treated for 17 weeks due to delayed improvement in total protein and white blood cell counts (Figure 2). This cat relapsed pleural FIP 13 weeks later and underwent further treatment at the time of writing.

Figure 2.
Treatment time scale and clinical outcome of 20 cats that entered the clinical study with the protease inhibitor GC376. The periods during which the cats were treated are indicated by solid lines. The date of the last day of treatment is given for six cats that have achieved permanent clinical remission. Cat 21 was still in treatment at the time of writing. The remaining 13 cats succumbed to non-neurological FIP or neurological FIP after completion of primary or secondary treatment within 0-7 weeks.

Response to initial treatment and indicators of favorable response

During the first 1-4 weeks of treatment, 19/20 cats showed a dramatic and progressive improvement in health. The exception was the CT16 cat, which responded by a drop in rectal temperature during the first 4 days of treatment. However, the fever returned and the health continued to deteriorate over the next 23 days and the cat was euthanized. The fever (> 38.9 ° C) in the other 19 cats disappeared within 24-48 hours, with an improvement in appetite, activity, growth and weight gain. Abdominal effusions were usually undetectable within 2 weeks. The residual thoracic effusion remaining after the initial therapeutic drainage after 3 days in the CT20 cat almost disappeared. Renal tumors in cats CT02 and CT13 also shrunk rapidly and were no longer palpable after 2 weeks. Enlarged mesenteric lymph nodes returned to normal size more slowly. The palpable colon thickening and associated ileo-cecal-colic formations responded the slowest and persisted in the CT03 cat despite treatment and a return to an otherwise normal state of health. Jaundice, a common finding in younger cats with effusive FIP, slowly resolved over 2 weeks or more, with a reduction in hyperbilirubinemia. Symptoms of ocular disease began to resolve within 48 hours and resolved within 1 week, regardless of initial severity (Figure 3).

Figure 3.
Appearance of CT08 cat eyes before treatment (a) and one week later (b). This cat developed severe neurological symptoms 3 weeks after the start of treatment.

Weight gain was a simple and accurate criterion for growth and health improvement. The weight of cat CT04, the oldest cat in the test, was used as a reference value for this parameter (Figure 4a). Cat CT04 showed a significant weight loss of 30%. She gained weight after each round of treatment, began to lose weight shortly before each relapse, and gained weight again after each treatment. After 9.3 months without treatment and after treatment (4.8 kg to 7.19 kg), she regained all her lost weight. All kittens with permanent remission during and after antiviral treatment gained weight continuously, indicating that normal growth continued with antiviral treatment (Figure 4b). One cat (CT15) and two cats (CT17, 20) were castrated without complications during disease remission.

Figure 4.
Antiviral treatment and weight changes. (a) Cat CT04, a 6.8-year-old castrated male who had dry feline infectious peritonitis (FIP), underwent four cycles of antiviral treatment with increasing duration, as shown by the dotted boxes. He lost weight before each relapse and gained weight after subsequent treatment. (b) Weight gains in four kittens aged 3.5-4.4 months during and after antiviral treatment are indicated by dots. The dotted rectangle indicates the length of antiviral treatment (12 weeks)

Lymphopenia was a common clinical symptom in cats with wet FIP (Figure 5) and tended to directly correlate with the severity of abdominal inflammation, as indicated by viscosity, presence of fibrin fibers, protein content, cell number, and yellowness of the effusion. Lymphopenia improved in the treatment of all cats with wet FIP except CT16, but did not help predict disease relapses that occurred afterwards (Figure 5a). In cats with dry FIP, lymphopenia was not as severe and was not as useful as other parameters in assessing response to treatment (Figure 5b).

Figure 5.
Mean and standard deviation (SD) of absolute lymphocyte counts in treated patients with (a) wet or (b) dry feline infectious peritonitis (FIP). (a) Twelve cats (empty ring) showing abdominal or thoracic effusion and treated for up to 12 weeks. The thirteenth cat (CT16, full ring) with abdominal effusion responded poorly to treatment. b) Seven cats with dry or wet form of FIP and subsequent treatment for 6 weeks.

Total plasma protein levels as an indirect indicator of globulin concentration were often increased on examination, but values were highly variable during the first 4 weeks and often increased transiently during effusion resorption. Cats that ultimately failed treatment tended to have higher total plasma protein concentrations at the start of treatment and tended to maintain higher levels during treatment than cats that successfully achieved permanent remission (Figure 6).

Figure 6.
Mean and SD of total plasma protein levels in 20 cats over a 12 week period. Thirteen cats suffered fatal relapses at different weeks of treatment (W) and seven cats achieved permanent remission after 12 weeks of treatment (W-SV)

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

Sequential ascites samples were taken from several identical cats during the first 6-25 days of antiviral treatment and tested for viral RNA levels by qRT-PCR. FIPV levels are often low or negative in the blood of cats with FIP and are highest in effusion cells. Therefore, cells from ascites or pleural effusions were the most reliable source of FIPV RNA. Cats CT15, CT16 and CT17 had 955, 1699 and 2937-fold higher levels of viral RNA, respectively, than CT02, which had the lowest viral load in pre-treatment effusion (Figure 7). Viral RNA levels decreased up to 1567463-fold over 2 weeks compared to pre-treatment values, except for the CT16 cat (Figure 8), which had the second highest pre-treatment viral RNA level among the 12 cats with effusion samples available for testing (Figure 7). The absence of a rapid decrease in viral RNA levels in the CT16 cat, together with severe lymphopenia, may explain why it did not respond to treatment. CT10 also had a slightly slower decrease in virus levels and relapsed twice after antiviral treatment. Notably, viral RNA levels in ascites cells in CT15, CT17, and CT18 cats declined most rapidly and were also among the five cats that experienced permanent remission of the disease. Whether the cause was a property of individual FIPV isolates or the form and severity of the host disease was not determined.

Figure 7.
Relative initial levels of feline infectious peritonitis virus (FIPV) RNA in patient effusions prior to antiviral therapy. Real-time quantitative PCR was performed on pre-treatment effusion patient samples. Relative baseline viral RNA levels as fold differences compared to viral levels before treatment with CT02, the cat with the lowest RNA levels. RNA transcript levels were calculated for each patient using the ∆Ct method with the beta-actin reference gene
Figure 8.
Reduction of feline infectious peritonitis virus RNA from sequential effusion samples during GC376 treatment in cats CT10, CT12, CT15, CT16, CT17, CT18 and CT23. Each point indicates a fold reduction in viral RNA levels compared to pre-treatment levels (day 0). Viral RNA levels were determined by real-time quantitative PCR using the ∆Ct method and beta-actin reference gene

Treatment failure due to recurrence of abdominal FIP or the occurrence of neurological disease

Thirteen of the 20 cats in the test eventually relapsed. One cat (CT16) did not show significant improvement and was euthanized 3 weeks after the start of the 4-week treatment regimen (Figures 2 and 8), 8), while the other 12 had a variable period of disease remission after primary or secondary treatment lasting 3-17. weeks (mean 7.8 weeks) (Figure 2). All but one of these 13 cats (CT09) were necropsied (Table 1). Eight of these cats were euthanized for severe neurological symptoms and five for recurrent abdominal disease (Figure 2). Three cats that underwent neurological disease (CT05, CT08, CT13) due to ocular FIP were secondary to the examination (Table 1). The earliest symptoms of neurological disease included fever, which persisted despite continued treatment, apathy, occasional muscle twitching of the ears and muscles, unusual swallowing movements, compulsive limb twitching, and loss of normal mentality, manifested by brief episodes of numbness or numbness. These symptoms persisted during treatment for several days or weeks, but eventually led to incoordination and tonic / clonic seizures. The incidence and rapid progression of neurological symptoms after discontinuation of treatment were more pronounced than during treatment (Figure 2).

Five cats (CT03, CT07, CT10, CT14 and CT16) had a recurrence of typical intra-abdominal lesions in the absence of neurological symptoms during or after treatment (Table 1). Four of them had ileocecal formations (CT03, CT07 and CT14) or an enlarged colonic lymph node (CT10) that had shrunk (CT03, CT10 and CT14) or were no longer palpable (CT07) after primary treatment. However, CT03 continued to suffer from severe constipation, strain, and toothpaste-like stools. The severity of colonic obstruction required resection of the colon, which alleviated clinical symptoms but did not prevent a recurrence of abdominal disease. All three cats that developed severe ileocecal infiltrates still showed evidence of this form of FIP at necropsy, and immunohistochemistry showed FIPV antigen in macrophages in granulomatous inflammation (Figure 9).

Figure 9.
An incision from the severely thickened wall of the resected colon from cat CT03. Immunoperoxidase (brown) stained for feline antigen of infectious peritonitis virus is observed in macrophages around the periphery of the granulomatous lesion. Persistence of the virus in the colon occurred during treatment and remission of other symptoms of the disease (eg effusive peritonitis)

Attempts to treat a neurological disease by increasing the dose of the drug and prolonging the duration of treatment

To alleviate neurological symptoms, we tried to increase the dose of GC376, thereby increasing its blood level and the amount of drug that crossed the blood-brain barrier. Cat CT01 showed effusion FIP and was initially treated with GC376 (10 mg / kg q12h SC for 9 days). The cat responded well, but on day 9 the fever returned and the dose was increased to 15 mg / kg every 12 hours for 5 days. The fever disappeared and treatment was stopped on day 14. Three days later, the fever returned along with indeterminate neurological symptoms consisting of muscle twitching, abnormal limb stretching, and abnormal swallowing movements. The cat was immediately re-administered a dose of 15 mg / kg every 12 hours of SC and its condition improved, but soon after it worsened with the return of fever and the same vague neurological symptoms with mild incoordination. The dose was then increased to 50 mg / kg every 12 hours of SC for 14 days and improved to near normal. Treatment was stopped, but neurological symptoms returned immediately. The cat was then treated for another four days with a dose of 50 mg / kg q12 h SC, during which the neurological symptoms improved again. However, the decision was made to stop the treatment completely. The cat's condition remained stable for 1 week and then developed extreme incoordination, dementia and tonic / clonic seizures. Euthanasia was performed and autopsy showed lesions only in the brain.

Cat CT12 responded well to treatment at 15 mg / kg q12 h SC; rectal temperature returned to normal within 48 hours and abdominal effusion disappeared within 2 weeks. The cat appeared normal after the second week of treatment, but then had a persistent fever of 38.9-40 ° C. The owners felt that the cat otherwise had normal activity and appetite, so the treatment continued at the same dosage. However, the fever persisted, mild signs of behavior change were observed, and the cat did not grow as expected. The cat continued treatment for another 15 weeks, during which the drug dose was temporarily reduced twice (ie to 10 mg / kg every 12 hours and 15 mg / kg every 24 hours) for several days, but the fever increased and the activity decreased each time. Dosing of 15 mg / kg was resumed every 12 hours. The cat continued to show signs of variable fever and unclear signs of behavior, but the owners were optimistic about the cat's appetite and activity level. The treatment of the cat was then stopped because further use of the drug for this purpose could not be justified. The cat's condition remained unchanged with persistent fever, solitary behavior and stunted growth for another 5 weeks. At week 22, severe neurological symptoms consisting of incoordination, dementia and seizures appeared and the cat was euthanized. Macroscopic and microscopic lesions were limited to the brain.

Viral resistance testing

The development of a drug-resistant virus was considered in the CT03 cat, which relapsed with abdominal lesions after an initial favorable response to the treatment of granulomatous colitis and mixed FIP. At the time of necropsy, granulomatous lesions were still present in the abdominal cavity and no macroscopic or microscopic lesions were found in the brain (Table 1). Therefore, disease recurrence was not associated with neurological disease and FIPV persistent antigen was identified in macrophages in granulomatous lesions. Sequence comparisons were made between 3CLpro from pre-treatment effusion and from the omentum taken at autopsy 95 days later. However, no amino acid substitutions were found in 3CLpro, suggesting that the presence of drug-resistant virus was not the cause of recurrent cat disease.

Pre-treatment 3CLpro viral RNA sequences were also compared with those obtained 25 days (CT16), 139 days (CT02), 149 days (CT12) and 231 days (CT10) later at necropsy. No differences in 3CLpro were observed during this period. The sequences also remained unchanged for CT02, CT16 and CT12 from the time of admission to necropsy. CT10 lung and spleen viral 3CLpro, which relapsed twice for 8 months and was re-treated, showed an Asp-to-Ser substitution at position 25 and a Lys-to-Asp substitution at position 260 compared to the pre-treatment abdominal fluid virus. . The exact effects of these mutations on protease function are currently being investigated. Genetic evolution of quasi-viral proteins has been reported to occur over time in patients chronically infected with RNA virus (HCV) and may lead to sporadic amino acid changes.

Occurrence of permanent clinical remissions

Seven of the 20 cats in the GC376 treatment study, all of whom underwent at least 12 weeks of continuous treatment, were categorized as potential treatment successes based on more than 12 weeks of disease remission after cessation of treatment (Figure 2). Six of these kittens had an acute abdominal effusion (CT15, CT17, CT18, CT21, CT23) or pleural (CT20) disease at 3.3-4.4 months of age and were treated continuously for 12 or 17 (CT21 cat) weeks (Table 1, figure 2). The seventh cat (CT04), a 6.8-year-old cross-castrated male with dry FIP restricted to the mesenteric lymph node, also achieved long-term remission, but only after four treatment cycles of increasing duration (Table 1, Figure 2).

Six of these long-lived cats had abnormalities in CBC, hematocrit, and total proteins at the start of treatment, but had completely normal blood levels at the time of treatment. However, the CT21 cat still had elevated plasma protein levels and an increased white blood cell count after 12 weeks and continued treatment for another 5 weeks. Plasma protein and white blood cell counts improved after another 5 weeks of treatment, but were still not within the reference range. Thirteen weeks after the end of treatment, the cat developed a typical FIP chest effusion with fever. Chest fluid was aspirated to improve respiration, and the cat began a second round of GC376 and at the time of writing was afebrile, active, and ate after 8 weeks of treatment. The treatment will last for 12 weeks if there are no signs of the disease again.

Side effects observed during and after treatment

Two side effects were observed during and after GC376 treatment. The drug often caused stinging / burning when injected. Subcutaneous edema occurred when too many injections were given at the same site, but they resolved rapidly. One cat (CT12) experienced deeply localized ulceration between the scapulae at approximately week 14 of the 18-week treatment period. However, no evidence of dermal FIP was observed at necropsy and was likely a response to continuous injections at the same site. A survey of seven long-lived cats showed appreciable focal subcutaneous thickening. In one cat, four calcified peas the size of peas appeared on X-rays. These lumps were surgically removed along with the surrounding fibrotic tissue. The other three long-term survivors have 1-3 small focal areas with permanent hair loss at the injection sites, which are covered by the surrounding fur (Figure 10). The owners and their veterinarians were asked to check these lesions regularly for any changes or the appearance of new lesions.

Figure 10.
Focal area of permanent hair loss caused by unwanted deposition of GC376 in the epidermis of cat CT21. These areas were usually covered with hair and were not visible from the outside

The most significant side effect associated with long-term treatment was juvenile teeth. Normal formation, growth and incision of permanent teeth were delayed in all four treated kittens aged 3.3-4.4 months. The ocular teeth, incisors, fourth premolar and molar were the least affected, while the second and third premolar were the most affected (Figure 11). Adult teeth appeared smaller than normlingual, and this, together with the delayed cutting, led either to the retention of the deciduous canines, the failure of the deciduous teeth or the partial incision of the abnormal permanent teeth to the concurrent deciduous teeth. No other anatomical or physiological defects were observed in any of the long-term survivors and no autopsy was observed.

Figure 11.
Adult teeth of a CT17 cat who was treated with GC376 for 12 weeks, starting at 4.4 months of age. The upper left milk canine is visible. The upper second and third premolars appear to be milky. The small permanent third premolars were partially cut lingually to the dairy third upper premolars. The gingiva surrounding the left canine and premolars is inflamed. Adult canines also appear smaller than usual. The permanent right canine and fourth upper premolar appear to have cut normally

Autopsy findings

The bodies of 12/13 cats that did not succeed in treatment were necropsied, including a rough and histological examination and immunohistochemistry of the affected tissues for FIPV antigen. Tissues collected and examined included representative sections of all major abdominal and thoracic organs, brain and eyes. The rough examination identified three different presentations. Five cats did not show significant signs of active FIP (CT01, CT02, CT05, CT08, C12), three had lesions corresponding to non-fusion FIP (CT07, CT10, CT13) and four had effusion peritonitis with multiorgan involvement (CT03, CT14, CT16, CT22). The histology of the five cats, which did not show sufficient evidence of the disease, showed mostly mild mononuclear infiltrates, usually perivascular inflammation in the eye, liver, intestinal wall and kidneys. Three cats with non-fusion FIP had mild to severe inflammation in many organs with the most severe lesions in the eye, mesenteric lymph nodes, kidneys, and lungs. Three cats with effusion FIP had severe pyogranulomatous inflammation in several abdominal organs, including the omentum, peritoneum, intestinal wall, mesenteric lymph nodes, liver, and spleen.

Severe inflammation typical of cerebral FIP was present in the brains of all but one (CT07) of the eight cats that underwent necropsy without significant signs of FIP or with non-fusion FIP. One cat without characteristic brain lesions had severe cerebral edema. In contrast, typical FIP lesions were absent in the brains of all three dissected cats with effusive FIP. Stereotypic FIP brain lesions were characterized by moderate to severe chronic meningoencephalitis and ventriculitis associated with periventricular necrosis of the parenchyma (Figure 12a). The fourth ventricle was the most severely affected, and meningitis was most often observed ventrally into the cerebellum and brainstem. Strong perivascular cuffs associated with vasculitis have often been observed. FIP antigen was demonstrated by immunoperoxidase staining in the brain of 6/7 cases of stereotyped cerebral FIP (Figure 12b). Tissues from 11 dissected cats were tested for the presence of FIPV RNA by qRT-PCR. All showed a positive result, thus determining the persistence of the virus in cats in which treatment was not successful.

Figure 12.
Photomicrographs of lesions in cat brain CT08. This cat developed a severe neurological disease during the initial treatment of GC376. (a) The fourth chamber contains protein fluid mixed with numerous neutrophils and macrophages that interfere multifocally with the surrounding dilute neuropil. Large cuffs of lymphocytes and plasma cells surround blood vessels (*) (hematoxylin staining, 20x magnification). (b) Several cells resembling peritoneal macrophages (marked small area in Figure 12a) show positive immunoreactivity for feline infectious peritonitis antigen (hematoxylin contrast dye, 600x magnification).

Discussion

Success in the treatment of GC376 in experimental FIPV infection motivated us to examine the efficacy of GC376 in naturally developed FIP. There are significant differences between experimental fusion abdominal FIP and naturally occurring disease. The experimental disease bypasses the critical initial stage, which begins in kittens by exposure to the harmless feline enteric coronavirus (FECV). Naturally occurring FIP is the result of specific mutants that arise after FECV infection, and FIP occurs in the presence of immunity to FECV. In contrast, experimental FIP is induced in cats that have not encountered coronavirus by intraperitoneal injection of a large dose of purified FIPV obtained from a laboratory cat. The naturally occurring disease is often subclinical for many weeks or months before observing external signs of the disease, while experimental symptoms of the disease appear within 2-4 weeks and progress rapidly. Naturally occurring FIP has various clinical forms, while experimental infection almost always has an abdominal effusion form. FIP in nature is also affected by the environment of disease-increasing cofactors, while experimental disease occurs in cats without external influences. Differences may explain why only a small proportion of cats naturally exposed to FIPV develop the disease, while 80-100% experimentally infected cats die. Our predictions proved to be correct, and naturally occurring FIP was much more difficult to treat than experimental disease. However, it should be emphasized that this experiment would not have been approved without the information obtained from pharmacokinetic studies, acute and chronic toxicity and efficacy studies performed in laboratory cats.

It was the first attempt to use a targeted antiviral drug against a systemic and highly fatal veterinary disease. Although no specific antiviral drugs for coronavirus infections in humans or animals are yet available, antiviral drugs for other human viral infections, such as HCV and HIV-1, have been developed for treatment and the use of these drugs has provided a solid basis for their application to animal diseases such as FIP. HCV mainly infects liver cells and causes a persistent viral infection in most people. However, only about 20-30% of them develop liver disease within a time horizon of 20-30 years. HCV infection can be eliminated by non-specific antiviral therapy (interferon and ribavirin) for 6-12 months in approximately half of the patients, and the recent introduction of direct-acting antiviral drugs for 3-6 months significantly increased the cure rate to more than 90% during treatment. HIV infection in humans leads to a prolonged asymptomatic condition and eventually to advanced HIV disease. HIV-1 infects T cells and macrophages and survives in a latent state. More than 30 antiretroviral drugs, most of which are used in combination with two or more drugs, have been used successfully to reduce the viral load to undetectable levels in the blood of HIV / AIDS patients. However, the virus returns after discontinuation of antiviral treatment and thus requires lifelong antiviral treatment. The spread of the virus to the brain, which is mainly mediated by virus-infected macrophages, and the subsequent development of neurological disease occur in more than 50% HIV infections. Therefore, neurological deterioration still remains an important problem at this time of antiviral treatment. These precedents for antiviral treatment of HCV and HIV-1 infections indicate that treatment outcome (viral clearance vs. viral persistence), duration of treatment (final vs. continuous) and the presence of neurological sequelae are strongly influenced by viral pathogenesis.

This study was limited to 20 cats with FIP, which represented the spectrum of age and forms of the disease. Although the number of cats treated was small, a surprising amount of information was gathered, such as how long to be treated, possible side effects, how to identify the clinical form of FIP that is most likely to respond to treatment, and potential indicators. treatment failure and its success. The clinical study was based on experience gained from pharmacokinetic and efficacy studies performed in laboratory cats. Based on experimental studies, the initial treatment period was set at 2 weeks, but was eventually extended to 12 weeks or more based on experience gained during testing. This final treatment period was close to 3-6 months used to treat HCV infection in people with direct-acting antiviral drugs. Based on experimental studies, difficulties in treating neurological forms of the disease have been expected. Side effects were acceptable and included injection burning and dermal and subcutaneous inflammation when too much drug was administered to the same sites. This phenomenon has previously been observed in laboratory cats. A more serious side effect not previously observed in laboratory cats was limited to kittens and included slowed development of adult teeth and retention or delayed loss of deciduous teeth.

GC376 treatment was successful in inducing significant remission of disease symptoms and regression of lesions in 19/20 cats. This result confirms our findings of a rapid reversal of clinical signs in laboratory cats with experimental FIP treated with GC376, and expanded our knowledge of the effects of the drug on a wide range of forms of naturally occurring FIP. The cats came from different parts of the United States and even Peru, confirming that the geographically diverse strains of FIPV were equally sensitive to this inhibitor. Significant reductions in viral RNA transcripts in effusions occurred within a few days of treatment, along with a rapid improvement in health. However, remission of the disease persisted for 3 months and longer in only 7/20 of these cats. The inability to achieve long-term remission of the disease was ultimately associated with the occurrence of neurological disease in the absence of extensive abdominal lesions or with recurrence / persistence of extensive abdominal lesions in the presence of histological lesions in the brain and / or eyes. These findings suggest that FIPV has a greater tendency to spread from body cavities to the brain than previously thought, especially if given sufficient time. This spread most likely involves infected macrophages that enter the brain through small blood vessels in the meninges and ependyma.

In cats that developed neurological disease, this occurred either during treatment (CT05, CT08, CT22), or 2 (CT01, CT02, CT09), 3 (CT13) or 6 (CT10) weeks after treatment. The most likely explanation for this delay, as well as some therapeutic benefit of higher doses, was that part of GC376 was still able to penetrate the brain. GC376 levels in cerebrospinal fluid represented only 3% plasma in the brain 2 hours after subcutaneous injection at a dose of 10 mg / kg (unpublished data). Although the relative concentrations of drug in the brain of these cats were low, they were still 21.4 times higher than the levels required to inhibit virus replication in tissue culture. Based on this finding, it was hypothesized that higher doses would allow higher amounts of drug to enter the brain. This assumption was supported by the experience of two cats that showed neurological symptoms. Increasing the dose of GC376 to 50 mg / kg every 12 hours in one cat (CT01) resulted in a marked improvement but did not eliminate the symptoms of brain disease. Prolongation of treatment by almost 3 months at 15 mg / kg every 12 hours appeared to delay the progression of neurological symptoms in the second cat (CT12), while attempts to reduce the total daily dose in this cat to 10 mg / kg every 12 hours or 15 mg / kg caused worsening of neurological symptoms every 24 hours. This suggests that doses of 15 mg / kg every 12 hours or higher allowed sufficient GC376 to cross the blood-brain barrier to slow but not eliminate neurological symptoms.

The high incidence of central nervous system (CNS) disease in this study was higher than previously reported and was unexpected because cats with signs of brain or spinal cord involvement were excluded from the study. CNS diseases have been shown to be much more likely in older cats with dry or mixed FIP than in young cats with wet FIP. This suggests that FIPV can enter the brain of many cats if given sufficient time. Peritoneal-type macrophages appear to play a role in CNS infection because FIPV-infected cells in the brain of cats with neurological FIP are more similar to peritoneal macrophages than to resident brain macrophages. This should come as no surprise, as macrophages migrate to a variety of tissues, including the brain, to perform immune surveillance and are also targets for a variety of infectious agents, such as FIPV and HIV-1. Infected macrophages play a major role in the spread of the virus to the brain in HIV patients, and detection of the virus in the brain is possible within a few weeks of infection. However, neurological damage usually occurs at a later stage. Anti-HIV drugs also reduce the frequency of severe neurological disorders, similar to that observed in this study on FIPV and GC376. There are also alternative explanations. It is possible that extra-CNS involvement may inhibit the development of brain diseases and vice versa. It is common for CNS disease to occur in the absence of visceral disease and vice versa. Suppression of virus replication in non-neuronal tissues may also increase the positive selection of mutants that are more neurotropic or neurovirulent. However, evidence of the latter would require large studies using laboratory cats.

Certain forms of FIP appeared to affect treatment success. The behavior of GC376 in the treatment of ocular FIP was paradoxical because this form responded extremely well to GC376. Although the eye lesions responded to treatment, all three cats with the eye eventually succumbed to brain diseases, which supported a close anatomical relationship between the eye and the CNS. Chronic ileocecal and colon involvement and growth retardation in older cats in this study were also a poor prognosis. In many of these cats, abdominal effusion appeared only as a terminal manifestation of their disease. Host factors also associated with a reduced response to antiviral therapy for other viral infections, such as HCV, include age, gender, cirrhosis or liver fibrosis, race, or body weight.

The development of resistance is a major problem for any antiviral drug, but FIPV is rarely transmitted from cat to cat, and drug resistance, if it occurs, would be a problem only for individual treated cats and not for the entire population. Although viral resistance to GC376 has not been observed for up to 20 passages in vitro, suggesting that resistance cannot be easily acquired, long-term and repeated in vivo treatment may be a stronger selection factor. However, viral resistance did not appear to be responsible for relapses of abdominal disease in the five cats treated. These cats had granulomatous formations, often in the colon and ileo-cecal-colic lymph nodes, which could provide a protected site for viruses to persist. The protection of pathogens in granulomas is a well-documented phenomenon for mycobacteria and applies to other pathogens such as viruses. Liver disease (cirrhosis) from HCV infection also increases the risk of relapse and requires longer treatment, which also suggests that viruses may be protected from drugs when they are in certain protected areas. The formation of "protective granulomas" involves a large number of chemokines and cytokines and upregulation of chemokine receptors, addressins, selectins and integrins. The persistence of pathogens in these sheltered sites may require a higher dose of the drug and a longer duration of treatment.

Treatment failures may also be the result of the host's inability to elicit a protective immune response during periods of viral replication. Such failure has been observed in HCV infection in humans. T-cell mediated immunity plays an important role in the protective immunity that occurs in about 20% acute HCV infection and in the same or greater proportion of FIPV infection. The possible synergies between T-cell-mediated viral clearance and antiviral therapy in cats with FIP have yet to be investigated. A combination of antiviral drugs and T-cell immune stimulants in the treatment of FIP, such as the combination of interferons and ribavirin in the treatment of HCV infection, could also be beneficial.

Sustained remission in 6/7 cats treated for 12 weeks or longer was to some extent predictable. These cats were 3.3-4.4 months old at the time of acute abdominal symptoms (C15, C17, C18, CT21, CT23) or thoracic effusion FIP (CT20). This made them younger than all but three other cats in the study (CT8, CT16, CT21) and more resembled 16-week-old laboratory cats with acute onset effusion FIP, which responded well to GC376. The disease, if acute, gives the virus little time to penetrate the brain or eyes. The acute nature of their disease may also have allowed the infection to permanently disrupt any protective immune response. The seventh cat, CT04, was extreme compared to these six younger cats. At age 6.8, CT04 was the oldest cat in a study that experienced significant weight loss (30%) and mesenteric lymph node disease. CT04 suffered relapses of the disease requiring re-treatment, but all relapses were identical to baseline and did not involve the CNS. Cats with this form of FIP are known to undergo spontaneous remission, suggesting that there is a tipping point between immunity and disease. Cats CT04 and CT21 demonstrated the wise decision to restart treatment in relapse, provided that the relapses did not involve the eyes or nervous system and still respond to drugs.

The determination of the minimum duration of treatment was based on a gradual prolongation of the duration of treatment based on a favorable response to treatment. Based on experimental studies, 2 weeks of treatment were expected to be sufficient; it was therefore used as a starting point. However, this study indicated that the minimum treatment period was close to 12 weeks, which was surprisingly close to the usual 12-week period required to treat people with HCV with protease inhibitors. However, the duration of HCV treatment can range from 8 to 24 weeks in different people. The CT21 cat was healthy, active on the outside and grew after 12 weeks of treatment, but the total protein and white blood cell counts still did not return to normal, as in the other six cats. Nevertheless, it was decided to discontinue treatment after 17 weeks due to the long period of normal health. Whether longer-term treatment could prevent a relapse of the disease 13 weeks after the end of treatment, we can only imagine, but it raises doubts about how long the treatment period should be for some cats. The question also arises as to how long the remission period must be in order for us to declare that there has been a recovery, and not just a long-term remission. The longest disease-free period at the time of writing was more than 11 months, with five additional cats showing no signs of infection for 5-9 months. Based on clinical and histological evidence of neurological disease at the time of fatal relapses, it appears that the virus eventually reaches the brain and may be the most important limiting factor in FIP antiviral therapy.

Although only one-third of cats have survived for a long time, the 20 cats in this study provide the basis for future studies with GC376 and other antiviral drugs that will follow. Not all cats will be treatable, but that shouldn't stop the effort. In this limited study, almost all treated cats returned to normal health, albeit for only a few weeks or months. It is important to be aware of the universality of viral pathogens and to take advantage of the pioneering development of drugs that are used clinically in the treatment of human diseases such as HIV / AIDS, hepatitis C, MERS, SARS, Ebola and influenza.

Conclusions

Inhibition of FIPV 3CLpro by GC376 was shown to be effective under the study conditions and led to a reduction in virus replication and remission of disease symptoms in cats with naturally occurring FIP outside the CNS. However, persistent remission in this study occurred earlier in kittens less than 18 weeks of age with acute wet FIP or in cats with dry FIP limited to mesenteric lymph node and is less likely to occur in cats older than 18 weeks with dry, mixed or the ocular form of FIP. Failure to achieve permanent remission was associated with either a high incidence of neurological disease during or after treatment or a recurrence of abdominal lesions. Antiviral therapy appeared to slow the progression of neurological disease but failed to reverse it at the dose used in this study. The cause of recurrence of extra-neurological disease during treatment has not been determined, but was not related to mutations in the protease-binding region.

Footnotes

Received: 3.8.2017

Additional material: Informed consent form of the owner.

Conflict of interests: YK, KOC and WCG have patent claims on protease inhibitors. Other authors do not represent any potential conflicts of interest in connection with the research, authorship or publication of this article.

Financing: The main support for this study was made possible by a grant from the Morris Animal Foundation, Denver, CO, USA. Additional funding for technical support and animal care was provided by Philip Raskin Fund, Kansas City, SOCK FIP, National Institutes of Health grant R01AI109039, and the Center for Pet Health, University of California, Davis, CA, USA.

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Read "Efficacy of a 3C-Like protease inhibitor in the treatment of various forms of acquired feline infectious peritonitis"

Brief Personal Biography - Niels C. Pedersen

The original biography, which was written by Niels Pedersen himself on January 31, 2021 at my request in our e-mail communication.
English version: Brief Personal Biography - Niels C. Pedersen

Niels C. Pedersen

Before college, I never thought about being a vet, even though I grew up on a poultry farm in Southern California and had all kinds of animals. In 1957, as a high school student, I moved to southern Nevada and worked as a volunteer on a local ranch with cattle that roamed large areas of state land. After joining the University of Nevada in Rene, my goal was to be a teacher of professional agriculture. In my second year, I was inspired by a course I took in the field of Veterinary Sciences, which was taught by veterinary researcher Dr. Donald Marble. He opened my eyes to a career in veterinary medicine, which I hadn't even thought about before his course. I completely fell for it and enrolled in a veterinary school at the University of California, Davis in 1963. I loved the veterinary school and considered studying veterinary medicine the greatest educational experience in biology and medicine available. My first experience with feline infectious peritonitis (FIP) came in 1964, when I was working with a doctoral student on a veterinary pathology program. The cause of FIP was not known at the time and there was no animal or human disease that resembled it. My first professional article, which I co-authored and was published in 1967, drew on this work. It was at the veterinary school that my interest in cat medicine grew, mainly because of my love for them and my experience with wild cats on my father's poultry farm. . In teaching, but also in clinical practice, I realized a great lack of knowledge about cats and their diseases. As an older student, I was most interested in infectious and immunological diseases with an emphasis on cats and dogs. This was a great opportunity for someone who originally wanted to be a cattle doctor. In 1967-68, I completed an internship in the field of medicine and surgery of small animals at Colorado State University and then traveled with my wife and young daughter to Australia, where I completed my doctoral studies at the National University of Australia, John Curtin School of Medical Research in the field of experimental pathology and immunology. My three years in Canberra have been the second greatest educational experience of my life and have led me to a lifelong fascination with the Australian outback, especially the flora of Western Australia. I received my PhD from ANU in late 1971 and returned to UC Davis Faculty of Veterinary Medicine in early 1972 to research cancer. This did not suit me very well, but fortunately my dean recognized my clinical abilities and asked me to join the clinical faculty as an assistant professor. I've stayed in Davis ever since. I retired in 2010 as Professor Emeritus. I remained active in research at school until 2020, when I retired definitively.

The years I spent at UC Davis as a faculty member are full of memories and experiences. I have been an active small animal internal medicine clinician for 17 years, teaching infectious diseases, immunological diseases and cat medicine for 21 years, and then spending most of my time on administration and research. I served as chair of the department and later was director of the Veterinary Genetic Laboratory and founder and director of the Center for Pet Health. My main research interests have been feline infectious diseases, which has enabled me to make a significant contribution to our understanding of feline leukemia (FeLV), calicivirosis, herpesvirus and feline immunodeficiency virus (FIV). However, I was most obsessed with feline coronavirus and its association with feline infectious peritonitis. I have found that feline coronavirus is related to canine and pig coronavirus, and that it exists as a ubiquitous and mostly non-pathogenic feline enteric virus (so-called feline enteric coronavirus - FECV). The most important finding was the relationship of FIP to FECV and the postulate that FIP is a specific mutation of FECV that is similar but unique to each cat. Gaining this knowledge was not easy, because the FIP revealed its secrets only bit by bit and with great reluctance gradually over several decades. We now have almost complete knowledge of how the FIP virus is formed and how it causes the various clinical forms of FIP. Although we understand how FIP viruses cause disease, all attempts to develop protective vaccines have failed. This has led to studies of how host and environmental factors affect the occurrence of FIP. Although we already understood this factor relatively well, and based on this knowledge, we were able to reduce the incidence of FIP, FIP continued to be the main killer of cats. Sometime around 2015, I realized that we would probably not be able to effectively prevent FIP, and that we should try to find a cure instead. The treatment option was based on the growing use of antiviral therapy for human diseases caused by herpesvirus, influenza virus, HIV and hepatitis C virus. Experience in the treatment of hepatitis C, which is also caused by RNA virus, was particularly important. Viral protease inhibitors have been particularly noteworthy in the treatment of infectious hepatitis C. My attention was drawn to an article on animal RNA virus protease inhibitors from researchers at Kansas State University. This led to the first important collaboration and discovery that a viral protease inhibitor called GC376 can cure FIP in about one-third of cats or more. Subsequent research into another class of antiviral drugs, called nucleoside analogues, for the Ebola virus has led to collaboration with scientists from Gilead Sciences on one of their compounds, GS-441524. Over the course of two years, two completely different antiviral drugs have been discovered that are able to cure FIP. The rest is history - In the last two years, thousands of cats have been cured from the FIP using GS-441524 worldwide.

In the life and career of a scientist, it is not often that he can be involved in almost every stage of learning about a new infectious disease such as FIP, and end his career by finding a cure. The first mention of the FIP dates back to the late 1950s, and there is no evidence that it existed much earlier. Therefore, in a sense, FIP in cats is similar to COVID-19 in humans, and the fact that both diseases are caused by coronavirus should not go unnoticed. Hopefully, researchers studying COVID-19 will not take 50 years to understand and find a cure for SARS-CoV-2 infection. I am satisfied with my career and my contribution to the health of cats on many fronts, not only the FIP. But the FIP has always been a worthy adversary that has caught my attention. There is still much to be learned, and I hope that the next generation will continue to study this fascinating disease.

-Niels C. Pedersen, January 31, 2021 Read "A Brief Personal Biography - Niels C. Pedersen"

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 ”

UC Davis professor claims that half a century of coronavirus study is neglected

Original article: UC Davis professor says his half-century of work studying the coronavirus is being neglected;(27.2.2020)
Translation: 18.1.2021

After half a century of studying the virus, a 76-year-old doctor was able to come up with a vaccine for cats two years ago.

DAVIS, Calif. - Coronavirus is associated with COVID-19 in the minds of many people around the world, but doctors say coronaviruses have been with us for decades.

Professor Dr. Niels Pedersen of UC Davis in California studied the virus for half a century. His focus? A specific strain occurring in cats causing feline infectious preitonitis (FIP).

"At one time it was a relatively unusual disease, but highly fatal for cats, full of mysteries and therefore very interesting," Pedersen explained. "So, among other things, I made it my life's work."

After half a century of studying the virus, a 76-year-old doctor was able to come up with a vaccine that is currently being used effectively to treat cats.

It is a drug that he thinks could be used to treat coronavirus in humans, but it is not a cheap affair for pet owners.

"The treatment consists of giving an injection for 12 weeks, which can cost up to $ 10,000, but there are thousands of people who have already treated their cats," he said.

However, Pedersen says his research and work are being ignored.

"It's also strange that much of what we've already learned through FIP in cats at this time can be applied to human COVID-19, but it's overlooked," Pedersen said.

Pedersen said that if a vaccine is currently being prepared for humans, public health officials do not necessarily have to rediscover the wheel.

"Knowledge about these vaccines can be used for all vaccines that will be developed for humans in the future," Pedersen explained.

Watch the conversation at  Facebook with Lena Howland. Read "Professor UC Davis claims that half a century of his work on the study of coronavirus is neglected"

DR. PEDERSEN’S AUTOBIOGRAPHY

Original article: DR. PEDERSEN'S AUTOBIOGRAPHY
Published 18.3.2014, Translation 29.1.2021

I was born in 1943. My father was a Danish emigrant and my mother was the daughter of Danish emigrants. However, I learned that I also have Swedish roots after my father's line. I spent the first 14 years of my life in Southern California (Azusa and San Dimas), where my parents rented and then owned several family poultry farms. Although wholesale and retail eggs were their main business, they also raised ducks, geese and turkeys for the holidays. My father liked cats, but not pets. He always kept a large number of them on the farm to keep the rodent population low and their diet was supplemented by blood eggs. I loved all sorts of animals and bred pigeons, pheasants, wild ducks. As a pet, I even had a goat and a rabbit. However, my greatest love was the cats.

My first feline companion was a white, short-haired female with brownish ears, called MoMo (the mother of a cat in my child's tongue). She lived with me for 14 years and regularly and later irregularly gave me kittens. I also befriended many wild cats and one of my tasks was to sit with a box of kittens when customers came to buy eggs. I found homes for many kittens and once in a while I got 25 or 50 cents. I got to know the character and personality of cats more than anyone else. My life changed in 1956, when a huge summer fire in the hills around our San Dimas farm caused the temperature around the chickens to rise to more than 53 ° C and we lost all the water used to cool them. Sixty thousand chickens died within a week as a result of the sunburn. This unfortunate incident was, in fact, a blessing, as small family poultry farms in Southern California were rapidly becoming unprofitable, and this event forced my father to leave poultry after 30 years. He monetized all the property, which had some value, and exchanged it for a small warm spring resort in southern Nevada, and since then my family has been a real middle class. The resort was located in a valley called Upper Muddy, which also housed a large cattle ranch. The high school was 20 miles from Overton and the school started by 6:30. I started high school as a freshman with great tremor and on the first day of school I immediately fell in love with my future wife (Gerie). A small high school and living in a Mormon farming community have given me all sorts of experiences I wouldn't have had if I attended a much larger high school in Southern California. Every capable boy competed in sports and there were many interesting courses and extracurricular activities. Each student had to participate in several activities. My greatest high school experience was with Future Farmers of America, and I eventually became an FFA civil servant.

In 1961, I was accepted to the University of Nevada in Rene for scholarships from the Union Pacific Railroad and the Max E. Fleishman Foundation with the intention of becoming a teacher of professional agriculture. I soon realized that this was probably not the best career choice, but I had no idea about anything else. At the end of the first year, however, I switched to Animal Sciences and in the second year I began to attend much more demanding subjects in preparation for a possible Ph.D. in this field. My life changed again when I enrolled in a veterinary course led by one of the two veterinarians who were responsible for the small veterinary program at UNR. I have never had any direct experience with veterinarians before. Dr. Marble started my interest in veterinary medicine and even allowed me to help him with some of his experimental operations. I was immediately caught up in it and at the last minute I applied to three veterinary schools, UC Davis, WSU and CSU. I was accepted to all three schools, but I decided to go to UC Davis, California, which was closer to my home. I'm a native Californian (and an adapted Nevada). I was also fortunate that my tuition was paid by the state of Nevada under the Western Interstate Compact for Higher Education (WICHE).

I entered veterinary school in September 1963 with the intention of becoming a general practitioner for cattle (my second and third loves are cows and poultry). I worked on several science projects over the weekends and holidays, and I fell even more in love with research. In August 1964, I married my high school love Gerie. In my spare time, I worked with one of the graduate pathology students on a new feline disease called feline peritonitis and co-authored my first scientific article on FIP in 1964. I was horrified by a lack of knowledge about cats and their diseases, but that didn't stop me. After graduating from veterinary school, I graduated in internal medicine for small animals and surgery at Colorado State University, which significantly affected my future life and career. Once again, I realized how little we knew about cat diseases, and I became even more aware of my lifelong affinity and understanding with this species. Prior to my internship in 1967, I was accepted as a graduate student at the John Curtin School of Medical Research (JCSMR), Australian National University, Canberra, ACT, Australia. Australia has fascinated me all my life and JCSMR has given me not only a great research experience, but also my first experience with this vast and beautiful country. After graduating, I worked briefly as a celebrity practitioner in Hollywood to earn money to get my family (I already had a daughter) to Australia. Studying research at the Department of Experimental Pathology at JCSMR was unmatched, but not entirely related to my future career. But I learned about experimental methods. In 1972 I received my Ph.D. from experimental pathology and immunology with honors in. My love for Australia, which I cultivated in books as a child, became a lifelong passion. My hobby is identifying and photographing Western Australian plants. At our house in Winters, California, I have a large collection of photos of the flowering shrubs of Western Australia.

Dr. As longtime director of the Center for Pet Health (CCAH), Pedersen led a team that raised money and designed his current home. The CCAH building was completed in 2006 at a cost of $ 16 million, all of which was obtained from private sources. It is considered one of the most beautiful and functional buildings in the complex and is home to five clinical services and four major research programs.

In early 1972, I returned to the United States for a research position at the School of Veterinary Medicine at UC Davis, where I studied animal cancer associated with retroviruses. This was my first experience with the feline leukemia virus, which we saw during my studies in many cats, but whose cause was unknown at the time. My first year in research did not go well for me and I was considering moving to a postdoctoral position in transplant immunology at the University of Washington. Before formalizing this position, however, the dean of the Veterinary School called me and asked me if I would consider accepting a position at an internal medicine clinic for small animals. Dean Pritchard was tired of hiring doctors with rich clinical experience but weak scientific skills. He decided to try to admit to the clinic people with great research experience and good clinical potential. I became the first of a large number of employees at UC Davis with a professional background of this kind. It turned out to be a very wise decision, as the clinical program at UC Davis soon became world famous not only for its high clinical level but also high level of research.

I have worked at UC Davis since 1972 and retired in October 2013 as Professor Emeritus. I spent the first 17 years in clinics, teaching and working in research. For the rest of my time at Davis, my career was much more about administration and research. After serving as chair of the department, I became the founder and director of the Center for Pet Health and a few years later the director of the Laboratory for Veterinary Genetics. This latter function has rekindled my interest in the role of genetics in disease. Research into infectious and immunological diseases in cats and dogs has accompanied me throughout my career. I am still working and continuing my first love, research. I have no plans to retire if my health allows it, because for me, retirement in research is much better than retirement.

Although I have studied many infectious diseases of cats, FIP remains my first love, if the term can be called a disease that is still so widespread and almost always fatal. I learned to look at the FIP as a "worthy adversary." It is undoubtedly one of the most complex and challenging infectious diseases in existence, and trying to find prevention and treatment is what drives me the most. I realize that the answers don't just come. It's not like researching human diseases that have no problems with funding, whether from individuals or organizations that have a personal interest in discovering treatments. I hope that I will continue to strive to keep the disease at the center of attention, and I can say that I am not alone. I have recently reviewed more than 100 clinical and scientific publications on FIP submitted by research groups around the world since 2008. No other cat disease has received so much attention.

I enclose a more detailed biography of my career with an emphasis on FIP research, which still engulfs me. My biggest career achievements have been in the field of feline infectious diseases such as FeLV, FIV, FCV, FECV and FIPV. My textbook on diseases in a multi-cat environment, which has become a classic today, is perhaps my best creative work. However, my wife, three daughters, a son and 7 (soon to be 8) grandchildren, all successful so far, are my greatest legacy. But I hope that at least for a while, my "paw prints" will remain visible through the literature on cat diseases. And something like pets I've always had cats - always only two and always males.

–Niels C. Pedersen

Note: When Dr. Pedersen mentions a more detailed career CV, meaning mainly a number of published articles that you will find in scientific journals.


This is a 1991 photo when I received the first Jean Holzworth Cat Research Award. Dr. Holzworth was for me the best feline medicine expert he had ever lived. She graduated from DVM at Cornell University and spent her entire life at Angell Memorial Animal Hospital in Boston. She wrote the first modern textbook on cat diseases and I considered her a good friend and inspiration. She convinced me of something I've always believed in - you can't be a great veterinarian for cats without deep and confidential knowledge of this species, including everything from anatomy and physiology to behavior. -DR. P.

Wife of Dr. Pedersen Gerie with the newest members of the SOCK FIP family - recently adopted two brothers Piper (right) and Frodo (left). Frodo and Piper are among the many former experimental cats that the Pedersen have adopted over the past 40 years.

Read “Autobiography of Dr. Pedersena ”
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