Miscellaneous questions frequently arising during antiviral drug treatment for FIP and aftercare

23.3.2022
NC. Pedersen, DVM PhD
Original article: Miscellaneous questions frequently arising during antiviral drug treatment for FIP and aftercare

Several issues often arise during FIP treatment. Before addressing these issues, it is important to mention the FIP treatment itself. Only antivirals that target specific viral proteins and inhibit FIP replication have been shown to have therapeutic effects. Currently, these include nucleoside analogs and RNA replication inhibitors GS-441524 (and a related prodrug Remdesivir), Molnupiravir (EIDD-2801) and the viral protease inhibitor GC376. Proper administration of these drugs has resulted in the cure of all forms of FIP in more than 90 % with minimal side effects. Most treatments are completed without complications. However, certain issues that are the subject of this article often arise.  

I pointed out the problems associated with unwanted sexual behavior in intact females and males treated with specific antivirals. The questions often come from countries where castration is either postponed or not common practice. They fear that the stress of castration and vaccines may affect the outcome of antiviral treatment. I believe that such concerns are exaggerated. If a cat is in treatment and in remission or is considered cured, it is okay to sterilize or neuter it, but preferably in the least stressful way possible. Cats can be neutered and sterilized quickly and efficiently and returned to their homes on the same day (castration) or within one day (sterilization) with minimal preoperative, operative and postoperative drug treatment and restrictions (eg cages, E-collars). Such operations will be less stressful for cats (and owners, which will then be reflected in their cats) than their sexual behavior. 

I am also not in favor of hormonal treatment to prevent unwanted sexual behavior in males or females, and I feel that effective castration and sterilization will be less stressful in the long run than such preventive measures. Therefore, if it is necessary to permanently change this behavior, surgical castration is more appropriate than chemical.  

Some owners seem to want to keep cured cats intact so that they can be used for breeding later. We know that FIP has both genetic and environmental components, which has led to the recommendation that purebred cats that breed FIP kittens should not be used for breeding. This should be even more true for cats that have been cured of the FIP.  

As far as vaccines are concerned, many already know that I am not a big fan of adult vaccines and the first annual booster vaccines because I feel that immunity is long-lasting. I also think that rabies vaccines cannot be used routinely in cats, whether in terms of public health or cats. Nevertheless, I accept that these views are not generally accepted and that the laws in several states require rabies to be vaccinated against rabbits, in some vaccination is not required and in others it is recommended but not required. I have not noticed the consequences of routine vaccinations in any of our cured cats. However, it is not something I would recommend for cats undergoing treatment. The immune system of these cats is responsible for other things than responding to vaccines.  

What are the indications for drugs other than specific antivirals for the treatment of FIP? During the initial illness, supportive (symptomatic) treatment may be required to keep the cats alive long enough for the antivirals to take effect. Drugs often used in this early stage usually include antibiotics (doxycycline / clindamycin), analgesics (opioids, gabapentin), anti-inflammatory drugs (corticosteroids, NSAIDS), immunostimulants (interferons, non-specific immunostimulants), and drugs. I have tried to avoid excessive use of these drugs except for temporary use and only if it is strongly justified, especially in severely ill cats during the first days. The most important goal of FIP treatment is to stop the replication of the virus in macrophages, which immediately stops the production of the numerous inflammatory and immunosuppressive cytokines that cause the symptoms of FIP. Although some drugs, such as corticosteroids (prednisolone) or NSAIDs (meloxicam), may inhibit inflammatory cytokines and cause clinical improvement, they are not curative. They can also mask the beneficial effects of GS treatment, which are often monitored to assess the effect and course of treatment. The response to antiviral treatment is also used for diagnostic purposes. The only drugs that completely suppress these harmful cytokines and cure FIP are antivirals such as GS-441524, molnupiravir or GC376, and related compounds. These antivirals cause a dramatic improvement in fever, activity, appetite, etc. within 24-48 hours. This improvement will be much greater than any improvement made with other drugs. Therefore, if the use of other drugs is not warranted, they should be discontinued as soon as the symptoms of FIP have steadily improved. 

I also do not believe in many supplements that are said to treat or prevent problems with the liver, kidneys, immune system or other organs. These supplements are expensive and have not been shown to be effective in what they claim. B12 injections only treat B12 deficiency, which is rare, and not anemia in FIP. The same goes for other vitamins. This also applies to a wide range of nutritional supplements and special diets for cats of many types. There is no essential ingredient in any of these supplements that could be provided by well-tested commercial cat food brands. There is also a possibility that some supplements interfere with the absorption of oral antivirals.  

How should cats be monitored after treatment and during the post-treatment observation period? From a technical point of view, no further blood tests are needed, especially if routine health assessments such as weight, appetite and temperature are continued during this period. Blood tests during this period do not change the outcome and can only increase the cost of treatment and increase the owner's stress. However, it is common for successfully treated cats to routinely test for blood during a 12-week post-treatment observation, usually every 4 weeks, but sometimes more frequently. In some cases, routine blood testing is continued for 12 weeks after treatment, even out of fear of possible relapse or recurrence. Relapses or new infections after a 12-week observation period are rare and are preceded by external signs of the disease, such as weight loss, lethargy, anorexia, poor coat and fever, which would be the best indicators for a blood test. Blood test panels also contain many values, and it is not uncommon for one or more values to be abnormal in healthy cats. Care must be taken not to over-interpret such abnormalities and to lead to excessive concern or additional testing in order to determine their significance. For example, a mild to moderate increase in one in three liver enzymes in a healthy cat is much less important than in another cat with symptoms of the disease. Read "Various issues common during FIP antiviral treatment and aftercare"

History of Feline infectious Peritonitis 1963-2022 – First description to Successful Treatment

Niels C. Pedersen
Center for Companion Animal Health, School of Veterinary Medicine, University of California, 944 Garrod Drive, Davis, CA, 95616, USA
Original article: History of Feline infectious Peritonitis 1963-2022 – First description to Successful Treatment
17.4.2022

Abstract

This article discusses the development of knowledge about feline infectious peritonitis (FIP) from its recognition in 1963 to the present and has been prepared to inform veterinarians, cat rescuers and carers, shelter staff and cat lovers. The causative agent of the feline coronavirus and its relationship to the ubiquitous and minimally pathogenic feline intestinal coronavirus, epizootology, pathogenesis, pathology, clinical signs and diagnostics are briefly mentioned. The main emphasis is placed on the risk factors influencing the incidence of FIP and the role of modern antivirals in successful treatment.

Introduction

Figure 1. Photo of the author and Dr. Jean Holzworthová (1915-2007) from 1991. Dr. Holzworth was the best feline veterinarian the author knew and was responsible for the first report of FIP as a specific disease. She spent her entire career at Angell Memorial Animal Hospital in Boston.

Feline infectious peritonitis (FIP) was described as a specific disease in 1963 by veterinarians at Angell Memorial Animal Hospital in Boston (Holzworth 1963) (Fig. 1). Pathology records from this institution and Ohio State University failed to identify earlier cases (Wolfe and Griesemer 1966), although identical cases were soon recognized worldwide. The initial pathological descriptions were of diffuse inflammation of the tissues lining the abdominal cavity and abdominal organs with extensive effusion of inflammatory fluid, after which the disease was eventually named (Wolfe and Griesemer 1966, 1971) (Figs. 2, 3). A second and less common clinical form of FIP, which presents with less diffuse and more widespread granulomatous lesions involving organ parenchyma, was first described in 1972 (Montali and Strandberg 1972) (Figs. 3,4). The presence of inflammatory effusions in the body cavity in the common form and the absence of effusions in the less common form led to the names wet (effusion, non-parenchymatous) and dry (non-effusion, parenchymatous) FIP.

The prevalence of FIP appears to have increased during the panzootic disease caused by feline leukemia virus (FeLV) in the 1960s–1980s, when many cases of FIP were found to be associated with FeLV (Cotter et al., 1973; Pedersen 1976a). Subsequent management of FeLV infection in owned cats through rapid testing and vaccination resulted in an increase in FIP cases. However, recent interest in breeding/rescue along with effective treatment has led to increased awareness of the disease and its diagnosis.

Figure 2. Gross necroptic appearance of the abdominal cavity of a cat with acute onset wet FIP. The abdomen is filled with several hundred ml of yellow viscous fluid, the omentum is reddened, edematous and contracted, and fibrin deposits (arrows) are visible on the surface of the spleen and the edges of the liver. A fiber of fibrin can be seen on the spleen
Figure 3. Appearance of an open abdomen at autopsy of a cat that died of a chronic form of effusive FIP. The abdomen is filled with a viscous, yellow-colored exudate, and the omentum is thickened and contracted. The main lesions are in the liver with numerous plaque-like structures (pyogranulomas) on the cover. More circumscribed lesions (granulomas), also oriented on the serous surface, look more fleshy and are elevated above the surface. These lesions also involve the underlying liver parenchyma and are more typical of dry FIP. This is an example of a case of FIP that transitions between the wet and dry forms (arrow).
Figure 4A – Gross section of kidneys of two cats with dry form of FIP. The lesions are superficial and extend into the underlying parenchyma.
Figure 4B - lesions of the dry form of FIP in organs such as the kidneys, cecum, colon and intestinal lymph nodes (Fig. 5) were grossly confused with renal lymphoma.
Figure 5. Gross enlargement of ileo-cecal-colic lymph nodes in a cat with dry FIP.

Etiological factor

The first attempts did not allow identifying the causative agent of FIP, but confirmed its infectious nature (Wolfe and Griesemer 1966). A viral etiology was established in 1968 using ultrafiltrates of infectious material (Zook et al., 1968). The causative virus was subsequently identified as a coronavirus (Ward 1970), which is closely related to enteric coronaviruses of dogs and pigs (Pedersen et al., 1978).

Confusion arose when feline enteric coronavirus (FECV) was isolated from the feces of healthy cats and proved to be indistinguishable from feline infectious peritonitis virus (FIPV) (Pedersen et al., 1981). Unlike FIPV, which readily induced FIP in laboratory cats, experimental infections with FECV were largely asymptomatic. The relationship between the two viruses became clear when FIPVs were found to be FECV mutants that arise in the body of every cat with FIP (Vennema et al., 1995; Poland et al., 1996).

FECV is ubiquitous in feline populations worldwide and is first shed in faeces from approximately 9–10 weeks of age, coinciding with the loss of maternal immunity (Pedersen et al., 2008 ;). The infection takes place via the faecal-oral route and targets the intestinal epithelium, and the primary signs of enteritis are mild or inconspicuous (Pedersen et al., 2008; Vogel et al., 2010). Subsequent faecal excretion occurs from the colon and usually stops after several weeks or months (Herrewegh et al., 1997; Pedersen et al., 2008; Vogel et al., 2010). Immunity is short-lived and repeated infections are common (Pedersen et al., 2008; Pearson et al., 2016). Over time, stronger immunity eventually develops and cats older than 3 years are less likely to shed the infection in their faeces (Addie et al., 2003). FECV is constantly subject to genetic drift into locally and regionally identifiable clades (Herrewegh et al., 1997; Pedersen et al., 2009).

FECV and FIPV are classified as biotypes of the feline coronavirus (FCoV) subspecies. The genomes of FECV and FIPV biotypes are related at >98 %, but with unique host cell tropism and pathogenicity (Chang et al., 2012; Pedersen et al., 2009). FECVs infect the mature intestinal epithelium, whereas FIPVs lose intestinal tropism and acquire the ability to replicate in monocytes/macrophages. The published names FECV or FIPV will be used here when discussing aspects of the disease specific to each biotype, while the term FCoV will be used when discussing features common to both biotypes.

Three types of mutations are involved in the biotype change of FECV to FIPV. The first type, which is unique to each cat with FIP (Poland et al., 1996), consists of an accumulation of missense and nonsense mutations in the c-terminus of the auxiliary 3c gene, often resulting in truncated 3c gene products (Pedersen et al., 2012 ; Vennema et al., 1995). The second type of mutation consists of two specific single nucleotide polymorphisms in the fusion peptide of the S gene, one or the other form being common to >95 % FIPV and absent in FECV (Chang et al., 2012). A third type of mutation, unique to each FIPV isolate and not found in FECV, occurs in and around the furin cleavage motif between the receptor binding domain (S1) and the fusion domain (S2) of the spike gene (S) (Licitra et al., 2013). These mutations have different effects on furin cleavage activity. Together and in an as yet undetermined manner, they are responsible for the shift of the tropism of the host cell from the enterocyte to the macrophage and for the profound change in the form of the disease.

FCoV, and therefore FECV and FIPV, exist in two serotypes identified by antibodies against the viral neutralizing epitope on the S gene (Herrewegh et al., 1998; Terada et al., 2014). Serotype I FCoVs are identified in cat sera and are prevalent in most countries. Serotype II FCoVs result from recombination with the S part of the canine coronavirus gene (Herrewegh et al., 1998; Terada et al., 2014) and are identified by canine coronavirus antibodies. Serotype II FIPVs are easily cultured in tissue culture, whereas serotype I FIPVs are difficult to adapt to growth in vitro. Serotype I and II FECVs were not grown in conventional cell cultures (Tekes et al., 2020).

FIPVs are found exclusively in activated monocytes and macrophages in affected tissues and effusions and are not secreted into the environment. Therefore, cat-to-cat (horizontal) transmission of FIPV is not the main mode of spread. Rather, FIP follows the pattern of an underlying enzootic FECV infection, with sporadic cases and occasional small outbreaks of disease (Foley et al., 1997). These clusters of cases can be mistaken for epizootics. The only report of an epizootic occurrence of FIP was associated with a single serotype II virus that appeared to develop in a shelter housing both dogs and cats (Wang et al., 2013). Horizontal transmission in this case followed an epizootic rather than an enzootic disease model, with infection spreading rapidly to cats of all ages and in close contact with the index case (Wang et al., 2013).

The low incidence of FIP cases in the population suggests that FIPV mutations arise infrequently. However, studies involving FECV infection in immunocompromised cats infected with FIV and FeLV suggest that FIP mutants may be common but only cause disease under certain circumstances. Nineteen cats infected with feline immunodeficiency virus (FIV) for 6 years and a control group of 20 littermates not infected with FIV were orally challenged with FECV (Poland et al., 1996). Cats in both groups remained asymptomatic for two months when two cats in the FIV-infected group developed FIP. In a second study, 26 young cats with enzootic FECV infection from a breeding colony with no history of FIP were contact-exposed to FeLV carriers (Pedersen et al., 1977). Two kittens in the group subsequently developed FIP 2–10 weeks after becoming FeLV viremic. The question remains, how long can FIPV viruses survive in the body before they are eliminated? According to one of the theories, they persist in the body for a certain time and become pathological only if immunity against them is impaired (Healey et al., 2022). This theory is supported by the way immunity to FeLV develops. Most cats resist FeLV by kitten age and develop robust and permanent immunity, but this occurs within a few weeks, during which the virus persists in a subclinical or latent state (Pedersen et al., 1982; Rojko et al., 1982). . Methylprednisolone given during this period, but not after, will abolish developing immunity and lead to a state of persistent viremia.

Epizootology

Epizootiology is the study of the occurrence, spread and possible control of animal diseases and the influence of environmental, host and agent factors. FIP is considered one of the most important infectious causes of death in cats, although there are no precise data on prevalence. It is estimated that 0.3–1.4 % deaths of cats presented to veterinary institutions are related to FIP (Rohrbach et al., 2001; Pesteanu-Somogyi et al., 2006; Riemer et al., 2016) and in some shelters and breeding stations up to 3.6–7.8 % (Cave et al., 2002). FIP is also described as an environmental disease with a higher incidence of multiple cats. Three-quarters of the FIP cases in the currently ongoing treatment study came from the field through foster carers/rescues and cat shelters, 14 % from kennels, and only 11 % from households.1

Studies based on cases observed in academic institutions have demonstrated the influence of age and gender on the incidence of FIP (Rohrbach et al., 2001; Pesteanu-Somogyi et al., 2006; Pedersen 1976a; Worthing et al., 2012; Riemer et al., 2016) . Three-quarters of the cases in these cohorts occurred in cats younger than 3 years of age, and few cases occurred after 7 years of age. This was also confirmed by a current and ongoing field study from the Czech Republic and Slovakia, in which it was found that more than 80 % cases of FIP occurred in cats under 3 years of age and only 5 % in cats older than 7 years (Fig. 6) .1 Earlier institutional studies differed on the effect of sex, but indications were that male cats were slightly more susceptible to FIP than female cats. This was also confirmed by current data from the field, which show a ratio of males to females of 1.3:1.1. It is unclear whether castration affects the incidence of FIP, with some reports suggesting that it may increase susceptibility (Riemer et al., 2016), while others do not report such a clear effect.1

Figure 6. Age of more than 607 cats from the Czech Republic and Slovakia at the time of diagnosis and treatment of FIP.1 Thirty percent of infections were in cats six months of age or younger, 50 % at one year of age, and 85 % at three years of age or younger.

Other environmental and viral risk factors have been implicated in the increased incidence of FIP, but their significance requires knowledge of disease occurrence in their absence. A possible baseline may have been provided by a study of enzootic FECV infection, which had been unrecognized for many years in a well-managed specific pathogen-free breeding colony (Hickman et al., 1995). This colony was kept in strict quarantine free of other infections and the standard of nutrition and husbandry was high. This colony produced hundreds of kittens each year before the first case of FIP was diagnosed. Such observations suggest that FIP may be a rare phenomenon in the absence of risk factors.

The importance of moving to a new home as a risk factor for FIP is only now being appreciated. Breeders, many of whom have not experienced any cases of FIP in their litters, are most concerned about the announcement that one of their kittens has developed FIP shortly after going to a new home. A recent study found that more than half of cats with FIP had experienced a change in environment, shelter or capture in the weeks before the illness.1 Cats are known to hide outward signs of stress, even when suffering from serious internal disease consequences. Even simple procedures such as changing the cage suppress immunity and reactivate latent herpes virus shedding and disease symptoms in cats (Gaskell and Povey, 1977). Stressful situations, even those that seem minor, can cause a decrease in lymphocyte levels and “sickness behavior” (Stella et al., 2013).

Differences in the genetic make-up of enzootic FCoV strains may also contribute to the prevalence of FIP in the population. Serotype II FIPVs are thought to be more virulent than serotype I and more likely to be transmitted from cat to cat (Lin et al., 2009; Wang et al., 2013). It is also possible that certain FECV clades are more susceptible to mutation to FIPV, which should be studied. The author also observed a disproportionately high proportion of cats with neurologic FIP in some regions, suggesting that genetic determinants in certain FCoV strains may be more neurotropic.

Immunodeficiencies associated with retroviruses are associated with susceptibility to FIP. Up to half of FIP cases during the peak of FeLV panzootic disease were persistently infected with FeLV (Cotter et al., 1973; Pedersen 1976a; Hardy 1981). FeLV infection causes suppression of T-cell immunity, which may inhibit the protective immune response to FIP. The importance of FeLV infection for the incidence of FIP has declined significantly since the 1980s, when carrier elimination and vaccination pushed FeLV back into the wild, where exposures are less severe and immunity is the usual outcome. Chronic feline immunodeficiency virus (FIV) infection has also been shown to be a risk factor for FIP in FECV-infected cats under experimental conditions (Poland et al., 1996). In one recent field study, FeLV infection was recognized in 2 % and FIV in 1 % cats treated for FIP.1

The incidence of FIP in purebred cats is reported to be higher than in random breeding cats, with some breeds appearing to be more susceptible than others (Pesteanu-Somogyi et al., 2006; Worthing et al., Genetic predisposition to FIP has been investigated in several Persian cat breeds and is estimated to account for half the risk of the disease (Foley et al., 1997). Some breeds, such as the Birman, are more susceptible to developing dry than wet FIP (Golovko et al., 2013). Attempts to identify specific genes associated with susceptibility for FIP in Burmese cats included several immune-related genes, but none reached the desired significance (Golovko et al., 2013).The largest study of genetic susceptibility to FIP showed that it is extremely polymorphic and reported consanguinity as a major risk factor. breeding (Pedersen et al., 2016).Specific polymorphisms in several genes have also been associated with high levels of FECV shedding among several breeding cat breeds (Bubeniko and et al., 2020).

In females, FIP, usually the wet form, may develop during pregnancy or in the perinatal period. This phenomenon resembles the suppression of immunity in pregnant women and the predisposition to certain infections (Mor and Cardenas 2010). It is not clear whether subclinical FIP is activated by pregnancy or by increased susceptibility to new infection. Maternal infection early in pregnancy results in fetal death and resorption, while later infections often result in abortion (Fig. 7). Kittens can be born healthy, but develop disease in the perinatal period and die. Some babies are born uninfected thanks to the effectiveness of the placental barrier between mother and fetus or thanks to the help of antiviral treatment (Fig. 8).

Figure 7. Aborted kittens from a dam that developed wet FIP late in pregnancy. Miscarriage was the first symptom of FIP, quickly followed by the classic symptoms of abdominal wet FIP. The mother was successfully cured of FIP with the antiviral GS-441524.
Figure 8. This mother developed symptoms of wet abdominal FIP 3 weeks after the onset of pregnancy and was successfully treated with GS-441524. Subsequently, she gave birth to a litter of four kittens by caesarean section, one of which died and three survived and grew up healthy. Treatment was given during the remaining 6 weeks of gestation and continued for 6 weeks during which the kittens were successfully nursed. GS-441524 had no apparent side effects on the mother or the kittens.

A possible increase in the number of cases of FIP was observed in cats older than 10 years in studies conducted 50 years ago (Pedersen 1976a). Slightly more than 3 % cases of FIP in a recent study occurred in cats 10 years of age and older and 1.5 % in cats 12 years of age and older (Fig. 6).1 The occurrence of FIP in the elderly often involves two different scenarios. The first scenario also involves exposure to FECV faecal excretion, but in a unique way. It is common for old cats to mate as kittens and live together in relative isolation unexposed to FECV for many years. One cat in the pair dies, is left alone, and a much younger companion obtained from a rescue organization, shelter, or kennel is brought into the household that has a high probability of excreting FECV. Older cats are also susceptible to the same FIP risk factors as younger cats, as well as other factors associated with aging. The first of these is the impact of aging on the immune system, with the most consequential being the deterioration of cellular immune function (Day 2010). Other risk factors associated with old cats include the debilitating and potentially immunosuppressive effects of diseases such as cancer and chronic diseases of the kidneys, liver, oral cavity and intestines. Some diseases in old cats can be mistaken for FIP or complicate the treatment of FIP if they are present at the same time.

Other risk factors that need further investigation include loss of maternal systemic immunity by separation at birth, early weaning and loss of lactogenic immunity, malnutrition, common kitten infectious diseases, early neutering, vaccination, congenital heart defects, and even a shelter fire (Drechsler et al.), 2011; Healey et al., 2022; Pedersen 2009, Pedersen et al. 2019).1 However, the most important positive risk factor remains the presence of FECV in the population (Addie et al., 1995). The prevalence of FIP in several Persian cat breeds was also related in one study to the proportion of cats that shed FECV at a given time and to the proportion of these cats that are chronic shedders (Foley et al., 1997). The importance of exposure to FECV supports the need to find ways to either prevent infection or reduce its severity. One of the first steps is a better understanding of FECV immunity (Pearson et al., 2019).

Pathogenesis

The first interface between FECV and the immune system is the lymphatic tissues of the intestine (Malbon et al., 2019, 2020). Although the downstream events leading to FIP are not fully understood, it is possible to speculate based on what is already known about FECV and FIPV infections, other macrophage-tropic infections, and viral immunity in general. During intestinal infection, FECV particles and proteins reach the local lymphatic tissues and are processed by phagocytic cells first into peptides and finally into amino acids. Some of these peptides will be recognized as foreign when arrayed on the cell surface, triggering innate (innate or non-specific) and adaptive (acquired or specific) immune responses (Pearson et al., 2016). FECVs also mutate to FIPV at the same time and in the same cell type. Some of these mutations will allow the virus to replicate in these or closely related cells of a specific monocyte/macrophage lineage.

The host cell for FIPV appears to be a specific class of activated monocytes found around venules on the surface of intestinal and thoracic organs, mesentery, omentum, uveal tract, meninges, choroid and ependyma of the brain and spinal cord, and freely in effusions. These cells belong to the activated (M1) class (Watanabe et al., 2018) and resemble a subpopulation of small peritoneal macrophages described in mice (Cassado et al., 2015). This type of cell arises from circulating bone marrow-derived monocytes that are rapidly mobilized from the blood in response to infectious or inflammatory stimuli. A similar-looking population of activated monocytes has been described around blood vessels in the retina affected by FIP (Ziolkowska et al., 2017). These cells stained for calprotectin, indicating their blood origin. Although FIPV infection occurs initially in smaller activated monocytes, viral replication is most intense in large, vacuolated, terminally differentiated macrophages (Watanabe et al., 2018). The virus released from these cells rapidly infects activated monocytes produced in the bone marrow and drawn to the site from the bloodstream.

The cellular receptor used by FECVs to infect intestinal epithelial cells has not yet been determined. The cellular receptor that FIPVs use to infect activated monocytes is also unknown. RNAs for conventional coronavirus receptors such as aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2) and CD209L (L-SIGN) were not upregulated in infected peritoneal cells of cats with experimental FIP, and CD209 (DC-SIGN) was significantly underexpressed (Watanabe et al., 2018). An alternative route of infection of activated monocytes may involve immune complexation of the virus and entry into cells by phagocytosis (Dewerchin et al., 2008, 2014; Van Hamme et al., 2008). Activated monocytes in lesions stain strongly positive for FIPV antigen, IgG and complement (Pedersen, 2009) and mRNA for FcγRIIIA (CD16A/ADCC receptor) is markedly increased in infected cells (Watanabe et al., 2018), supporting infection through immune complexation and alternative receptors related to phagocytosis.

Macrophage pathogens are intracellular and elimination of infected cells occurs through lymphocyte-mediated killing. The first line of defense is non-specific lymphocytes, and if they fail, an adaptive immune response to FIPV follows through specific T-lymphocytes. If infected activated monocytes and macrophages fail to be contained and eliminated, they may disseminate locally in the abdominal cavity, possibly from lymph nodes in the lower intestinal region and the site of FECV replication. Spread locally and to distant sites via the bloodstream is by infected monocyte cells (Kipar et al., 2005).

FIP occurs in two basic forms, wet (effusive, nonparenchymatous) (Figures 2 and 3) or dry (noneffusive, parenchymatous) (Figures 4 and 5), with wet FIP accounting for 80 % cases.1 The term "wet" refers to a characteristic fluid discharge in the abdomen or chest (Wolfe and Griesemer 1966, 1971). Wet FIP lesions are dominated by inflammation reminiscent of immediate or Arthus-type hypersensitivity (Pedersen and Boyle, 1980), whereas dry FIP lesions resemble delayed-type hypersensitivity reactions (Montali and Strandberg 1972; Pedersen 2009). The wet and dry forms of FIP therefore reflect competing influences of antibody and cell-mediated immunity and associated cytokine pathways (Malbon et al., 2020, Pedersen 2009). Immunity to FIPV-infected cells, which is the norm, is thought to involve strong cell-mediated responses (Kamal et al. 2019). Dry FIP is thought to occur when cell-mediated immunity is partially effective in suppressing infection, and wet FIP when cellular immunity is ineffective and humoral immune responses predominate.

FIP is considered unique among macrophage infections because it is viral, but the dry form shares many clinical and pathogenic features with feline diseases caused by systemic mycobacterial (Gunn-Moore et al., 2012) and fungal infections (Lloret et al., 2013). . Similarities in pathogenesis also exist between wet FIP and antibody-enhanced viral infections such as dengue fever and dengue hemorrhagic shock syndrome (Pedersen and Boyle 1980; Rothman et al., 1999; Weiss and Scott 1981).

Host responses are thought to solely determine the outcome of FIPV infection and the resulting forms of disease. However, macrophage-tropic pathogens have evolved their own unique defense mechanisms against the host (Leseigneur et al., 2020). One of the mechanisms is the delay of programmed cell death (apoptosis). Delayed apoptosis allows sustained microbial replication and eventual release of more infectious agents, as has also been described in FIPV-infected macrophages (Watanabe et al., 2018). FIPV can also control the recognition and killing of infected activated monocytes by specific or non-specific T-cells. The cell surface targets for T-cells that kill infected cells are likely FIPV proteins (antigens) expressed on major histocompatibility complex class I (MHC-I) receptors. However, surface expression of viral antigens by MHC-I receptors was not detected on FIPV-positive cells collected from FIP tissues or effusions (Cornelissen et al., 2007). DC-Sign has been proposed as a receptor for FIPV (Regan and Whitaker, 2008), but RNA for DC-Sign is markedly underexpressed by infected peritoneal cells, whereas RNA for Fc (MHC-II) receptors is markedly overexpressed and RNA for MHC -I is reduced (Watanabe et al., 2018). This suggests that the normal mode of infection of host cells may be altered by FIPV to favor infection by phagocytosis instead of binding to specific viral receptors on the cell surface, fusion with the cell membrane, and internalization.

Pathology

Detailed descriptions of the gross and microscopic lesions in the wet form of FIP were first described by Wolfe and Griesemer (1966, 1971). The disease is characterized by vasculitis involving venules in the tissues lining the abdominal or thoracic cavity, organ surfaces, and supporting tissues such as the mesentery, omentum, and mediastinum. The inflammatory process leads to effusions in the abdominal or chest cavity up to a volume of one liter or more (Fig. 2, 3). The underlying lesion is a pyogranuloma, which consists of a focal accumulation of activated monocytic cells in various stages of differentiation, interspersed with non-degenerate neutrophils and sparse numbers of lymphocytes. Pyogranulomas are superficially oriented and appear grossly and microscopically as single and coalescent plaques (Fig. 2).

FIPV antigen is immunohistochemically (IHC) observed only in activated monocytes in lesions and effusions (Litster et al., 2013). Large vacuolated terminally differentiated macrophages are particularly rich in virus (Watanabe et al., 2018), reminiscent of the lepromatous form of leprosy (deSousa et al., 2017). Lymph nodes located near the sites of inflammation are hyperplastic and enlarged.

The relationship between dry and wet FIP was first described in 1972 in a report of cases of unknown etiology with similar pathology (Montali and Strandberg 1972). As the authors state, "this pathological syndrome was characterized by granulomatous inflammation in various organs, but mainly affected the kidneys, visceral lymph nodes, lungs, liver, eyes and leptomeninges". Tissue extracts of these lesions induced wet FIP in laboratory cats, confirming that wet and dry FIP are caused by the same agent.

The gross and microscopic pathology of dry FIP resembles that of other macrophage-tropic infections such as feline systemic blastomycosis, histoplasmosis, coccidioidomycosis (Lloret et al., 2013), tuberculosis and leprosy (Gunn-Moore et al., 2012). Lesions of dry FIP mainly involve the abdominal organs (Figs. 5, 6) and are rare in the thoracic cavity (Montali and Strandberg 1972; Pedersen 2009). Lesions are less widespread and focal than in wet FIP, with a tendency to extend from the serous surfaces into the parenchyma of the underlying organs (Figs. 5, 6). The target of the host immune response are small aggregates of infected monocytic cells associated with venules, similar to pyogranulomas in wet FIP, but surrounded by dense accumulations of lymphocytes and plasma cells and variable fibrosis. The florid hyperemia, edema, and microhemorrhage associated with wet FIP are mostly absent, therefore significant effusions in the body cavities are absent. The host response to foci of infection gives the lesions a gross tumor-like appearance (Figs. 5, 6). Infected activated monocytes in the central focus of infection are less dense and contain lower levels of virus than in the wet form (Pedersen 2009;), a feature of the tuberculoid form of leprosy (de Sousa et al., 2017). Lesions in some places, for example on the wall of the large intestine, can cause a dense surrounding zone of fibrosis, which resembles classic tuberculosis granulomas. Transitional forms also exist between wet and dry forms in a small number of cases and are mostly recognizable at autopsy (Fig. 3).

Ocular and neurological FIP are classified as forms of dry FIP (Montali and Strandberg 1972). However, pathology in the uveal tract and retina and in the ependyma and meninges of the brain and spinal cord is intermediate between wet and dry FIP (Fankhauser and Fatzer 1977; Peiffer and Wilcock 1991). This can be explained by the effect of the blood-ocular and blood-brain barrier in protecting these areas from systemic immune reactions.

Clinical characteristics of FIP

The five most common symptoms in cats with FIP, regardless of clinical form and frequency of occurrence, are lethargy, loss of appetite, enlarged abdominal lymph nodes, weight loss, fever, and deteriorating coat.1 These symptoms can appear quickly, within a week, or they can exist for many weeks or even months before a diagnosis is made. The course of the disease tends to be more rapid in cats with wet FIP than with dry FIP, and growth retardation is common in young cats, especially those with more chronic disease. 20 % cats with fever as the main symptom are eventually diagnosed with FIP (Spencer et al., 2017).

The wet form of FIP occurs in approximately 80 % cases, more often in younger cats, and tends to be more severe and more rapidly progressive than the dry form. Abdominal effusion (ascites) is four times more common than pleural effusion, with abdominal distension (Fig. 9) and dyspnea being common symptoms. Pyrexia and jaundice are more common symptoms in cats with wet than dry FIP (Tasker, 2018).

Figure 9.  Adult longhair cat with chronic abdominal wet FIP. The cat was in acceptable health except for mild weight loss, lethargy, poor coat quality, and occasional low-grade fever. Abdominal distension was not noted for some time, and the abdominal fluid contained relatively low protein and white blood cell counts.
Figure 9. A young cat who presented with rapid onset of high fever, loss of appetite, abdominal distension and abdominal fluid with a high protein and white blood cell content.

Most cats with dry FIP present with disease symptoms limited to the abdomen and/or chest. The most common clinical signs of dry FIP are palpable or ultrasound-identifiable masses in the kidney (Fig. 4), cecum, colon, liver, and associated lymph nodes (Fig. 5). Lesions of dry FIP usually spare the thoracic cavity and rarely occur in the skin, nasal passages, pericardium, and testes as part of a wider systemic disease.

Neurological and ocular disease are the sole or secondary features of 10 % of all FIP cases and are 10 times more often associated with dry than wet FIP (Pedersen 2009). The neurological and ocular forms of FIP have been classified as forms of dry FIP, but it may be more appropriate to classify them as distinct forms of FIP resulting from the modifying effects of the blood-ocular and blood-brain barriers behind which they occur. These barriers have a strong impact on the nature of eye and central nervous system (CNS) disease and response to antiviral therapy.

Clinical signs of neurologic FIP involve both the brain and spinal cord and include posterior weakness and ataxia, generalized incoordination, seizures, mental dullness, anisocoria, and varying degrees of fecal and/or urinary incontinence (Foley et al., 1998; Dickinson et al., 2020) ( Fig. 10). Extreme intracranial pressure can lead to sudden herniation of the cerebellum and brainstem into the spinal canal and spinal shock syndrome. Prodromal symptoms include compulsive wall or floor licking, litter eating, involuntary muscle twitching, and reluctance or inability to jump to high places. Eye involvement may precede or accompany neurological disease. Neurological FIP is a common phenomenon with antiviral therapy, either occurring during treatment of non-CNS forms of FIP or as a manifestation of disease relapse after treatment cessation (Pedersen et al., 2018, 2019; Dickinson et al., 2020).

Figure 10. A young cat with dry FIP and neurological impairment. The cat is lethargic, emaciated and with poor fur. The fur in the perineal area is wet and stained from urinary incontinence.
Figure 11. This cat's right iris staining was the first sign of FIP-associated uveitis. There is a slight haze in the anterior chamber, and there are fibrin deposits rich in red blood cells on the inside of the cornea. The pupils are also unequal (anisocoria).
Figure 12. A young cat with ocular FIP presenting as anterior uveitis in the right eye with secondary glaucoma causing globe enlargement. The iris has changed color due to inflammation, the vessels at the base of the iris are congested, and there is turbidity of aqueous humor and inflammatory products on the back of the cornea. Intraocular pressure is usually low in uncomplicated uveitis but elevated in cats with glaucoma.
Figure 13. This young cat had anterior uveitis, but her FIP therapy with GS-441524 was delayed, allowing glaucoma to develop in both eyes. Treatment cleared the underlying uveitis and greatly improved external health, but secondary glaucoma and blindness persisted.

Eye involvement is usually obvious and is confirmed by ophthalmoscopic examination of the anterior and posterior chambers. Ocular FIP affects the iris, ciliary bodies, retina, and optic disc to varying degrees (Peiffer and Wilcock, 1991; Ziółkowska et al., 2017; Andrew, 2000). The earliest symptom is often a unilateral change in the color of the iris (Fig. 11). The anterior chamber may appear cloudy and may show high protein levels and water turbidity on refraction. Inflammatory products in the form of activated macrophages, red blood cells, fibrin markers and small blood clots are washed into the anterior chamber. This material often adheres to the back of the cornea as keratic precipitates (Fig. 12). The disease can also affect the retina in tapetal and non-tapetal areas and lead to retinal detachment. Intraocular pressure is usually low, except in cases complicated by involvement of the ciliary body and glaucoma (Fig. 12, 13).

FIP Diagnosis

Signaling, environmental history, clinical signs, and physical examination findings often point to FIP (Tasker, 2018). A thorough physical examination should include body weight and temperature, coat and body condition, manual palpation of the abdomen and abdominal organs, gross assessment of cardiac and pulmonary function, and a cursory examination of the eyes and neurological system. Strong suspicion of an effusion in the abdominal or thoracic cavity may warrant confirmatory aspiration and even in-house fluid analysis as part of the initial examination.

Abnormalities in the complete blood count (CBC) and basic serum biochemical panel are important factors in the diagnosis of FIP (Tasker, 2018; Felten and Hartmann, 2019) and monitoring of antiviral therapy (Pedersen et al., 2018, 2019; Jones et al., 2021). ; Krentz et al., 2021) (Fig. 14). Total leukocyte counts are most likely high in cats with wet FIP, but low counts can occur with severe inflammation. A high leukocyte count is often associated with neutrophilia, lymphopenia, and eosinopenia. Mild to moderate non-regenerative anemia is also frequently seen in both wet and dry FIP. Total protein is usually elevated due to elevated globulin levels, while albumin values tend to be low (Fig. 14). This results in an A:G ratio that is often lower than 0.5-0.6 and is considered one of the most consistent indicators of FIP. However, a low A:G ratio can occur in situations where both albumin and globulin are within the reference range or in other diseases. Therefore, the A:G ratio should not be the only FIP indicator and should always be evaluated in the context of other FIP indicators (Tasker, 2018; Felten and Hartmann, 2019). Serum protein values obtained from most serum chemistry panels are usually adequate. Serum protein electrophoresis can provide additional information, especially if protein values from serum chemistry are questionable (Stranieri et al., 2017).

Figure 14. Complete blood count (CBC) (a) of a young cat with acute wet abdominal FIP. Although the leukocyte count was not elevated, relative but not absolute neutrophilia, relative and absolute lymphopenia, relative and absolute eosinopenia, and unresponsive anemia were noted, indicated by low red blood cells, hematocrit, and hemoglobin with a normal reticulocyte count.
Figure 14. Serum biochemical examination (b) of a young cat with acute wet abdominal FIP. Relevant values in the serum chemistry panel were elevated total protein, low albumin, high globulin, low albumin/globulin (A:G) ratio, and elevated total and direct bilirubin. Liver enzymes were normal except for mildly elevated AST and BUN and creatinine were normal, indicating the absence of significant liver or kidney disease. Globulin values are not always given, but a reasonable estimate can be calculated by subtracting the albumin level from the total protein.

Overreliance on CBC and serum biochemistry abnormalities can lead to diagnostic uncertainty when absent, despite the fact that no test value is consistently abnormal in all cases of FIP (Tasker, 2018)1. The biggest differences are between the clinical form of the disease, with leukocytosis and lymphopenia being more common in cats with wet than with dry FIP (Riemer et al., 2016). Hyperbilirubinemia is common in cats with FIP, but especially in cats with wet FIP (Tasker, 2018). The author also found that many cats with primary neurological FIP show minor or no blood abnormalities. Blood test values for FIP also vary from study to study (Tasker, 2018).

A complete analysis of the effusion is important to diagnose wet FIP and to rule out other potential causes of fluid accumulation (Dempsey and Ewing, 2011). It includes color (clear or yellow), viscosity (thin or viscous), presence of precipitates, ability to form a partial clot on standing, protein content, leukocyte count, and differential. The nature of the fluid may vary depending on the duration of the disease and its severity. Effusions in cats with more severe disease usually have protein values close to serum values, are more viscous, contain more leukocytes, are more yellow in color, and have a greater ability to form partial clots on standing. Chronic effusions tend to be less inflammatory in nature, with lower protein and leukocyte counts, less viscous and clearer. These values can be determined on the spot in most clinics. The clotting factor is determined by comparing the fluid collected in the serum and in the anticoagulant tubes after standing. Color and viscosity can be approximated and protein levels can be estimated using a handheld refractometer to determine total solids. Cells are pelleted from the fluid and analyzed on a fast-stained slide using light microscopy, and the leukocyte count and differential are estimated. Cells include nonseptic neutrophils, small and medium-sized mononuclear cells, and large vacuolated macrophages (Fig. 15). It is important to note that effusions can occur in a variety of conditions, such as heart failure, cancer, hypoproteinemia, and bacterial infections. Effusions in these other diseases usually have different identifying features.

Figure 15. Stained smear of peritoneal cells centrifuged from the abdominal fluid of a cat with wet FIP and examined on a fast-stained slide by light microscopy. The predominant cells are large strongly vacuolated macrophages, smaller differentiated activated monocytes and neutrophils. The greatest concentration of viral particles is in the intracytoplasmic vacuoles of macrophages (arrows).
Figure 16. Positive result of the Rivalt test. A small sample of abdominal or thoracic fluid is carefully dropped into a small beaker filled with diluted acetic acid (8 ml of distilled water and 1 drop of concentrated acetic acid). Inflammatory proteins almost immediately precipitate and sink to the bottom (positive). Less inflammatory fluids will form diffuse precipitates (questionable) or diffuse freely in solution (negative).

A positive Rivalt test on abdominal or chest fluid is often used to diagnose FIP as a cause of effusion, and a negative test tends to rule it out (Fischer et al., 2010) (Fig. 16). However, the test may be positive in inflammatory effusions of another cause and negative in some cats with FIP. Therefore, Rivalt's test is most helpful in combination with other clinical findings of FIP and should not replace a thorough fluid analysis (Felten and Hartmann, 2019).

Serum total and direct bilirubin levels are often elevated, especially in cats with wet FIP (Fig. 14), and may be associated with jaundice and bilirubinuria. Hyperbilirubinemia in FIP is not caused by liver disease (Tasker, 2018), but rather by vasculitis, microhemorrhage, hemolysis, and destruction of damaged red blood cells by macrophages locally and in the liver. The released hemoglobin is finally metabolized to bilirubin, which is then conjugated in the hepatocytes and excreted in the urine. Glucuronidation is essential for bilirubin excretion, and genetic disorders affecting glucuronidation in humans prevent its excretion (Kalakonda et al., 2021). Cats as a species are deficient in the enzymes required for glucuronidation, making it difficult to excrete substances such as bilirubin (Court and Greenblatt 2000).

Although FIP can affect the kidneys and liver, it is not severe enough to cause significant loss of kidney or liver function. However, serum tests for blood urea nitrogen (BUN) and creatinine as indicators of kidney disease and alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyltransferase (GGT) as indicators of liver disease are often mildly elevated in cats with FIP, especially with a more acute and serious disease (Fig. 14). Therefore, slightly abnormal test values should not be interpreted excessively if other clinical signs of liver or kidney disease are not present, while their significant increase should point to the possibility of concurrent and possibly predisposing diseases of these organs.

Serum can also be tested for other markers of systemic inflammation, such as increased levels of alpha-1-acid glycoprotein (AGP) (Paltrinieri et al., 2007) and feline serum amyloid A (fSAA) (Yuki et al., 2020). They may also prove useful in monitoring response to antiviral therapy (Krentz et al., 2021).

Radiography can be helpful in identifying chest and abdominal effusions. Abdominal ultrasound can reveal a smaller amount of effusion, identify enlarged mesenteric and ileo-cecal-colic lymph nodes, thickening of the colonic wall and lesions in organs such as the kidneys, liver and spleen (Lewis and O'Brien 2010). It may also be useful in examining the chest for lesions and assisting with needle aspiration or biopsy.

Antibody titers against FCoV have decreased since the first report nearly 50 years ago (Pedersen 1976b). The reference antibody test uses indirect fluorescent antibody staining (IFA) IFA titers ≥ 1:3200 in FIP cats are higher than most FECV-exposed cats (1:25–1:400). Newer tests often use ELISA procedures for rapid in-house or laboratory testing, but are qualitative rather than quantitative. IFA antibody titers decrease during successful antiviral treatment in many cats, but remain high in others (Dickinson et al., 2020; Krentz et al., 2021). Sequential titers can show a gradual increase in titers during the development of FIP (Pedersen et al., 1977), but previous serum samples are rarely available for comparison. Like most tests, FCoV antibody levels should not be used as the sole criterion to diagnose or rule out FIP (Felten and Hartmann, 2019) or to assess treatment success (Krentz et al., 2021).

Reverse transcriptase polymerase chain reaction (RT-PCR) is the primary means of identifying FCoV RNA in inflammatory effusions, fluids, or affected tissues (Felten and Hartmann, 2019). Accessory gene 7b RNA is present at the highest levels in tissues, fluids or exudates infected with FECV or FIPV, making it the most sensitive target for detecting low levels of virus (Gut et al., 1999). RT-PCR for FIPV S gene mutations is often used in samples that are positive for 7b RNA to be specific for FIPV (Felten et al., 2017). Other studies suggest that RT-PCR assays for FIPV-specific S gene mutations have similar specificity for FIP, but at the cost of a significant loss of sensitivity (Barker et al., 2017). A decrease in sensitivity is associated with an increase in the number of false negative results. False-negative RT-PCR tests also occur in samples that do not contain sufficient numbers of infected macrophages or in cats with very low levels of virus. False-negative results are especially common when testing whole blood.

Immunohistochemistry (IHC) detects feline coronavirus nucleocapsid protein in formalin-fixed tissues with high sensitivity and specificity, but is not as popular as RT-PCR (Litster et al., 2013; Ziółkowska et al., 2019). Specimens for IHC must contain intact infected macrophages (Fig. 17), which requires careful separation of cells from effusions and mounting them on slides, or formalin-fixed, paraffin-embedded diseased tissues that show lesions compatible with FIP. The coronavirus antigen in macrophages within a typical FIP lesion or fluid is seen only in FIP, giving IHC a high level of specificity.

Figure 17. Histological section from the thickened colon of a cat with the intestinal form of FIP. The thickened wall contained foci of macrophages (square area), which stained positive (brown-red) for FIPV nucleocapsid protein by immunoperoxidase.

A thorough ophthalmological examination is necessary to diagnose the characteristic changes of FIP (Pfeiffer and Wilcock 1991; Andrew, 2000). A sample of aqueous humor from the anterior chamber of an inflamed eye may also be useful for cytology, PCR and IHC.

Neurological FIP is often diagnosed using contrast-enhanced magnetic resonance imaging (MRI) and is often associated with cerebrospinal fluid (CSF) analysis (Crawford et al., 2017; Tasker, 2018; Dickinson et al., 2020). However, these are expensive procedures that are not always available and carry a certain risk for the cat. MRI lesions include obstructive hydrocephalus, syringomyelia, and herniation of the foramen magnum with contrast enhancement of the meninges of the brain and spinal cord and ependyma of the third ventricle, mesencephalic aqueduct, and brainstem. CSF shows an increased number of proteins and cells (neutrophils, lymphocytes, monocytes/macrophages) and, if present, can be reliable material for PCR or IHC examination.

Neurologic and/or ocular forms of FIP are often confused with systemic feline toxoplasmosis, and many cats with FIP are empirically treated for toxoplasmosis before a diagnosis of FIP is made. Fortunately, the availability of effective treatment for FIP has curtailed this practice. Systemic toxoplasmosis is much less prevalent than FIP, and fewer than 1 % cats with FIP were serologically positive in one field study.1 Therefore, testing or treatment for toxoplasmosis should only be considered once FIP has been adequately diagnosed.

Antiviral treatment as a diagnostic tool

Figure 18. A cat with FIP at the start of treatment with GS-441524 (a) and after 1 week (b). The answer is quick, the fever will disappear within 24-48 hours and the general state of health will improve significantly within 1-2 weeks. This type of response is often used to confirm a diagnosis of FIP.

Situations commonly occur where clinical findings point to FIP but doubts remain. Then there is a choice of performing several diagnostic tests, which may not lead to a more definitive diagnosis. An alternative diagnostic approach is treatment with a suitable antiviral for 1-2 weeks in the correct dose for the suspected form of FIP.2 Treatment often produces clinical improvement in as little as 24-48 hours and this rapidly progresses over the next 2 weeks and the total duration of treatment (Fig. 18). No response to test treatment and/or deterioration in health would indicate the need for further investigation of the cause(s) of ill health.

FIP Treatment

Before 2017, there was no cure for FIP, and treatment was mainly aimed at alleviating the symptoms of the disease (Izes et al., 2020). Such supportive treatment was aimed at maintaining good nutrition, controlling inflammation (corticosteroids), changing immune responses (interferons, cyclophosphamide, chlorambucil) and inhibiting key cytokine responses (pentoxifylline and other TNF-alpha inhibitors). Nutritional supplements that were supposed to help specific organ functions were also commonly used, such as one (Polyprenyl Immunostimulant) that was supposed to improve immunity and prolong survival in cats with dry but not wet FIP (Legendre et al., 2017). The effect of good supportive care on survival could not be determined because most cats were euthanized after diagnosis or within days or weeks. The survival rate for even the mildest forms of dry FIP and the most permanent treatment in one study was only 13 % at 200 days and 6 % at 300 days (Legendre et al., 2017).

Many commercially available drugs and compounds inhibit FIPV infection or replication in vitro, some of which are drugs known to inhibit specific HIV or hepatitis C proteins, while others work by inhibiting normal cellular processes that the virus usurps for its own life cycle (Hsieh et al., 2010; Izes et al., 2020; Delaplace et al., 2021). These various drugs and agents include cyclosporine and related immunophilins, several nucleoside and protease inhibitors, vioporin inhibitors, pyridine N-oxide derivatives, chloroquine and related compounds, ivermectin, several plant lectins, ubiquitin inhibitors, itraconazole, and several antibiotics. However, the concentrations required to inhibit viral replication in vitro often approach toxic values for cells. It has also been difficult to transfer favorable in vitro conclusions to animals, and studies in sick cats have rarely followed. Ribavarin inhibits FIPV replication in vitro, but was not effective as a treatment for experimental FIP (Weiss et al., 1993). The efficacy of chloroquine was tested in laboratory cats infected with FIPV, but clinical outcomes in treated cats were only slightly better than untreated ones and hepatotoxicity was demonstrated (Takano et al., 2013). A 3-month-old kitten with chest wet FIP treated with itraconazole and prednisolone developed neurological FIP and was euthanized after 38 days of treatment (Kameshima et al., 2020). Mefloquine also inhibited FIPV replication at low concentrations in cultured feline cells without cytotoxic effects, and preliminary pharmacokinetic studies in cats appeared favorable (Yu et al., 2020), but evidence of its safety and efficacy in clinical trials in cats with FIP has yet to be established. published.

A breakthrough in the treatment of FIP occurred in 2016-2019 when antiviral drugs were reported that target specific FIPV proteins essential for replication. The first of these drugs was GC376, a major protease inhibitor (Mpro ) FIPV (Kim et al., 2016; Pedersen et al., 2018). Protease inhibitors prevent the formation of individual viral proteins by inhibiting their cleavage from polyprotein precursors. GC376 was able to cure all experimentally infected cats and 7 of 21 cats with naturally occurring wet and dry FIP, but was less effective for cats with ocular or neurological signs (Pedersen et al., 2018). The second of these drugs was GS-441514, the active part of the prodrug remdesivir (Gilead Sciences; Murphy et al., 2018; Pedersen et al., 2019). GS-441524 is an adenosine nucleoside analog that blocks FIPV replication by inserting a nonsense adenosine into the developing viral RNA. GS-441524 was also able to cure all experimentally infected cats (Murphy et al., 2018) and 25/31 cats with naturally occurring wet and dry FIP (Pedersen et al., 2019). It has also been shown to be effective at higher doses in several cats with ocular and neurological FIP (Pedersen et al., 2019) and is now the drug of first choice for cats with neurological FIP (Dickinson et al., 2020). GS-441524 has cured thousands of FIP cats from around the world over the past three years, with an overall cure rate of just over 90 % (Jones et al., 2021).1

Although the ability of GC376 and GS-441524 to treat cats has been known for several years, neither is currently legally available in most countries. The rights to GC376 have been purchased by Anivive, but it has not yet been launched.3 Potential conflicts with the development of remdesivir for the treatment of COVID-19 in humans led Gilead Sciences to withhold rights to GS-441524 for animal use, prompting the creation of an unapproved source for GS-441524 from China (Jones et al, 2021).1,2,4 Remdesivir is rapidly metabolized in the body to GS-441524 and has been approved for the treatment of FIP in some countries.2 GS-441524 can also be administered orally in higher doses and is currently commonly used in practice (Krentz et al., 2021).1

The efficacy of drugs such as GC376 and GS-441524 on FIP cats, the use of which preceded the COVID-19 pandemic, has been recognized by researchers investigating related SARS-CoV 2 inhibitors (Yan et al., 2020; Vuong et al., 2021). Remdesivir, an injectable drug called glaucoma (Gilead), has been used worldwide to reduce mortality from COVID-19 (Beigel et al., 2020). GC373, the active form of the prodrug GC376, has undergone simple modifications to increase efficacy and oral bioavailability (Vuong et al., 2021). The GC373-related drug, nirmatrelvir, has been successfully tested against early COVID-19 infections and has been approved for the treatment of early COVID-19 and marketed as paxlovid (Pfizer). Paxlovid consists of two medicines, nirmatrevir and the HIV protease inhibitor ritonavir. Ritonavir is not a significant inhibitor of SARS-CoV 2, but is reported to prolong the half-life of Mprowhen used in combination (Vuong et al., 2020). Nirmatrelvir and paxlovid have not been tested in cats with FIP at present, but based on experience with the closely related drug GC376, oral treatment of some forms of FIP may be important in the future.

Two other nucleoside analogs, EIDD-1931 and EIDD-2801 (Painter et al., 2021), have been investigated for the treatment of multiple RNA virus infections in humans and animals. EIDD-1931 is the experimental designation for beta-D-N4-hydroxycytidine, a compound widely studied since the 1970's. Beta-D-N4-hydroxycytidine is metabolized to a ribonucleoside analog, which is incorporated into RNA instead of cytidine and leads to fatal mutations in the viral RNA strand. The compound is an inhibitor of a wide variety of human and animal RNA viruses, including all known coronaviruses. EIDD-1931 was modified to increase oral absorption and was termed EIDD-2801 (molnupiravir) (Painter et al., 2021). Molnupiravir is deesterified in the body to its active ingredient, beta-D-N4-hydroxycytidine. Therefore, EIDD-1931 and molnupiravir are analogous to GS-441524 and remdesivir. Molnupiravir is marketed for the home treatment of primary COVID-19 under the names Lagevrio (Merck, USA) or Molnulup (Lupine, India).

Both EIDD-1931 and EIDD-2801 have been shown to be effective in inhibiting FIPV in tissue culture (Cook et al., 2021), and EIDD-2801 is currently used to treat some cases of FIP in the field.5,7 The effective concentration of 50 % (EC50) for EIDD-1931 against FIPV is 0.09 µM, EIDD-2801 0.4 µM and GS-441524 0.66 µM (Cook et al., 2021). The percentage cytotoxicity at 100 μM for these compounds is 2.8, 3.8 and 0.0. Thus, EIDD-1931 and -2801 are slightly more inhibitory to viruses, but more cytotoxic than GS-441524. Resistance to GS-441524 has been reported in some cases of FIP (Pedersen et al., 2019) and to remdesivir in patients with COVID-19 (Painter et al., 2021), but these isolates remain sensitive to molnupiravir (Sheahan et al., 2020). This may prove useful in combating resistance to GS-441524 in cats and humans and in developing multidrug therapy to prevent the development of resistance.

What will full approval of medicines like molnupiravir and paxlovid mean for cats? Full human approval should allow veterinarians in most countries to legally procure medicinal products authorized for human consumption for direct use in animals, provided that the guidelines for use in non-food producing animals are followed.6 This requires a reformulation of a medicine made for humans and purchased at a price for humans. Hopefully, antivirals similar or identical to those approved for humans will be licensed exclusively for animals and sold at a much lower price, but this is likely to take years.

Commercial and policy issues that limit the current use of antivirals such as GS-441524 in animal diseases such as FIP are for current cat owners and feline support groups who have already bypassed the current drug approval system and its emphasis on overriding human needs, irrelevant (Jones et al., 2021; Krentz et al., 2021). Advocates of FIP treatment are currently found around the world and often associate under the expanded FIP Warrior brand. Members of these groups often act as intermediaries between owners, veterinarians and antiviral suppliers and often provide advice to those who are unable to obtain veterinary treatment assistance. Some of these groups, such as FIP Warriors Czech Republic / Slovakia7, have placed their experience with FIP treatment on the Internet, where they provide much-needed information about current antiviral treatment.

Current situation of FIP treatment

The current drug of choice for the treatment of FIP is the adenosine nucleoside analog GS-441524, which was first published in the scientific literature under experimental conditions (Murphy et al., 2018) and later against naturally occurring disease (Pedersen et al., 2019). Although initial experimental and field studies of GS-441524 were conducted in collaboration between researchers at Gilead Sciences and the University of California, Davis, the relationship between Remdesivir and GS-441524 and the onset of the COVID-19 pandemic in 2019 led Gilead Sciences to eventually did not grant rights to use GS-441524 to animals on the grounds that it could interfere with the development of Remdesivir for human use.4 Objections to this decision have been raised directly by the company and in several internet forums.4 Subsequent pressure from cat owners, cat rescue groups and cat lovers, along with opportunistic Chinese drug manufacturers, quickly created an alternative unapproved source of GS-441524, its market and treatment network.4  This network has largely bypassed veterinarians, most of whom have decided to wait for the drug to be legalized (Jones et al., 2021). The result of this relationship was an almost seamless transition of FIP treatment with GS-441524 from the laboratory to a rapidly expanding worldwide network of groups, under the umbrella of FIP Warriors (Jones et al., 2021).4,7 

The sale and use of GS-441524 in practice for the treatment of FIP began almost immediately with the first publication of the results of field trials (Pedersen et al., 2019) (Fig. 19).

Figure 19.  Graph of the monthly development of treatment of cats from the Czech Republic and Slovakia since August 2019. This graph comes from the FIP Warrior CZ / SK website.1 These figures reflect the experiences of other FIP Warrior groups around the world. Since 2019, when the first field study GS-441524 was published (Pedersen et al. 2019), thousands of cats have been successfully treated for FIP worldwide. The winter peaks of the disease reflect a late spring and summer increase in the number of kittens born and a high incidence of FIP, which usually begins at 3 to 6 months of age (Fig. 6). This chart is from the FIP Warrior CZ / SK website.1
Figure 20. The main participants in the GS-441524 treatment. This chart is from the FIP Warriors CZ / SK website.1

The fact that GS-441524 is not legally approved for use in animals has prevented many veterinarians from recognizing or participating in this treatment. Only 25 % cats in the CZ / SK treated group received veterinary support during treatment (Fig. 20), although more veterinarians may have been involved in the diagnosis of the disease. Interestingly, this number was higher than the 8.7 % treated cats in the United States that received veterinary care (Jones et al., 2021). However, participants in CZ / SK studies and similar groups around the world are not without medical experience, as many of them are engaged in temporary care / rescue and have had considerable direct and indirect veterinary experience with cat diseases and their treatment and castration programs.

From the first laboratory studies and research of Chinese manufacturers, it was known that GS-441524 can be absorbed orally, although with less efficiency (Kim et al. 2016).9 The first sellers of GS-441524 further investigated this fact and found that effective blood levels could be achieved by increasing the amount administered orally compared to injection.8 Supplements have often been added to GS-441524 oral capsules or tablets, claiming that they increase absorption or have additive therapeutic benefits (Krentz et al., 2011). Most major retailers of GS-441524 now offer oral versions, and oral therapy is becoming increasingly popular either as a single treatment or in combination with GS-441524 (Figure 21). The success of GS-441524 oral therapy did not differ significantly from GS-441524 injection therapy (Figure 22).

Figure 21. Comparison of the use of oral (tablets or capsules) and injectable (subcutaneous) forms of GS-441524 for the treatment of FIP in cats from the Czech Republic and Slovakia. This chart is from the FIP Warriors CZ / SK website.1
Figure 22. There is no significant difference in treatment success with oral administration of GS-441524 compared to subcutaneously administered GS, but the actual amount (mg) of drug administered orally in each dose is up to twice the amount contained in the same dose of GS injection. This chart is from the FIP Warriors CZ / SK.1 website

The recommended dosing schedule for GS-441524 based on published data from field studies (Pedersen et al., 2019) was 4 mg / kg, subcutaneous (SC), daily (q24h), ie 4 mg / kg, SC, q24h. This recommended starting dose for cats with wet or dry FIP without ocular or neurological symptoms tended to increase to 6 mg / kg SC q24h over time (Fig. 23). 8 mg / kg SC q24h is the current recommended dose for cats with ocular symptoms and 10 or 12 mg / kg SC q24h for cats with neurological symptoms.

Figure 23. Daily dose of GS-441524, which was used to treat FIP in cats from the Czech Republic and Slovakia. The usual starting dose was 6 mg / day, with some cats requiring higher doses based on response to treatment, form of the disease and recurrence after treatment appeared to be successful. GS-441524 oral formulations are usually labeled to match the dosage used for the injectable drug, but contain up to twice the labeled amount. This chart is from the FIP Warrior CZ / SK website.1

The optimal duration of treatment, as determined in the initial clinical study, is 84 days (Pedersen et al., 2019). In some cases of acute wet FIP in younger cats, healing has been achieved in 6-8 weeks, but some cats need more than 84 days. As shown in Figure 24.72 % cats were treated for 81-90 days, 19 % longer and only 9 % were treated shorter. Unfortunately, there is no simple and accurate test to determine the moment of cure, and the decision to stop treatment is based on a complete return to health and normal blood test values. Cats treated for much longer than 100 days were usually those requiring a GS dose higher than 12 mg / kg per day by injection or equivalent oral dose, cats that relapsed during the 12-week post-treatment observation period, cats with neurological disease or cats that have become resistant to GS-441524.   

Figure 24. Duration of treatment with GS-141524 in 352 cats successfully treated for all forms of FIP. This chart is from the FIP Warriors CZ / SK website.1
Figure 25. Initial treatment was successful in 88.1 % cats and 6.2 % cats died or were sacrificed either due to insufficient response to treatment, financial reasons or treatment side effects. Another 5.7 % cats relapsed after initial treatment and approximately the same number of cats either recovered or died after further treatment. This chart is from the FIP Warriors CZ / SK.1 website

The treatment success rate for all forms of FIP in cats from the Czech Republic and Slovakia is 88.1 % in the first treatment, but when cats that relapsed after the first treatment and recovered after the second treatment (3.1 %) were included, the overall success rate was more as 91 % (Fig. 25). This cure rate is identical to the cure rate of other groups of FIP fighters (Jones et al., 2021). Treatment success did not differ between cats with wet or dry FIP and without ocular or neurological impairment (Fig. 26). However, the cure rate in cats with ocular and neurological impairments was lower, at 80 % compared to 92 % in all other forms of FIP (Fig. 26).

Figure 26.  Cure rate of cats with wet or dry FIP without ocular or neurological symptoms and cats with ocular or neurological disease as the main feature of their disease. This chart is from the FIP Warriors CZ / SK website.1
Figure 27.  The condition of the cats one year after the successful completion of treatment with GS-441524. This chart is from the FIP Warriors CZ / SK website.1

Cats that have been successfully treated for FIP have been followed for 4 to 5 years, including cases reported in the first field studies. There have been no recurrences or recurrent cases of FIP in this group of first field trials. Data on annual survival are available from a much larger population of the CZ / SK study, which shows that 90.5 % cats are still healthy one year after the end of treatment (Fig. 27). Only 1.3 % of these cats died from causes other than FIP and 8.2 % cohort is currently in an unknown medical condition. The low proportion of cats that died of unknown causes within a year of treatment and their positive response to treatment suggest that FIP has been diagnosed correctly.

EIDD-2801 (molnupiravir) is currently being used in the field for the main treatment and for the treatment of GS-441524-resistant cats.5,7,9 EIDD-1931, the active form of EIDD-2081, needs to be further researched because it is no longer covered by patent protection and is thus easily approved for use in animals if it is found to be truly safe and effective.5 Nirmatrelvir, an oral form of GC373 and a closely related GC376, still needs to be studied for the treatment of FIP.

Acknowledgement

I am indebted to Ladislav Mihok and his collaborator from "FIP Warriors Czech Republic / Slovakia" for allowing me to share data from their website. This website contains the most important, comprehensive and organized collection of data on FIP antiviral treatment today. The website also contains useful information and advice on starting, conducting and monitoring current treatment. The collection of cats and their data is continuously and regularly updated and at the time of writing this article included more than 600 cats with FIP.

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Footnotes

  1. FIP Treatment - Czechia / Slovakia. Basic data, 2022. https://docs.google.com/spreadsheets/d/e/2PACX-1vRAnj_FV_fteWIW1HXsROLuJ7YY1-i_Sf81BCmM9JT9LbCT2mcnwD1rL9IBsLCTB1U59CcnalOGjFqq/pubhtml?gid=1340189982&single=true  (Accessed 4 April2022).
  2. Hughes D, Howard G, Malik R, 2021. Treatment of FIP in cats with Remdesivir. Clinical review, 2021. The Veterinarian. https://www.turramurravet.com.au/wp-content/uploads/2021/07/FIP-Article_The-Veterinarian.pdf (Accessed 5 March 2022).
  3. Anonymous. Thanks to Cats, One Promising Coronavirus Treatment is Already in Development-The GC376 story. 2021,  https://anivive.com/coronavirus (Accessed 4 April 2022)
  4. Zhang S (2020) A Much-Hyped COVID-19 Drug Is Almost Identical to a Black-Market Cat Cure. The Atlantic. https://www.theatlantic.com/science/archive/2020/05/remdesivir-cats/611341/ (Accessed 4 April 2022).
  5. Pedersen NC, 2021. The long history of Beta-d-N4-hydroxycytidine and its modern application to the treatment of Covid-19 in people and FIP in cats. https://ccah.vetmed.ucdavis.edu/sites/g/files/dgvnsk4586/files/inline-files/Molnuparivir%20as%20a%20third%20antiviral%20drug%20for%20treatment%20of%20FIP%20v13_1.pdf  (Accessed 4 April 2022).
  6. American Veterinary Medical Association. Guidelines for veterinary prescription drugs. 2022 https://www.avma.org/resources-tools/avma-policies/guidelines-veterinary-prescription-drugs (Accessed 4 April 2022).
  7. FIP Warriors CZ / SK. https://www.fipwarriors.eu/en/ (accessed 15 April 2022).
  8. Pedersen NC, Jacque N, 2021. Treatment with oral formulations of GS-441524. https://sockfip.org/2021-treatment-with-oral-formulations-of-gs-441524/  (Accessed 11 December 2021).
  9. Pedersen NC, Jacque N. 2021. Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524. https://ccah.vetmed.ucdavis.edu/sites/g/files/dgvnsk4586/files/inline-files/Approaches-to-drug-resistance-in-cats-treated-with-GS-441524-for-FIP-v3.pdf (Accessed 16 April 2022).
Read "History of feline infectious peritonitis 1963-2022 - from the first mention to successful treatment"

The history of Save Our Cats and Kittens over four decades and where we go from here

Niels C. Pedersen, DVM, PhD
December 2021
Original article: The history of Save Our Cats and Kittens over four decades and where we go from here

Niels C. Pedersen

Those who have followed my career know that I have many interests in addition to infectious diseases of cats. However, I am best known for feline medicine and diseases that plague multi-cat environments. This interest in infectious diseases started in 1965 as a second-year veterinary student but evolved after I joined the faculty of the UC Davis School of Veterinary Medicine in 1972. My first appointment was to help win President Nixon’s war on cancer. This war emphasized potential viral causes of cancer, in particular retroviruses and human leukemias. This was my entry back into the world of feline leukemia virus (FeLV). Of course, my interest was more on FeLV infection as it applied to cats than any application to human cancers. It became rapidly apparent that FeLV infection was a serious panzootic (pandemic) of cats that had unknowingly spread from feral to pet cats in the preceding decades and would account for one-third of mortality in cats in the 1960s and 70s. Cat lovers quickly mobilized once the virus was discovered and started raising money to support FeLV research. The original SOCK was created by a group of amazing cat lovers led by Vince, Connie and Dorothy Campanile and friends. SOCK it to leukemia became the rallying cry of the group and I was privileged to join forces with them from their beginning to end. Thereafter, donations from cat lovers and not federal research funds provided the bulk of our research into FeLV infection at UC Davis. This research led to an understanding of how FeLV became a pandemic of pet cats, how it caused a wide range of diseases, and how it could be controlled. FeLV infection of pet cats was brought under control in the 1970’s and 1980’s through rapid diagnostic tests and vaccination. The conquest of FeLV infection was one of the highlights of veterinary research of the period, and perhaps one of the most important contributions of modern feline medicine in the 20th century. SOCK it to leukemia had ultimately worked itself out of existence with over $1M dollars raised towards the ultimate conquest of FeLV infection. FeLV infection still exists in nature, where it remains a problem for a small number of younger cats coming into foster/rescues and shelters from the field.

During this same period, another highly fatal disease was rearing its head. Feline infectious peritonitis (FIP) was first reported in 1963 by veterinarians from the Angell Memorial Animal Hospital in Boston. It was later found to be closely linked to FeLV infection and the hope was that it would largely disappear with control of FeLV. This did not prove true and FIP soon replaced FeLV as a major infectious cause of deaths in cats up to this time. As a result, the torch was passed from SOCK it to leukemia to SOCK it to FIP. This was also a natural progression for my research. FIP was my first “love” from the time I helped research the first cases of FIP at UC Davis as a veterinary student in 1965. My interest in FIP only took second stage for a brief period in the 1980s with my work on HIV/AIDS and subsequent discovery of feline immunodeficiency virus (FIV). FIP has been my major research interest for the last three decades.

I am pleased to have had the support of SOCK FIP over these later years. One of our greatest discoveries at UC Davis was how an innocuous and ubiquitous feline enteric coronavirus (FECV) ends up causing such a highly fatal disease as FIP. Our theory that the virus of FIP arose as an internal mutation of FECV was first met with great skepticism but is now universally accepted. The internal mutation theory has led to a much better understanding of the conditions under which FIP occurs and how the FIP virus causes disease. Unfortunately, no one, including us, was able to find a successful vaccine for FIP. This failure led to my interest in curing rather than preventing FIP using modern antiviral drugs, which I became familiar with during the HIV/AIDS pandemic. The capstone of my almost 50-year experience with FIP was the discovery of two antiviral drugs that could cure FIP. Thousands of cats from round the world have been cured of FIP with antiviral drugs researched at UC Davis over the last 3 years. Our discoveries at UC Davis could have been impossible without the significant long-term financial and moral support of SOCK FIP and cat owners who have donated money.

The discovery of a cure for FIP has once again brought SOCK FIP to a logical ending, just as the conquest of FeLV infection ended the need for the original SOCK. Although I am retired, I continue to work with cat owners and caregivers on how to use antiviral drugs to treat FIP and will maintain my relationship with SOCK FIP as a consultant on FIP treatment and a lifelong member. Admittedly, there is still research to be done with FIP, mainly in the areas of disease prevention. Hopefully, others will take up this and other areas of FIP research. The question now is how SOCK can best improve the health of our cats and kittens. SOCK FIP is in the process of evaluating a broader mission than just FIP. This mission may or may not involve fund raising for research and could be more informational. We welcome suggestions on how the long history of SOCK’s can be used to improve the health of our cats and kittens. Read "Four Decades Save Our Cats and Kittens and What's Next"

Origin of abdominal or thoracic effusions in cats with wet FIP and causes of their persistence during treatment

Niels C. Pedersen, DVM, PhD
Pet Health Center
University of California, Davis
24.9.2021

Original article: Origin of abdominal or thoracic effusions in cats with wet FIP and reasons for their persistence during treatment


Origin of FIP exudates. Sweat in wet FIP comes from small vessels (venules) that line the surface of the abdominal and thoracic organs (visceral) and walls (parietal), mesentery / mediastinum, and omentum. The spaces around these vessels contain a specific type of macrophages that come from monocyte progenitors that constantly recirculate between the bloodstream, the interstitial spaces around the venules, the afferent lymph, the regional lymph nodes, and back into the bloodstream. Other sites of this recirculation are located in the meninges, brain ependyma, and uveal eye tract. A small proportion of these monocytes develop into immature macrophages (monocyte / macrophage) and eventually into resident macrophages. Macrophages are constantly looking for infections.

FIPV is caused by a mutation in feline enteric coronavirus (FECV) present in lymphoid tissues and lymph nodes in the lower intestine. The mutation changes FECV cell tropism from enterocytes to peritoneal-type macrophages. Monocytes / macrophages appear to be the first cell type to be infected. This infection causes more monocytes to leave the bloodstream and begin to turn into macrophages, which continue the cycle of infection. [2]. Monocytes / macrophages do not undergo programmed cell death as usually expected, but continue to mature into large virus-loaded macrophages. These large macrophages eventually undergo programmed cell death (apoptosis) and release large amounts of virus, which then infects new monocytes / macrophages. [1]. Infected monocytes / macrophages and macrophages produce several substances (cytokines) that mediate the intensity of inflammation (disease) and immunity (resistance). [1,2].

Inflammation associated with FIP leads to three types of changes in the venules. The first is loss of vascular wall integrity, micro-bleeding, and leakage of plasma protein rich in activated complement clotting and activation factors and other inflammatory proteins. The second type of damage involves thrombosis and blocking blood flow. The third injury occurs in more chronic cases and involves fibrosis (scarring) around the blood vessels. Variations in these three events determine the amount and composition of exudates according to the four Starling forces that determine the movement of fluids between the bloodstream and interstitial spaces. [3].

The classic effusion in wet FIP is mainly due to acute damage to the vessel walls and leakage of plasma into the interstitial spaces and finally into the body cavities. Protein that escapes into the interstitial spaces attracts additional fluids, which can be exacerbated by blocking venous blood flow and increasing capillary pressure. This type of effusion, known as exudate, also contains high levels of protein, which is involved in inflammation, immune responses and blood clotting.

This fluid also contains a large number of neutrophils, macrophages / monocytes, macrophages, eosinophils and a lower number of lymphocytes and red blood cells. This classic type of fluid has the consistency of egg white and forms weak clots containing a high amount of bilirubin. Bilirubin does not originate from liver disease, but rather from the destruction of red blood cells that escape into interstitial tissue cells and are taken up by monocytes / macrophages and macrophages. Red blood cells break down and hemoglobin is broken down into heme and globin. Globin is further metabolized to biliverdin (greenish color) and finally to bilirubin (yellowish color), which is then excreted by the liver. However, cats lack the enzymes used for conjugation and are therefore ineffective in removing bilirubin from the body. [4]. This leads to the accumulation of bilirubin in the bloodstream and gives the effusion a yellow tinge. The darker the yellow tint, the more bilirubin is in the effusion, the more severe the initiating inflammatory response and the more severe the resulting bilirubinemia, bilirubinuria and jaundice.

The opposite extreme of the classic and more acute effusion in FIP are effusions arising mainly from chronic infections and blockage of venous blood flow and consequent increase in capillary pressure. High capillary pressure results in effusion that is more distant to interstitial fluid than plasma, has a lower protein content, is watery rather than sticky, clear or slightly yellow in color, is not prone to clotting, and has a lower number of acute inflammatory cells such as neutrophils. There are also FIP effusions that are among these extremes, depending on the relative degree of acute inflammation and chronic fibrosis. These transient types of fluids are commonly referred to in the veterinary literature as modified transudate, but this is a misnomer. The modified transudate begins as a transudate and changes as it persists and causes mild inflammation. Low protein and cell effusions in FIP arise as exudates and not as transudates and do not conform to this description. The more correct term is "modified exudate" or "variant exudate outflow".

How long do sweats usually last in cats treated with GS-441524 or GC376? The presence of abdominal effusions often leads to a large dilation of the abdomen and is confirmed by palpation, hollow needle aspiration, X-ray or ultrasound. Cats with thoracic effusions are most often presented with severe shortness of breath and are confirmed by radiological examination and aspiration. Chest effusions are almost always removed to relieve shortness of breath and recur slowly compared to abdominal effusions. Therefore, abdominal effusions are usually not removed unless they are massive and do not interfere with respiration, as they are quickly replaced. Repeated drainage of abdominal effusions can also deplete proteins and cause harmful changes in fluid and electrolyte balance in severely ill cats.

Chest effusions disappear faster with GS-441524 treatment, with improved breathing within 24-72 hours and usually disappearing in less than 7 days. Abdominal effusions usually decrease significantly within 7-14 days and disappear within 21-28 days. The detection of exudates that persist after this time depends on their amount and method of detection. Small amounts of persistent fluid can only be detected by ultrasound.

Persistence of exudates during or after antiviral treatment. There are three basic reasons for the persistence of exudates. The first is the persistence of the infection and the resulting inflammation at a certain level, which can be caused by inappropriate treatment, poor medication or drug resistance. Inadequate treatment may be the result of incorrect dosing of the wrong drug or the acquisition of virus resistance to the drug. The second reason for fluid persistence is chronic venous damage and increased capillary pressure. This may be due to a low-grade infection or residual fibrosis from an infection that has been removed. The third reason for persistence is the existence of other diseases, which can also manifest as exudates. These include congenital heart disease, in particular cardiomyopathy, chronic liver disease (acquired or congenital), hypoproteinemia (acquired or congenital) and cancer. Congenital diseases causing effusions are more common in young cats, while acquired causes and cancer are more commonly diagnosed in older cats.

Diagnosis and treatment of persistent effusions. A thorough examination of the fluid, as described above, is a prerequisite for diagnosis and treatment. If the fluid is inflammatory or semi-inflammatory and the cell pellet is positive by PCR or IHC, the reason for the persistence of the infection must be determined. Was the antiviral treatment performed correctly, was the antiviral drug active and its concentration correct, was there evidence of acquired drug resistance? If the fluid is inflammatory and PCR and IHC are negative, what other diseases are possible? Low protein and non-inflammatory fluids that are negative for PCR and IHC indicate a diagnosis of residual small vessel fibrosis and / or other contributing causes such as heart disease, chronic liver disease, hypoproteinemia (bowel disease or kidneys). Some of the disorders causing this type of effusion may require an exploratory laparotomy with a thorough examination of the abdominal organs and a selective biopsy to determine the origin of the fluid. The treatment of persistent effusions will vary greatly depending on the end cause. Persistent effusions caused by residual small vessel fibrosis in cats cured of the infection often resolve after many weeks or months. Persistent discharges caused in whole or in part by other diseases require treatment for these diseases.

Identification and characteristics of persistent effusions. The presence of fluid after 4 weeks of GS treatment is unpleasant and is usually detected in several ways depending on the amount of fluid and its location. Large amounts of fluid are usually determined by the degree of abdominal dilation, palpation, X-ray and abdominal aspiration, while smaller amounts of fluid are best detected by ultrasound. Persistent pleural effusion is usually detected by X-rays or ultrasound. Overall, ultrasound is the most accurate means of detecting and semiquantitatively determining thoracic and abdominal effusions. Ultrasound can also be used in combination with thin needle aspiration to collect small and localized amounts of fluid.

The second step in examining persistent effusions is to analyze them based on color, protein content, white and red blood cell counts, and the types of white blood cells present. Fluids generated primarily by inflammation will have protein levels close to or equal to plasma and a large number of white blood cells (neutrophils, lymphocytes, monocytes / macrophages and large vacuolated macrophages). Fluids produced by increased capillary pressure are more similar to interstitial fluid with proteins closer to 2.0 g / dl and cell counts <200. The Rivalt test is often used to diagnose FIP-related effusions. However, this is not a specific test for FIP, but rather for inflammatory effusions. It is usually positive for FIP effusions that are high in protein and cells, but is often negative for very low protein and cell effusions. The effluents that are between these two types of effusions will be tested either positively or negatively, depending on where they are in the spectrum.

The third step is the analysis of exudates for the presence of FIP virus. This usually requires 5 to 25 ml or more of fluid. For fluids with a higher protein and cell count, a smaller amount may suffice, while for fluids with a low protein and cell count, a larger amount is required. The freshly collected sample should be centrifuged and the cell pellet analyzed for the presence of viral RNA by PCR or cytocentrifuged for immunohistochemistry (IHC). The PCR test should be for FIPV 7b RNA and not for specific FIPV mutations, as the mutation test does not have sufficient sensitivity and does not provide any diagnostic benefits [5]. Samples that are positive by PCR or IHC provide definitive evidence of FIP. However, up to 30 % samples from known cases of FIP may have a false negative test either due to an inappropriate sample and its preparation, or because the RNA level of the FIP virus is below the level of detection. It is also true that the less inflammatory the fluid, the lower the virus levels. Therefore, effusions with lower protein and white blood cell levels are more likely to be tested negative because viral RNA is below the detection limit of the test.

References

[1] Watanabe R, Eckstrand C, Liu H, Pedersen NC. Characterization of peritoneal cells from cats with experimentally-induced feline infectious peritonitis (FIP) using RNA-seq. Vet Res. 2018 49 (1): 81. doi: 10.1186 / s13567-018-0578-y.

[2]. Kipar A, Meli ML, Failing K, Euler T, Gomes-Keller MA, Schwartz D, Lutz H, Reinacher M. Natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection. Vet Immunol Immunopathol. 2006 Aug 15; 112 (3-4): 141-55. doi: 10.1016 / j.vetimm.2006.02.004. Epub

[3] Brandis K. Starling's Hypothesis, LibreTexts. https://med.libretexts.org/Bookshelves/Anatomy_and_Physiology/Book%3A_Fluid_Physiology _(Brandis)/04%3A_Capillary_Fluid_Dynamics/4.02%3A_Starling%27s_Hypothesis

[4]. Court MH. Feline drug metabolism and disposition: pharmacokinetic evidence for species differences and molecular mechanisms. Vet Clin North Am Small Anim Pract. 2013; 43 (5): 10391054. doi: 10.1016 / j.cvsm.2013.05.002

[5]. Barker, EN, Stranieri, A, Helps, CR. Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis. Vet Res 2017; 48: 60. Read "Origin of abdominal or thoracic effusions in cats with wet FIP and causes of their persistence during treatment"

Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524

Niels C. Pedersen, Nicole Jacque, 3.11. 2021
Original article: Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524

Abbreviations:
SC - subcutaneous
IV - intravenous
IM - to the muscle
PO - per os - orally
SID - once a day
BID - 2x this
q24h - once every 24 hours
q12h - once in 12 hours

Introduction

Antiviral resistance is well documented in diseases such as HIV / AIDS and hepatitis C. In some cases, this resistance is present in the infecting virus, but is more often due to long-term drug exposure. Resistance to GC376 [1] and GS-441524 [2] has also been documented in cats with naturally acquired FIP. Resistance develops based on mutations in regions of the viral genome that contain targets for the antiviral drug. For example, several amino acid changes (N25S, A252S or K260N) were detected in the GIP376-resistant FIPV isolate (3CLpro). [3]. A change in N25S in 3CLpro was found to cause a 1.68-fold increase in 50 % GC376 inhibitory concentration in tissue cultures [3]. Resistance to GC376, although recognized in initial field trials, has not yet been described. GC376 is less popular in the treatment of FIP and is not recommended for cats with ocular or neurological FIP. [1].

Natural resistance to GS-441524 was observed in one of 31 cats treated for naturally occurring FIP [2]. One of the 31 cats in the original GS-441524 field study also appeared to be resistant, as viral RNA levels did not decrease throughout the treatment period and the symptoms of the disease did not abate. Although this virus has not been studied, resistance to GS-5734 (Remdesivir), a prodrug of GS-441524, has been established in tissue culture by amino acid mutations in RNA polymerase and corrective exonuclease. [4].

Resistance to GS-441524 has been confirmed in a number of cats that have been treated for FIP with GS-441524 in the last 3 years, especially among cats with neurological FIP [5]. Resistance to GS441524 is usually partial and higher doses often cure the infection or significantly reduce the symptoms of the disease during treatment. Interestingly, resistance to GS-441524 has also been found in patients with Covid19 treated with Remdesivir [12]. An immunocompromised patient developed a prolonged course of SARS-CoV-2 infection. Remdesivirus treatment initially alleviated symptoms and significantly reduced virus levels, but the disease returned with a large increase in virus replication. Whole genome sequencing identified an E802D mutation in nsp12 RNA-dependent RNA polymerase that was not present in pre-treatment samples and caused a 6-fold increase in resistance.

Although the history of molnupiravir and its recent use in the treatment of FIP has been described [6], there are currently no studies documenting natural or acquired resistance to molnupiravir. Molnupiravir has been shown to function as an RNA mutagen causing several defects in the viral genome [7]while remdesivir / GS-441524 is a non-binding RNA chain terminator [8], which suggests that its resistance profile will be different.

Overcoming resistance to GS-441524

Drug resistance can only be overcome in two ways: 1) by gradually increasing the dose of the antiviral to achieve drug levels in body fluids that exceed the resistance level, or 2) by using another antiviral that has a different mechanism of resistance, either alone or in combination. So far, the first option has been chosen, which has proved effective in many cases. However, resistance to GS-441524 may be complete or so high that increasing the dose is no longer effective. In such cases, the second option is increasingly used. Currently available alternatives to GS-441524, although still from an unapproved market, are GC376 and molnupiravir.

Antiviral drug treatment regimens for resistance to GS-441524

GC376 / GS-441524


The combined GS / GC regimen has been shown to be effective in cats treated with GS-441524 at doses up to 40 mg / kg without cure due to resistance to GS-441524. It is better to intervene as soon as resistance to GS-441524 is detected, which will allow the cat to be cured sooner and at lesser cost to the owner.

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

GS / GC dosage: The dose of GS (SC or PO equivalent) in combination antiviral therapy is the same as the dose needed to adequately control the symptoms of the disease. This is usually the last dose used before the end of treatment and relapse. To this dose of GS-441524, GC376 is added at a dose of 20 mg / kg SC q24h regardless of the form of FIP. This is sufficient for most cats, including many cats with neuro FIP, but some will need higher doses. If remission of clinical signs is not achieved or blood tests are of concern, the dose of GC376 is increased by 10 mg / kg up to 50 mg / kg SC q24h.

Duration of treatment: An eight-week combination GC / GS treatment is recommended, which is added to previous GS monotherapy. Some cats were cured at 6 weeks of combination therapy, but relapse is more likely than at 8 weeks.

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

Molnupiravir

Molnupiravir has been reported to be effective in monotherapy in cats with FIP by at least one Chinese retailer GS-441524 [9], but there are no reports of its use in cats with resistance to GS-441524. However, resistance to GS-441524 is unlikely to spread to molnupiravir. The fact that it has been found to be effective as an oral medicine also makes it attractive for treatment alone, as many cats resistant to GS-441524 have suffered from injections for a very long time.

A field study of molnupiravir reportedly consisted of 286 cats with various forms of naturally occurring FIP, which were examined in pet clinics in the United States, the United Kingdom, Italy, Germany, France, Japan, Romania, Turkey and China. Among the 286 cats that participated in the trial, no deaths occurred, including seven cats with ocular (n = 2) and neurological (n = 5) FIP. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats remained healthy 3-5 months later, a period during which cats that were not successfully cured would be expected to relapse. These data provide convincing evidence of the safety and efficacy of molnupiravir in cats with various forms of FIP. However, we hope that this field study will be written in the form of a manuscript, submitted for review and published. Nevertheless, it is now sold to cat owners with FIP. At least one other major retailer of GS-441524 is also interested in using molnupiravir for FIP, indicating a demand for further treatment of cats with FIP antivirals.

Molnupiravir dosage: The safe and effective dosing of molnupiravir in cats with FIP has not been established based on closely controlled and monitored field studies such as those performed for GC376 [1] and GS-441524. [2]. However, at least one seller from China in his flyer for a product called Hero-2801 [9] provided some pharmacokinetic and field trials of Molnuparivir in cats with naturally occurring FIP. This information does not clearly state the amount of molnupiravir in one of their “50 mg tablets” and the actual dosing interval (q12h or q24h?). The dose used in this study also appeared to be too high. Fortunately, the estimated starting dose of molnupiravir in cats with FIP can be obtained from published studies on EIDD-1931 and EIDD-2801. [15] in vitro on cell cultures and laboratory and field studies GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 μM / μl against FIPV in cell culture, while the EC50 of GS-441524 is approximately 1.0 μM / μl. [18]. Both have a similar oral absorption of approximately 40-50 %, so an effective subcutaneous (SC) dose of molnupiravir would be approximately half the recommended starting dose of 4 mg / kg SC q24h for GS441524. [14] or 2 mg / kg SC q24h. The oral (PO) dose would be doubled to account for less effective oral absorption per 4 mg / kg PO q24h dose. The estimated initial effective oral dose of molnupiravir in cats with FIP can also be calculated from the available Covid-19 treatment data. Patients treated with Covid-19 are given 200 mg of molnupiravir PO q12h for 5 days. This dose was, of course, calculated from a pharmacokinetic study performed in humans, and if the average person weighs 60-80 kg (70 kg), the effective inhibitory dose is 3,03.0 mg / kg PO q12h. The cat has a basal metabolic rate 1.5-fold higher than humans, and assuming the same oral absorption in both humans and cats, the minimum dose for cats according to this calculation would be 4.5 mg / kg PO q12h in neocular and non-neurological forms of FIP. If molnupiravir crosses the blood-brain and blood-brain barriers with the same efficacy as GS-441524 [3,18], the dose should be increased to 1,51.5 and 2,02.0-fold to ensure adequate penetration into aqueous humor and cerebrospinal fluid for ocular cats (88 mg / kg PO, q12 h), respectively. neurological FIP (~ 10 mg / kg PO, q12 h). These doses are comparable to those used in ferrets, where 7 mg / kg q12h maintains sterilizing blood levels of the influenza virus drug (1.86 μM) for 24 hours. [10]. Doses in ferrets of 128 mg / kg PO q12h caused almost toxic blood levels, while a dose of 20 mg / kg PO q12h caused only slightly higher blood levels. [10].

Molnupiravir / GC376 or Molnupiravir / GS-441524

Combinations of molnupiravir with GC376 or GS-441524 will be used more and more frequently, not only to synergy or complement their individual antiviral effects, but also as a way to prevent drug resistance. Medicinal cocktails have been very effective in preventing drug resistance in HIV / AIDS patients [11]. However, there is currently insufficient evidence on the safety and efficacy of the combination of molnupiravir with GC376 or GS-441524 as initial treatment for FIP.

Case studies


Rocky - DSH MN Neuro FIP


A 9-month-old neutered domestic shorthair cat obtained as a rescue kitten had several weeks of seizures with increasing frequency, ataxia and progressive paresis. The blood tests were unremarkable. FIP treatment was started at a dose of 15 mg / kg BID GS-441524, which decreased to SID for about a week. The cat showed improvement, seizures stopped, and mobility increased within 24 hours of starting treatment. Within 5 days of treatment, the cat was able to move again. However, approximately 2 weeks after the start of treatment, the cat experienced loss of vision, decreased mobility, recovery of seizures and difficulty swallowing. Dose adjustments of levetiracetam and prednisolone were made, as well as a change in the composition of GS-441524, followed by a temporary improvement in motility and swallowing and a reduction in seizures, but overall the cat's condition worsened. The dose of GS-441524 was gradually increased to 25 mg / kg, with little or no improvement. At this point, GS was taken orally at a dose of 25 mg / kg (estimated to be approximately 12.5 mg / kg) and within 3 days, the cat began to move, improved vision, and stopped seizures with increased energy and appetite. Improvement in cats continued for approximately 4 weeks with oral administration of GS-441524, then stopped for approximately 3 weeks before rapidly progressing paresis. Oral doses up to 30 mg / kg SC equivalent have been tested but have no effect. GS-441524 was then injected at a dose of 20 mg / kg and the cat was able to move again within 4 days with good appetite and energy. After 2 weeks, a dose of GC376 20 mg / kg BID was added to the dosing regimen. The cat terminated 6 weeks of the GS441524 and GC376 combination therapy and then discontinued the treatment. Although the cat has certain permanent neurological deficits, its condition is stable, it has good mobility, appetite and activity for 9 months after the end of antiviral treatment.

Rocky's video: https://www.youtube.com/watch?v=RXB_NnfcMOY

Bucky - DSH MN Neuro / Eyepiece FIP


A four-month-old neutered domestic shorthair cat obtained as a rescue kitten was presented with a monthly history of lethargy and a progressive history of ataxia, hind limb paresis, spades, uveitis, anisocoria, and urinary and stool incontinence. Blood tests were mostly uncommon, with the exception of mild hyperglobulinemia. The A / G ratio was 0.6. The cat was treated with 10 mg / kg GS-441524 SC SID for 3 weeks. Activity, mentation and uveitis improved within 72 hours of starting treatment. During the first 2 weeks, a slow improvement in mobility and eye symptoms was observed, but then a plateau was reached. After 3 weeks, the dose of GS-441524 was increased to 15 mg / kg GS-441524 SC SID due to persistent neurological and ocular deficits. In addition, enlargement of the left eye due to glaucoma was noted at this time and the eye continued to swell until it was removed at week 8 of treatment.
Due to persistent weakness / lack of pelvic coordination and increasing lethargy, dose GS-441524 was increased to 20 mg / kg SC SID [or equivalent oral dose] at week 9 and 20 mg / kg SC BID was added to the regimen a few days later. GC376. Significantly increased activity and willingness to jump on elevated surfaces occurred within 48 hours of starting GS376 treatment. The combination treatment of GS-441524 and GC376 was maintained for 8 weeks. The cat has residual incontinence problems after treatment, but is otherwise clinically normal 6 months after treatment.

Boris - Maine Coon MI wet eye FIP


The five-month-old intact (uncastrated) Maine Coon cat, obtained from the breeder, had lethargy, anorexia, abdominal ascites, cough, anemia and neutrophilia. No biochemical analysis was performed to establish the diagnosis. The cat was treated with 6 mg / kg GS-441524 SC SID for 8 weeks. After six weeks of treatment, X-rays revealed nodules in the lungs, and after 8 weeks, hyperglobulinemia persisted. The GS-441524 dose was then increased to 8 mg / kg SC SID for 4 weeks. There was little improvement in blood tests and X-rays and the dose of GS-441524 was increased to 12 mg / kg SC SID over 4 weeks, followed by an increase to 17 mg / kg over 11 weeks, 25 mg / kg over 4 weeks and 30 mg / kg for 4 weeks. After 25 weeks of treatment, ultrasound revealed pleural abnormalities on the left side and X-rays showed no improvement in the pulmonary nodules. In addition, uveitis and retinal detachment have been reported in the right eye. Pulmonary aspirates that showed FIP-compliant inflammation were collected. After 33 weeks of treatment, 20 mg / kg SC BID GC376 was added to the regimen and the combined treatment of GS-441524 and GC376 was continued for 12 weeks. Increased activity was noted over several days. Over the course of 5 weeks, the weight gain accelerated, the cough subsided and the energy level increased. Blood tests showed an improvement in the A / G ratio, and chest X-rays showed a reduction in the lungs. After 84 days of combination antiviral therapy, the A / G ratio was 0.85 and the cat appeared clinically normal. The cat is currently 3 months after treatment.

References

  1. Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M, Meadows JM, Chang KO. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2018; 20 (4): 378-392.
  2. Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. efficacy and safety of the nucleoside analog GS-441524 for the treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019; 21 (4): 271-281.
  3. Perera KD, Rathnayake AD, Liu H, et al. Characterization of amino acid substitutions in feline coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease inhibitor. J. Vet Microbiol. 2019; 237: 108398. doi: 10.1016 / j.vetmic.2019.108398
  4. Agostini ML, Andres EL, Sims AC, et al. Coronavirus susceptibility to the antiviral remdesivir (GS5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI: 10.1128 / mBio.00221-18.
  5. Pedersen NC. 2021. The neurological form of FIP and GS-441524 treatment.
    https://sockfip.org/the-neurological-form-of-fip-and-gs-441524-treatment/
  6. Pedersen NC. The long history of beta-d-n4-hyroxycytidine and its modern application to treatment of covid019 in people and FIP in cats. https://sockfip.org/the-long-history-of-beta-d-n4-hydroxycytidineand-its-modern-application-to-treatment-of-covid-19-in-people-and-fip-in- cats /.
  7. Agostini, ML et al. Small-molecule antiviral beta-dN (4) -hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance. J. Virol. 2019; 93, e01348.
  8. Warren, TK et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531, 381–385.
  9. FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
  10. Toots M, Yoon JJ, Cox RM, Hart M, Sticher ZM, Makhsous N, Plesker R, Barrena AH, Reddy PG, Mitchell DG, Shean RC, Bluemling GR, Kolykhalov AA, Greninger AL, Natchus MG, Painter GR, Plemper RK . Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Sci Transl Med. 2019; 11 (515): eaax5866.
  11. Zdanowicz MM. The pharmacology of HIV drug resistance. Am J Pharm Educ. 2006; 70 (5): 100.doi: 10.5688 / aj7005100
  12. Gandhi, S, Klein J, Robertson A, et al. De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: A case report. medRxiv, 2021.11.08.21266069AID
Read "Alternative treatment of cats with FIP and natural or acquired resistance to GS-441524"

The long history of Beta-d-N4-hydroxycytidine and its modern application to treatment of Covid-19 in people and FIP in cats.

Niels C. Pedersen DVM, PhD
Original article: The long history of Beta-d-N4-hydroxycytidine and its modern application to treatment of Covid-19 in people and FIP in cats.

Beta-d-N4-hydroxycytidine is a small molecule (nucleoside) that was studied in the late 1970s in the former Soviet Union as part of biological weapons research [2]. The weaponization of diseases such as smallpox was a worldwide threat, but the danger of using the smallpox virus for this purpose was too great. Smallpox was eradicated from the world, virtually all stocks were destroyed and further research was banned. This led the US and the Soviet Union to research other RNA viruses as biological weapons and antivirals to defend against them. The Venezuelan equine encephalomyelitis virus (VEEV) was one of the first viruses to be seriously considered for use as a biological weapon. [3]. VEEV is transmitted to humans by mosquito bites and causes high fever, headaches and encephalitis with swelling that can be fatal. Beta-d-N4-hydroxycytidine has been found to inhibit not only VEEV replication but also a wide range of alphaviruses, including Ebola, chikungunya, influenza virus, norovirus, bovine diarrhea virus, hepatitis C virus and respiratory syncytial virus. [3-8]. The first reports of an inhibitory effect of beta-d-N4-hydroxycytidine on human coronavirus NL63 date back to 2006 [9]. Recent studies have confirmed its inhibitory effect on a wide range of human and animal coronaviruses [8].

An important part of the recent history of beta-d-N4-hydroxycytidine is associated with the Emory Institute for Drug Development (EIDD) [1], where he received the experimental designation EIDD-1931. The US government has provided significant financial support for the study of antivirals against alphaviruses in institutions such as Emory since 2004. [10]. In 2014, the Defense Threat Reduction Agency provided institutional support to find an antiviral compound against VEEV and other alpha-coronaviruses. "N4-Hydroxycytidine and its derivatives and antiviral uses" were included in U.S. Patent Application 2016/106050 A1 of 2016 [11]. Additional funding in 2019 was provided by the National Institute of Allergies and Infections for fellowship of the esterified beta-d-N4-hydroxycytidine precursor (EIDD-2801) for the treatment of influenza. [10]. The stated purpose of the chemical changes of EIDD-2801 was to increase its oral bioavailability, which would ultimately allow beta-d-N4-hydroxycytidine to be administered as pills and not as injections. In 2019/2020, the focus of research changed from influenza to SARS-CoV-2 [2]. The commercialization of EIDD-2801 was entrusted to Emory's Ridgeway Biotherapeutics subsidiary, which subsequently worked with Merck on a lengthy and costly FDA approval process. The current version of EIDD-2081 for field testing was named Molnupiravir.

Beta-d-N4-hydroxycytidine, the active substance in Molnupiravir, exists in two forms as tautomers. In one form, it acts as a cytidine with a single bond between the carbon and the N-OH group. In its other form, which mimics uridine, it has an oxime with a double bond between the carbon and the N-OH group. In the presence of beta-d-N4-hydroxycytidine, viral RNA-dependent RNA polymerase reads it as uridine instead of cytidine and inserts adenosine instead of guanosine. Switching between forms causes inconsistencies during transcription, which results in numerous mutations in the viral genome and a cessation of viral replication. [8].

Merck's commitment to conditional and full FDA approval of Molnuparivir continues. In its statement, Merck stated: [12] "In anticipation of the results of the MOVe-OUT program, Merck manufactures Molnupiravir at its own risk. Merck expects to produce 10 million therapeutic doses by the end of 2021, with more expected to be produced in 2022. Merck is committed to providing timely access to Molnupiravir worldwide, if authorized or approved, and plans to introduce access to tiered prices based on World Bank admission criteria that reflect countries' relative ability to fund their pandemic health response. As part of its commitment to extend the global approach, Merck has previously announced that it has entered into non-exclusive voluntary licensing agreements for Molnupiravir with established generic manufacturers to accelerate the availability of Molnupiravir in more than 100 low and middle income countries (LMICs) following approval or emergency approval by local regulatory agencies. . " This "generosity" is unlikely to apply to use in animals.

Drugs to inhibit the current Covid-19 pandemic have been the subject of accelerated field trials in the last two years, and one of them, Remdesivir, has been approved for use in hospitalized patients in record time. Last year, Molnupiravir was submitted for conditional approval as an oral medicinal product for home treatment of the infection at an early stage. [12]. However, anti-coronavirus compounds have been developed previously for another common and serious feline disease, feline infectious peritonitis (FIP). These drugs include a protease inhibitor (GC376) [13] and an RNA-dependent RNA polymerase inhibitor (GS-441524), which is an active ingredient of Remdesivir [14]. The success of antiviral drugs in the treatment of FIP prompted a recent study by EIDD-1931 and EIDD-2801 for their ability to inhibit FIPV in tissue cultures. [15]. The effective EC50 concentrations for EIDD-1931 against FIPV are 0.09 μM, EIDD-2801 0.4 μM and GS441524 0.66 μM [15]. The percentage of cytotoxicity at 100 μM is 2.8, 3.8 and 0, respectively. Therefore, EIDD-1931 and EIDD-2801 are slightly more effective at inhibiting viruses, but also more cytotoxic than GS-441524. These laboratory studies suggest that EIDD-1931 and EIDD-2801 are excellent candidates for the treatment of FIP.

Although EIDD-1931 and EIDD-2801 are a great promise for the treatment of FIP, there are several obstacles that will make the legal use of these compounds unlikely in the near future. GS-441524, the active form of Remdesivir and patented by Gilead Sciences, was investigated for use in cats with FIP shortly before the Covid-19 pandemic. FIP research [14] therefore stimulated the potential use of Remdesivir against Ebola and not SARS-like coronavirus [14]. Although these studies were conducted in collaboration with scientists from Gilead Sciences, the company refused to grant GS-441524 rights to treatment in animals as soon as it became clear that there was a much larger market for Covid-19 in humans. [16]. Similarly, my attempts over the past 2-3 years at Emory, Ridgeback Biotherapeutics, and Merck Veterinary Division to investigate EIDD-1931 and EIDD2801 for the treatment of FIP in cats have either remained unanswered or rejected, no doubt for similar reasons why Gilead refused to grant rights for GS-441524. However, the great worldwide need for FIP treatment quickly supported the unapproved market for GS-441524 from China. The same need to treat FIP has recently aroused interest in Molnupiravir, also from China.

Situation with EIDD-1931 vs. EIDD-2801 / Molnupiravir and GS-441524 vs. Remdesivir raises the question of why some medicines are being converted to prodrugs for marketing purposes [17]. Remdesivir was reportedly esterified to increase antiviral activity, although studies in cats showed that GS-441524 and Remdesivir had similar viral inhibitory activity in tissue culture. [18]. However, Remdesivir was found to be poorly absorbed by the oral route and was therefore conditionally approved for injectable use only. EIDD-2801 was designed to increase the oral absorption of EIDD-1931, although previous research has shown that EIDD-1931 is well absorbed orally without esterification. [6]. The motives for the commercialization of Remdesivir instead of GS-441524 for human use have been scientifically questioned, as it appears to be better in several ways without further modification. [17]. Why EIDD-2801 was chosen for commercialization, when EIDD-1931 would be cheaper, 4 times more effective against viruses and one third less toxic than EIDD-2801 [15]? The strength of patent rights and the longevity of patents may be more important factors in these decisions. [16,17,19].

One of the problems in the treatment of FIP in cats is the blood-eye and blood-brain barriers, which become very important when the disease affects the eyes and / or the brain. [13, 14, 20]. This problem has been largely overcome in the treatment of ocular and neurological forms of FIP with GS-441524 by gradually increasing the dose to increase blood levels and thus drug concentrations in the ventricular fluid and / or brain. [20]. GC376, one of the most effective antivirals against FIP virus in culture [17], is not effective against ocular and neurological FIP due to the inability to get enough drug to these sites, even if the dose is increased several times[14]. Fortunately, it appears that EIDD-1931 can reach effective levels in the brain, as indicated by studies in horses with VEEV infection. [3]. Drug resistance is another problem that now occurs in some cats treated with GS-441524, especially in individuals with the neurological form of FIP. Long treatment procedures and difficulties in transporting enough drug to the brain support the development of drug resistance.

The short-term and long-term toxic effects of the drug candidate on the test person or animal are crucial. GS-441524 showed lower toxicity in cell cultures than GC376, EIDD-1931 and EIDD-2801 [15]. Most important, however, is the toxicity that manifests itself in vivo. GC376 is one of the drugs with the highest coronavirus inhibitory effect [15], but slows the development of adult teeth when given to young kittens [13]. No serious toxicity was observed during nearly three years of field use of GS-441524, reflecting the complete absence of cytotoxic effects in vitro at concentrations up to 400 µM. [18]. However, EIDD-1931 and EIDD-2801 show significant cytotoxicity at 100 μM [15]. Therefore, the ability of EIDD-1931 to make fatal mutations in RNA has been raising a number of questions for some time. [8, 21, 22]. This was the main reason why the application for the treatment of diseases was still delayed. However, the current recommended duration of treatment with Covid-19 Molnupiravir is only 5 days at the initial stage of treatment. [10]. However, the recommended duration of FIP treatment with GS-441524 is 12 weeks [14], which represents a much longer time for the manifestation of toxicity. Therefore, close observation of cats during treatment with EIDD-1931 or EIDD-2801, whether short-term or long-term, will be important.

All existing antiviral drugs have led to the development of drug resistance through mutations in the viral genome. Although Remdesivir appears to be less susceptible to such mutations compared to drugs used in viral diseases such as HIV / AIDS, resistance is well documented. [23-25]. Resistance to GS-441524 in cats treated for FIP was observed at a higher frequency, especially in cats with neurological FIP, where it is more difficult to deliver sufficient drug to the brain [13, 14, 20]. Resistance to GS-441524 in cats is also likely to be a major problem, as cats with FIP are often treated for 12 weeks or longer, while Remdesivir (and Molnupiravir) are recommended for only five days during the initial viremic stage of Covid-19. [16]. The problem of drug resistance in HIV / AIDS treatment is effectively addressed by using a cocktail of different drugs simultaneously with different resistance profiles. Mutants resistant to one drug will immediately inhibit other drugs, thus preventing their positive selection during treatment. Inhibition of resistance is particularly strong when the two drugs attack different proteins involved in virus replication. For example, GC376 is a protease inhibitor [13], while GS-441524 acts on an RNA-dependent RNA polymerase [18]. However, GC376 is not as well absorbed across the blood-brain barrier. Although the necessary research has not yet been performed, there appears to be no cross-resistance between GS-441524 and Molnupiravir and is as effective as GS-441524 in crossing the blood-brain barrier. [3]. This makes Molnupiravir (or 5-hydroxycytidine) an important contribution to the future treatment of FIP.

As expected, Molnupiravir has recently been tested on cats with FIP by at least one Chinese retailer, GS-441524, and preliminary results are available on the FIP Warriors website. [26]. Field trials included 286 cats with various forms of naturally occurring FIP observed at pet clinics in the United States, the United Kingdom, Italy, Germany, France, Japan, Romania, Turkey, and China. The 286 cats that participated in the study, including seven cats with ocular (n = 2) and neurological (n = 5) FIP, did not die. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats were healthy after 3-5 months, a period during which relapses would be expected to relapse unsuccessfully. These data provide convincing evidence of the safety and efficacy of Molnupiravir in cats with various forms of FIP. However, we hope that this field study will be written in manuscript form, submitted for review and published. Either way, Molnupiravir is already marketed to owners of cats with FIP. At least one other major retailer of GS-441524 is also interested in using Molnupiravir for FIP, indicating a demand for additional antiviral drugs for cats with FIP.

Safe and effective dosing for Molnupiravir in cats with FIP has not been published. However, at least one vendor from China provided certain pharmacokinetic and field test data for Molnuparivir in cats with naturally occurring FIP in a leaflet for the product Hero-2081. [26]. However, this information does not clearly indicate the amount of Molnupiravir in one of their "50 mg tablets" and the actual dosing interval (q12h or q24h?). Fortunately, the estimated starting dose of molnupiravir for cats with FIP can be obtained from published in vitro cell culture studies of EIDD-1931 and EIDD-2801. [15] and laboratory and field studies GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 μM / μl against FIPV in cell culture, while the EC50 of GS-441524 is about 1.0 μM / μl. [18]. Both have a similar oral absorption of about 40-50 %, so the effective subcutaneous (SC) dose for Molnupiravir would be approximately half the recommended 4 mg / kg SC every 24 hours of the initial dose for GS441524. [14] or 2 mg / kg SC q24h. The per-os (PO) dose would be doubled to account for less effective oral absorption at a dose of 4 mg / kg PO every 24 hours. The estimated initial oral dose of molnupiravir for cats with FIP can also be calculated from the available Covid-19 treatment data. Patients treated for Covid-19 are given 200 mg of molnupiravir PO q12h for 5 days. This dose was evidently calculated from a pharmacokinetic study performed in humans, and if the average person weighs 60-80 kg (70 kg), the effective inhibitory dose is 3,03.0 mg / kg PO q12h. The cat has a basal metabolic ratio 1.5 times higher than humans, and assuming the same oral absorption in both humans and cats, the minimum dose for cats according to this calculation would be 4.5 mg / kg PO every 12 hours. Assuming that molnupiravir crosses the blood-brain barrier and the blood-brain barrier as efficiently as GS-441524 [3,18], the dose would be increased ~ 1.5 and ~ 2.0-fold to allow adequate penetration into the aqueous humor and cerebrospinal fluid for cats with ocular (~ 8 mg / kg PO, q12 h) or neurological FIP (~ 10 mg / kg PO, q12h). The treatment will last 10-12 weeks and the monitoring of the response to treatment will be identical to GS-441524 [14, 20]. These recommendations are based on published data assumptions and further experience with Molnupiravir will be required in this area. Molnupiravir is unlikely to be safer and more effective than GS-441524 in the treatment of FIP, but a third antiviral drug may be particularly useful in preventing resistance to GS-441524 (as a cocktail of antivirals with different resistance profiles) or in treating cats that no longer respond. good on GS-441524. It is largely unknown whether Molnupiravir will be without long-term toxic effects, as the active substance N4-hydroxycytidine is an extremely potent mutagen. [21] and the duration of FIP treatment is much longer than with Covid-19 and there is a likelihood of major side effects.

It is a pity that EIDD-1931 (N4-hydroxycytidine), the active substance in Molnupiravir, has not received much attention in the treatment of FIP cats than Molnupiravir. EIDD-1931 has a 4-fold greater inhibitory effect against the virus than Molnupiravir (EC50 0.09 vs. 0.4 μM) and the percentage of cytotoxicity is slightly lower (2.3% vs. 3.8% at 100 μM) [15]. N4-hydroxycytidine is also efficiently absorbed orally [3], which was downplayed in the development of EIDD-2801 (Molnupiravir). This scenario is identical to the GS-441524 vs. Remdesivir, the second of which, Remdesivir, was chosen for commercialization, although current research suggests that GS-441524 would be the best candidate.[17].

References

  1. Painter GR, Natchus MG, Cohen O, Holman W, Painter WP. Developing a direct acting, orally available antiviral agent in a pandemic: the evolution of molnupiravir as a potential treatment for COVID-19 [published online ahead of print, 2021 Jun 18]. Curr Opin Virol. 2021; 50: 17-22. doi: 10.1016 / j.coviro.2021.06.003
  2. Halford B. An emerging antiviral takes aim at COVID-19. c & en topics. 2020. https://cen.acs.org/pharmaceuticals/drug-development/emerging-antiviral-takes-aim-COVID19/98/web/2020/05.
  3. Painter GR, Richard A. Bowend RA, Bluemling GR et al. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal Venezuelan equine encephalitis virus infection. Antiviral Res. 2019, 171: 104597
  4. Costantini, VP, Whitaker, T., Barclay, L., Lee, D., McBrayer, TR, Schinazi, RF, Vinje, J., 2012. Antiviral activity of nucleoside analogues against norovirus. Antivir. Ther.17 (6), 981–991. https://doi.org/10.3851/imp2229.
  5. Ehteshami, M., Tao, S., Zandi, K., Hsiao, HM, Jiang, Y., Hammond, E., Amblard, F., Russell, OO, Merits, A., Schinazi, RF, 2017. Characterization of beta-dN (4) -hydroxycytidine as a novel inhibitor of chikungunya virus. Antimicrob. Agents Chemother. 61 (4) e02395-02316. https://doi.org/10.1128/aac.02395-16.
  6. Stuyver, LJ, Whitaker, T., McBrayer, TR, Hernandez-Santiago, BI, Lostia, S., Tharnish, PM, Ramesh, M., Chu, CK, Jordan, R., Shi, J., Rachakonda, S ., Watanabe, KA, Otto, MJ, Schinazi, RF, 2003. Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob. Agents Chemother. 47 (1), 244–254.
  7. Yoon J., Toots M, Lee S, Lee ME, et al., 2018. Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses. Antimicrob. Agents Chemother. 2018, 62 (8): https://doi.org/10.1128/aac.00766-18
  8. Urakova N, Kuznetsova V, Crossman DK, Sokratian A, Guthrie DB, Kolykhalov AA, et al. β-d-N4Hydroxycytidine is a potent anti-alphavirus compound that induces a high level of mutations in the viral genome. J Virol. 2018, 92: e01965 – e01917. doi: 10.1128 / JVI.01965-17.
  9. Pyrc, K., Bosch, BJ, Berkhout, B., Jebbink, MF, Dijkman, R., Rottier, P., van der Hoek, L., 2006. Inhibition of human coronavirus NL63 infection at early stages of the replication cycle . Antimicrob. Agents Chemother. 2006, 50 (6): 2000–2008. https://doi.org/10.1128/aac.01598-05.
  10. Whitfill T. A likely new treatment for Covid-19 was made possible by government-funded innovation. STAT +. https://www.statnews.com/2021/10/05/government-funding-backed-molnupiravir-possible-newcovid-19-treatment/.
  11. Painter, GR, Guthrie, DB, Bluemling, G., Natchus, MG N4-Hydroxycytidine and Derivatives and Antiviral Uses Related Thereto. US Patent Application, 2016, 2016/106050 A1.
  12. Merck news release, October 1, 2021. https://www.merck.com/news/merck-and-ridgebacksinvestigational-oral-antiviral-molnupiravir-reduced-the-risk-of-hospitalization-or-death-by-approximately -50percent-compared-to-placebo-for-patients-with-mild-or-moderate /.
  13. Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M, Meadows JM, Chang KO. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2018, 20 (4): 378-392.
  14. Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. efficacy and safety of the nucleoside analog GS-441524 for the treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019, 21 (4): 271-281.
  15. Cook SE, Vogel H and D. Castillo D. A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus. 2021. bioRxiv 2020.07.09.195016; doi: https://doi.org/10.1101/2020.07.09.195016
  16. Zhang S. A Much-Hyped COVID-19 Drug Is Almost Identical to a Black-Market Cat Cure. May 8, 2020 Shutterstock / The Atlantic, https://www.theatlantic.com/science/archive/2020/05/remdesivir-cats/611341/.
  17. Yan VC, Muller FL. Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment. ACS Medicinal Chemistry Letters. 2020, 11 (7): 1361-1366 DOI: 10.1021 / acsmedchemlett.0c00316
  18. Murphy BG, Perron M, Murakami E, Bauer K, Park Y, Eckstrand C, Liepnieks M, Pedersen NC. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies.Vet Microbiol. 2018, 219: 226-233.
  19. Common Dreams. Public citizen. Press release, August 4, 2020, https://www.commondreams.org/newswire/2020/08/04/public-citizen-scientists-gilead-and-federalscientists-have-neglected
  20. Dickinson PJ. Coronavirus Infection of the Central Nervous System: Animal Models in the Time of Covid-Front. Vet. Sci. 2020, 23: https://doi.org/10.3389/fvets.2020.584673
  21. Zhou S, Hill CS, Sarkar S, et al., Β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 through lethal mutagenesis but Is also mutagenic to mammalian cells. J Infect Dis. 2021, 224: 415–419, https://doi.org/10.1093/infdis/jiab247.
  22. Cohen J, Piller C. Emails offer look into whistleblower charges of cronyism behind potential COVID-19 drug. ScienceInsider-Health. 2020, https://www.science.org/news/2020/05/emails-offer-look-whistleblowercharges-cronyism-behind-potential-covid-19-drug.
  23. Agostini ML, Andres EL, Sims AC, et al. Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI:
    10.1128 / mBio.00221-18.
  24. Szemiel AM, Merits A, Orton RJ, In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. Plos Path, 2021, https: //doi.org/10.1371/journal.ppat.1009929.
  25. Martinot M, Jary A, Fafi-Kremer S, et al., Emerging RNA-Dependent RNA Polymerase Mutation in a Remdesivir-Treated B-cell Immunodeficient Patient With Protracted Coronavirus Disease 2019, Clinical Infectious Diseases, 2020 ;, ciaa1474, https: / /doi.org/10.1093/cid/ciaa1474
  26. FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
Read "A long history of Beta-d-N4-hydroxycytidine and its modern application for the treatment of Covid-19 in humans and FIP in cats."

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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FIP treatment with oral forms GS-441524

Niels C. Pedersen, Nicole Jacque,
3.10.2021
Original article: FIP treatment with oral formulations of GS-441524

Introduction

Initial field testing of GS-441524 for FIP treatment involved subcutaneous administration. This route of administration was based on previous pharmacokinetic (PK) studies performed in laboratory cats. The intravenous and subcutaneous routes of injection yielded similarly high blood levels, which were maintained at virus-inhibiting concentrations for more than 24 hours. Oral administration has been found to lead to blood levels that peak after 2 hours, but reach only about 40 % peak levels of subcutaneous and intravenous administration (Pedersen NC, unpublished data, 2018). However, dogs that have a longer intestinal tract developed for omnivores can absorb up to 85 % GS441524 orally. [1, 5]. Dogs are often used as a surrogate for humans in oral absorption studies, so oral absorption in humans is also likely to be higher than in cats. 

Chinese suppliers of GS-441524 copied the diluent, drug concentration, and subcutaneous route used in the original published field study. Mutian was the first company to offer GS441524 on an unapproved market. Mutian was also the first company to investigate and offer an oral formulation. Mutian researchers found that effective blood levels of GS-441524 could only be achieved by increasing the concentration of the drug in their oral preparations. Other companies (eg Aura, Lucky) subsequently offered their own versions of the orally administered drug GS-441524. However, as of September 2021, Mutian no longer lists GS oral preparations (in any form) on its website. Aura, Lucky and Capella are currently the most widely used oral forms of GS441524 in the United States.

Current brands of capsules / tablets are sold as nutritional supplements and their labels list several common harmless chemical compounds and medicinal herbs, with GS-441524 not being listed at all. This is probably so that manufacturers avoid customs controls. Regardless of the list of ingredients, GS-441524 is the active ingredient in all oral products. The exact concentration of GS-441524 in the various oral preparations is kept secret by the vendors, but it is clearly higher (1.5-2-fold?) Than would be required if the drug was administered subcutaneously. 

Initially, we were critical of the oral route for two reasons. First, oral forms were more wasted by what was initially a rare and expensive resource. Second, published research on oral absorption of nucleosides (GS-441524 is a nucleoside) documents a concentration limit or ceiling for oral absorption [2-5]. Results with nucleoside-related EIDD-1931 showed a decrease in bioavailability from 56 to 36 % with increasing oral dose [6]. This limitation would theoretically make it difficult to achieve the extremely high blood levels needed to treat some forms of FIP (e.g., neurological) and / or to overcome the problem of acquired drug resistance. Oral bioavailability can also be significantly reduced by certain substances in the diet, and cat owners are known to use a large number of dietary supplements, some of which could adversely affect treatment. 

More and more owners and veterinarians appear to be using GS-441524 oral therapy for some or all of their treatment. The cost of GS-441524 oral products has been steadily declining and improving over the last two years. The problem of injection site reactions together with the more effective oral preparations GS-441524 have stimulated oral treatment and more and more cats are being treated with oral drugs either partially or completely. 

Composition and labeling

Most established oral preparations are small tablets that are easier to administer than larger capsules. Newer formulations, such as Sweeper, offer a soluble film form of GS-441524 to avoid "pill" difficulties in some cats. 

The actual amount of GS-441524 in the tablet / capsule and the recommended dosage of the oral medication will vary considerably depending on the form of the FIP, the vendor and the experience of the owner and the FIP treatment groups. Therefore, the actual amount (mg) of GS-441524 in a tablet or capsule is usually not reported. Instead of the actual amount of GS-441524 in a tablet or capsule, the seller's dosage is often based on the number of tablets needed per kg of weight, e.g. 1 tablet / kg orally (P0) every (q) 24 hours (h) for cats with wet or dry FIP and without ocular or neurological impairment. The amount of GS-41524 in one such tablet administered after 24 hours corresponds to a dose of 4-6 mg / kg SC after 24 hours, but the actual amount of GS in one tablet can be doubled as in 1 ml of injectable GS to compensate for the reduced bioavailability when administered oral route. 

In addition, one supplier (Aura / Spark) has tablets labeled for q12h administration and another for q24h dosing. 1 tablet / kg after 12 hours contains half the amount of GS-441524 (probably 4-6 mg) as 1 tablet / kg after 24 hours (probably 10 mg) - the reason is that dosing after 12 hours prevents a decrease in blood concentration 24 hours ago . However, effective blood levels after a single dose of PO or SC are maintained for 24 hours or longer in both cases. At doses corresponding to 10-15 mg / kg SC q24h, a further advantage of q8h or q12h over q24h may be an advantage, as it can help bypass the absorption ceiling. Therefore, in cats with doses corresponding to 10-15 mg / kg SC q24h or higher, a dose division of q8h or q12h is often used.

Dosage

The recommended starting dose of GS-441524 for cats with wet or dry FIP and without neurological or ocular symptoms is 4-6 mg / kg SC q24h. The injection dose for cats with ocular disease is 8 mg / kg SC q24h and for cats with neurological disease 10 mg / kg SC q24h. If a cat is started on wet FIP and then develops eye disease, the dose is immediately increased to 8 mg / kg SC q24h and if neurological symptoms develop, it is increased to 10 mg / kg SC q24h. Failure to treat FIP at doses higher than 15 mg / kg SC q24h indicates drug resistance. Doses of PO corresponding to 4-6, 8 and 10 mg / kg SC q24h are 10, 16 and 20 mg / kg PO q24h. (Note: some oral preparations are designated as SC equivalents, but in fact contain up to twice the reported mg GS) The recommended duration of treatment is 12 weeks, with dose increases if considered necessary. However, it is known that some cats can be cured in 6 weeks with any form of GS-441524, several in 8-10 weeks and almost all in 12 weeks. Young cats with abdominal wet FIP tend to respond the fastest, cats with dry FIP slower and cats with neurological FIP the slowest. Therefore, it is a "universal" recommendation to treat any cat with FIP, regardless of form, for at least 12 weeks. The daily dose in the form of PO can be divided into q12h, which may be advantageous in higher dose treatment to avoid an absorption ceiling. SC and PO treatment can be alternated q12h to avoid large injection volumes.

Oral GS dosing is less accurate than for injections. Tablets are difficult to separate because they tend to break, so halving is often the best thing to do. If the calculated dose after use falls within the indicated doses in the tablets, it is always recommended to round up to the nearest half of the tablet. 

Serving

All oral brands have similar instructions for the administration of capsules or tablets. Half an hour of fasting before and after administration is generally recommended. A small amount of treats can encourage cats to take the tablets, and many cats consume them when they are placed on a plate wrapped in treats (e.g., Churu). 

Costs

The price of oral GS has dropped significantly over the last year. Nevertheless, the relative price of the oral GS-441524 is 20-40 % higher (depending on the supplier) than its injectable version.  

Factors affecting oral and injectable administration 

Cats currently experiencing vomiting / regurgitation and diarrhea are generally considered unsuitable candidates for oral treatment with GS-441524. Therefore, cats with severe gastrointestinal disease are often injected at least until these problems are resolved. Most people, especially in the past, have started injecting GS-441524. The injection form is cheaper and the dosage is more precisely controlled. Absorption of GS-441524 is also more reliable by the subcutaneous route than by the oral route, which is often a critical factor in the initial treatment of cats that are initially seriously ill and unstable. Whether a cat will continue to inject GS-441524 is often conditioned by the owner's ability to inject as efficiently as possible, the cat's willingness to adapt to the pain of the injection, and the occurrence of injection wounds (lesions) at the injection site. Oral medications are often a welcome relief for both the owner and the feline patient in such situations. Some substances administered orally may interfere with the absorption of GS-441524. Therefore, you should avoid the inclusion of other medications and nutritional supplements unless they are necessary for the treatment of FIP. 

Comparison of the success of treatment with injection and oral GS-441524  

Assuming that the dosage is accurately calculated and properly adjusted, the success rate of the oral drug GS-441524 currently reflects the success rate of the injectable drugs. Nevertheless, differences in responses between oral and injectable GS-441524 have been reported. A small number of cats did not respond well to oral GS-441524 as initial treatment or led to relapses during injection replacement. Alternatively, switching cats to oral GS-441524 at the equivalent dose resulted in resolution of the disease, which did not respond well to injections. It is difficult to attribute these dramatic differences in formulation response as GS-441524 administered subcutaneously or orally enters the bloodstream and eventually the tissues. This is more likely to be due to the fact that the brands of GS-441524 injectable or oral medicine used prior to such a change were not good or that there were problems with absorption or administration. Indeed, there have been many cases where switching to another oral or injectable brand immediately resulted in improved response. 

It was originally thought that only the injectable form of GS-441524 could achieve the extremely high levels of blood and cerebrospinal fluid needed to effectively treat neurological disease, especially in situations where the virus developed varying degrees of drug resistance. However, oral markers such as Aura / Lucky have been effective in many cats with neurological FIP. This also applied to some cats that did not respond to the extremely high doses of GS441524 injection. More and more cats with neurological FIP are being treated exclusively with the oral form of GS. This is due either to greater experience with oral treatment in severe cases of FIP, or probably to higher quality oral products. 

An overview of currently available oral form brands GS-441524 

Note: The GS label and content reflect information provided by suppliers and have not been independently verified. 

Mutian - This is the original and most famous brand of the oral form GS-441524. It has been sold in several different forms, including several tablets and capsules. At the beginning of 2021, Mutian switched to the form of tablets, designated as 200 mg or 50 mg "Mutian" or "Xraphconn" - these deliver an equivalent SC dose of 10, respectively. 2.5 mg GS-441524. The tablets are significantly larger (8.5 mm diameter) than tablets from other suppliers. Recently, a new capsule formulation is rarely available. As of September 2021, Mutian's website no longer offers the option of PO. For all oral forms of Mutian, the supplier states the dosage: 100 mg / kg “Mutian” for wet / dry FIP, 150 mg / kg Mutian for ocular FIP and 200 mg / kg for neurological FIP. 

Aura / Spark - Aura is a long-established brand and is sold in tablets that are given every 12 or 24 hours. They are sold in versions q12h and q24h, but there is no difference in composition (ie extended release, etc.) between the two versions. The actual amount of GS-441524 in each tablet is not reported, but the label and effective dose are as follows:

MarkingInjectable equivalentDosage instructions
Aura 12h-1kgapprox. 2.5 mg / kgWet / dry: 1 tablet per kg twice a day
Ocular: 1.5 tablets per kg twice a day
Neurological: 2 tablets per kg twice a day  
Aura 24h – 1 kgapprox. 5 mg / kgWet / dry: 1 tablet per kg per day
Ocular: 1.5 tablets per kg per day
Neurological: 2 tablets per kg per day         
Aura 12h – 3 kgapprox. 7.5 mg / kgWet / dry: 1 tablet per 3 kg twice a day
Ocular: 1.5 tablets per 3 kg twice a day
Neurological: 2 tablets per 3 kg twice a day         
Aura 24h – 2 kgapprox. 10 mg / kgWet / dry: 1 tablet per 2 kg twice a day
Ocular: 1.5 tablets per 2 kg twice a day
Neurological: 2 tablets per 2 kg twice a day         

The equivalent oral dose for> 10 mg / kg daily GS injection is increased proportionately. The tablets can be combined regardless of the 12 / 24h label using an effective injection dose - for example, a 2.5 kg cat with a wet FIP could take one tablet 24h - 2 kg and one tablet 12h - 1 kg per day.

Lucky - Lucky tablets are designated 24h - 1 kg (equivalent dose 5-6 mg / kg SC) or 24h - 2 kg (equivalent dose approximately 10-12 mg / kg SC) and are said to have the same composition as comparable Aura tablets, although they have a different Face. For FIP without ocular or neurological symptoms, you should give one 1 kg tablet daily per kg cat weight or one 2 kg tablet for every 2 kg, rounded to the nearest half tablet. Multiply the number of tablets per day by 1.5 for ocular or 2 for neurological forms. 

MarkingInjectable equivalentWet / dry FIP dosing instructions (dosing doubles for neuro / ocular FIP)
Lucky 24h - 1 kgapprox. 5-6 mg1 tablet per kg per day
Lucky 24h - 2 kgapprox. 10-12 mg1 tablet per 2 kg per day

Capella - Capella produces two tablet sizes, 1 kg (dose 5-6 mg SC equivalent) and 2 kg (dose 10-12 mg SC equivalent). For FIP without ocular or neurological symptoms, you should give one 1 kg tablet daily per kg cat weight or one 2 kg tablet for every 2 kg and round up to the nearest half tablet. Multiply the number of tablets per day by 1.5 for ocular or 2 for neurological forms. 

Kitty Care - This is another low-cost brand that now offers both injectable and oral GS-441524. Each tablet is assumed to contain the equivalent of a 6 mg SC dose of GS-441524. 

Hero 16 -It is a well-known brand, which is supplied in easy-to-apply and divisible tablets intended for administration in a dose of one tablet per 2 kg body weight, such as Capella 2 kg tablets. Each tablet probably contains 16 mg of GS-441524. 

Rainman - This brand is popular in China and seems to have a good reputation in the countries where it is used. It is sold in 1 kg and 2 kg tablets, which are believed to contain the equivalent of 5-6 mg and 10-12 mg SC GS-441524. 

Mary - Mary is sold in capsules that probably contain the equivalent of 6 mg SC GS-441524

Additional brands- Panda, Pany, Sweeper, Sweeper movie

Reference studies on GI uptake of nucleosides similar to GS-441524 and GS-441524

  1. Thomas L. A precursor to remdesivir shows therapeutic potential for COVID-19. https://www.news-medical.net/news/20210209/A-precursor-to-remdesivir-showstherapeuticpotential-for-COVID-19.aspx.
  2. Painter GR, Bowen RA, Bluemling GR, et al. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection. Antiviral Res. 2019; 171: 104597. doi: 10.1016 / j.antiviral.2019.104597
    After oral administration EIDD-1931 is quickly absorbed as evidenced by plasma T-max-values ranging between 0.5 and 1.0 h.Exposures are high (C-ma-xvalues range between 30 and 40μM) and are dose dependent, but significantly less than dose proportional. The observation of decreasing bioavailability with increasing dose may indicate capacity limited absorption, a phenomenon that has been reported for other nucleosides (de Miranda et al., 1981). EIDD-1931, like most endogenous nucleosides and xenobiotic nucleoside analogs, is a highly polar, hydrophilic molecule (cLog P = −2.2) and therefore likely to require functional transporters to cross cell membranes. This dependence would explain the capacity limited uptake seen in the pharmacokinetic studies done using the CD-1 mice. Earlier reports also indicated that nucleoside uptake into mouse intestinal epithelial cells is primarily mediated by sodium dependent concentrative nucleoside transporters (Cass et al., 1999; Vijayalakshmi and Belt, 1988).
  3. Cass, CE, Young, JD, Baldwin, SA, Cabrita, MA, Graham, KA, Griffiths, M., Jennings, LL, Mackey, JR, Ng, AM, Ritzel, MW, Vickers, MF, Yao, SY, 1999 .Nucleoside transporters of mammalian cells. Pharm. Biotechnol. 12313–12352
  4. de Miranda, P., Krasny, HC, Page, DA, Elion, GB, 1981. The disposition of acyclovir indifferent species. J. Pharmacol. Exp. Ther. 219 (2), 309–315
  5. Vijayalakshmi, D., Belt, JA, 1988. Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities. Biol. Chem. 263 (36), 19419–19423.
  6. Yan VC, Khadka S, Arthur K, Ackroyd JJ, Georgiou DK, Muller FL. Pharmacokinetics of Orally Administered GS-441524 in Dogs. bioRxiv, doi: https://doi.org/10.1101/2021.02.04.429674
  7. FIP Warriors CZ / SK, https://www.fipwarriors.eu/, https://www.facebook.com/groups/fipczsk
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A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus

9.7.2020
SE Cook, H. Vogel, D. Castillo, M. Olsen, N. Pedersen, BG Murphy
Original article: A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus

Abstract

Feline peritonitis (FIP), caused by a genetic mutant of feline enteric coronavirus known as FIPV, is a deadly disease in cats for which no FDA-approved vaccine or treatment is currently available. The spread of FIPV in affected cats leads to a number of clinical symptoms, including cavitation effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and perivasculitis with or without central nervous system and / or eye involvement. There has been a critical need for effective and approved antiviral therapies against coronaviruses, including FIPV and zoonotic coronaviruses such as SARS-CoV-2, caused by COVID-19. For SARS-CoV-2, preliminary evidence suggests that there may be potential clinical and pathological features common to feline coronavirus disease, including enteric and neurological impairment. We examined 89 selected antiviral compounds and identified 25 compounds with antiviral activity against FIPV, which represent different classes of drugs and mechanisms of antiviral action. Based on successful combination therapy strategies in human patients with HIV infection or hepatitis C virus, we have identified drug combinations targeting different phases of the FIPV life cycle that lead to a synergistic antiviral effect. Similarly, we suggest that combination anti-cancer therapy (cACT) with multiple mechanisms of action and penetration into all potential anatomical sites of viral infection should be applied to the treatment of other coronaviruses, such as SARS-CoV-2.

Author Summary

We tested in vitro antiviral activity against FIPV in 89 compounds. The antiviral activity of these compounds consisted either of a direct effect on viral proteins involved in viral replication or an indirect inhibitory effect on normal cellular processes usurped by FIPV to promote viral replication. Twenty-five of these compounds showed significant antiviral activity. We have also found that certain combinations of these compounds are more effective than monotherapy alone.

Dictionary

In shortEnglish expressionSlovak translation
PIprotease inhibitorprotease inhibitor
NPInucleoside polymerase inhibitornucleoside polymerase inhibitor
NNPInon-nucleoside polymerase inhibitora non-nucleoside polymerase inhibitor
CPEcytopathic effect cytopathic effect
cACTcombined anti-coronaviral therapycombined anticoronavirus therapy
cARTcombined anti-retroviral therapycombination antiretroviral therapy
CRFK cellsCrandell-Rees Feline Kidney cellsCrandell-Rees feline kidney cells

Introduction

Feline infectious peritonitis (FIP) is a highly fatal disease without an effective FDA-approved vaccine or treatment. Although the pathogenesis is not fully understood, FIP is generally thought to be the result of specific mutations in the viral genome of the minimally pathogenic and ubiquitous feline enteric coronavirus (FECV) that result in the virulent FIP virus (FIPV) [1–3]. These FECV mutations lead to a change in the tropism of the virus-infected host cell from intestinal enterocytes to peritoneal-type macrophages. FIPV productive macrophage infection, targeted extensive anatomical dissemination, and immune-mediated perivasculitis lead to the highly fatal systemic inflammatory disease FIP [4]. As a result of viral dissemination, FIP may present with clinical signs reflecting inflammation at various anatomical sites, which may potentially include the abdomen and intestines, the thoracic cavity, the central nervous system, and / or the eyes [5-8]. Due to its high mortality, FIP remains a devastating viral disease in cats and a challenge in making an accurate etiological diagnosis with a current lack of available and effective treatment options [7, 9]. The development of an effective FIP vaccine has been complicated by the role of antibody-dependent amplification (ADE) in the pathogenesis of FIP, where the presence of non-neutralizing anti-coronavirus antibodies has been shown to exacerbate FIP [10–12].

In mammals, coronaviruses infect and generally cause disease of the intestinal tract or respiratory system of infected hosts [13]. However, FIP often manifests itself as a multisystem inflammatory disease syndrome due to the widespread spread of FIPV-infected macrophages. The recent pandemic occurrence of SARS-CoV-2 in infected human patients results in various disease syndromes, collectively referred to as COVID-19. Although SARS-CoV-2 has overt tropism for respiratory epithelium leading to interstitial pneumonia, recent evidence suggests that COVID-19 may also present as a digestive disease and clinically manifest as diarrhea [14, 15]. The tropism for these tissues reflects the membrane expression of the ACE2 protein, the cellular target of SARS-CoV-2 [16]. Furthermore, SARS-CoV-2 has been shown to be able to infect and cause inflammatory disease in tissues outside the intestinal tract and respiratory tract, including the brain, eyes, reproductive organs, and cardiac myocardium [17-21]. Brain stem neuroinvasion and subsequent encephalitis caused by SARS CoV-2 may contribute to respiratory failure in patients with COVID-19 [20,22]. Experimentally, SARS CoV-2 is able to create a productive infection in cats [23]. Therefore, feline FIPV infection and SARS CoV-2 infection in human patients are more similar than originally thought.

There is an immediate and critical need for available and effective antiviral therapies for the treatment of these coronavirus diseases. FIPV-infected cats could serve as a translational model and provide useful insights useful for SARS-CoV-2-infected patients with COVID-19. Recent antiviral clinical trials in both experimental and naturally infected FIPV cats have provided hope for the treatment and cure of FIP with GS-441524, the nucleoside analog and metabolite of the prodrug Remdesivir (Gilead Sciences) or GC-376, a 3C-like FIPV protease inhibitor (Anivive) [24– 26]. Remdesivir, a prodrug of GS-441524, has recently been shown to be promising in the treatment of human patients infected with SARS-CoV-2 [27,28]. Despite these recent clinical successes, these antiviral compounds have yet to be approved and are not currently available for clinical veterinary use in cats with FIP.

Identifying and developing effective antiviral therapies can be costly and time consuming. Targeted screening and reuse of drugs already approved by the FDA or approved for research use can play an effective role in drug discovery. Using putative antiviral compounds selected on the basis of their proven efficacy in the treatment of other RNA viruses, we identified a subset of compounds with potent anti-FIPV activity and characterized their in vitro safety and efficacy profiles. Based on the great success of combination antiretroviral therapy (cART) against HIV-1 and combination treatment of hepatitis C virus [29], we have developed methods to identify effective combination therapies against FIPV. Initial monotherapies against HIV-1, such as azidothymidine (AZT), often led to viral escape mutations. The concomitant use of multiple antiviral compounds appears to block this adaptive viral evolutionary mechanism, as the development of HIV-1 is effectively arrested by modern cARTs [30]. The success of cART is the result of a pharmacological focus on multiple stages of the virus life cycle simultaneously, while achieving a synergistic antiviral effect [31].

Given the impressive success of cART, it could appear that the current targeting of FIPV at different stages of the viral life cycle by combined anti-coronavirus therapy (cACT) may offer a higher level of lasting and more complete success, compared to the monotherapies themselves. The inclusion of an antiviral agent in cACT capable of penetrating the blood-brain (BBB) ​​and blood-eye barriers, and reaching pharmacologically relevant tissue concentrations, may facilitate the eradication of FIPV throughout the system. We describe a set of in vitro assays that facilitate rapid screening and identification of effective anti-coronavirus compounds. Active antiviral agents with different mechanisms of action and presumed distribution in the body were combined into cACT and tested for compound synergy. We hypothesized that the combined use of two or more effective antiviral monotherapies with different mechanisms of action would facilitate the identification of synergistic combinations providing excellent anticoronavirus efficacy compared to their use alone. Identification of successful cACT may also provide guidelines for the treatment of other emerging viral diseases, such as SARS-CoV-2.

The results

Compound testing

To identify compounds with anti-FIPV activity, a group of 89 compounds were tested in vitro (Additional table 1) from different classes of drugs and with different presumed mechanisms of action. Test compounds included nucleoside polymerase (NPI) inhibitors, non-nucleoside polymerase inhibitors (NNPIs), protease inhibitors (PIs), NS5A inhibitors, a set of novel anti-helicase chemical "fragments", and a set of compounds with unspecified mechanisms of action. Of this group of 89 compounds, a total of 25 different compounds were shown to have antiviral activity against FIPV, including NPI, PI, NS5A inhibitors, and two compounds with unspecified mechanisms of action (referred to as "others", Figure 1). These successful antivirals included toremifene citrate, daclatasvir, elbasvir, lopinavir, ritonavir, nelfinavir mesilate, K777 / K11777, grazoprevir, amodiaquin, EIDD 1931, EIDD 2801 and GS-441524 from three different Chinese manufacturers (Table 1). We tested several nucleoside analog compounds provided by Gilead Sciences structurally related to nucleoside analogs GS-441524 and Remdesivir for their antiviral properties and found several with potential (contained in the 25 identified compounds above), but did not follow these substances further. Thirteen antiviral agents were selected for further analysis. This total includes the previously identified 3-C protease inhibitor, GC-376 (Anivive).

Name of the compoundDrug categoryEC50 (µM)
GC376PI0.04
EIDD 1931NPI0.09
ElbasvirNS5A Inhibitor0.16
EIDD 2801NPI0.4
GS-441524+NPI0.66
K777 / K11777PI0.67
Toremifene citrateOther*5
AmodiaquineOther**6.5
LopinavirPI8.08
RitonavirPI8.7
GrazoprevirPI12.13
Nelfinavir mesylatePI13.47
Table 1.
EC50 of compounds with anti-FIPV activity.
PI = Protease inhibitor; NPI = Nucleoside polymerase inhibitor
+ MedChem Express, HY-103586
* Selective estrogen receptor modulator
** 4-Aminoquinoline
Figure 1
Test compounds by mechanism of action
(A) All test compounds
(B) Compounds found to have anti-FIPV activity in in vitro assays
Figure 2
An example of a test matrix using crystal violet staining to identify anti-FIPV activity at 10μM.
The upper left row are control wells with CRFK cells only and without drug or FIPV. The upper right row is a positive control using GS-441524 with the known complete protection of CRFK cells from FIPV-induced cell death. The entire bottom row of wells represents CRFK cells infected with FIPV and without treatment. The remaining rows are test wells, with the left half evaluating cytotoxicity at 10μM (no FIPV infection) and the right half evaluating anti-FIPV activity at 10μM for any given compound. Loss of staining indicates loss of cells. Daklatasvir and velpatasvir demonstrated anti-FIPV activity, as evidenced by increased crystal violet staining (relatively intact cell monolayers) compared to control wells containing only FIPV (bottom row of the plate). Drugs 32, 33 and 34 showed absent to minimal antiviral activity, while drug 34 (Ravidasvir) also showed cytotoxicity at 10 μM based on the dramatic well cleaning observed in the left half of the FIPV-free matrix.

Determination of antiviral activity

Antiviral activity (EC50) was determined for 10 antiviral compounds. For these compounds, the EC50 ranged from 0.04μM to 13.47μM (Table 1, Figure 3). One of the antiviral agents, Daclatasvir, showed unacceptable cytotoxicity at 20 μM and was excluded from further testing. GS-441524 originating in China (MedChemExpress, HY-103586) was shown to have a comparable EC50 compared to previously published values for GS-441524 originating from Gilead Sciences [25].

Figure 3
Representative examples of non-linear EC50 regression assays for compounds with anti-FIPV activity.

Serial dilutions of each compound with anti-FIPV activity were performed to identify half-maximal effective concentration (EC50). The GS-441524 results reported herein represent a compound derived from MedChemExpress.

Cytotoxicity safety profiles

Cytotoxicity safety profiles (CSPs) were determined for ten different antiviral compounds in CRFK cells. At 5 μM, the seven test compounds showed essentially no cytotoxicity, while two of the antivirals, amodiaquine and toremifene, had 11 and 12% cytotoxicity, respectively (Fig. 4; Table 2). The 50% cytotoxic concentration (CC50) for GC376 is reported as> 150μM [32]. Interestingly, based on the Promega CellTox-Green Cytotoxicity assay, the cytotoxicity of both EIDD compounds was essentially undetectable up to 100μM. However, visual inspection of the EIDD wells just prior to fluorescent dye application and matrix reading revealed differences in cell morphology (cytopathic effect) between untreated CRFK cells and treated cells. Untreated CRFK cells showed adherent spindle morphology in a single monolayer, while EIDD wells showed a marked decrease in confluence compared to variable cell morphology, including cell rounding (cytopathic effect). The discrepancy between the subjective visual evaluation of the EIDD wells and the fluorescence assay is a mystery. It is possible that an overall reduction in the number of cells in the EIDD wells led to the loss and degradation of the nucleic acid necessary for fluorescent binding and detection in the CellTox assay.

CompoundDrug category5μM10μM25μM50μM100μM
ElbasvirNS5A Inhibitor0.670.461.424.9
K777 / K11777PI0.610.292.3916
Toremifene citrateOther *1222233539
AmodiaquineOther **1112192326
LopinavirPI0.6715131618
RitonavirPI0.490.8423326
GrazoprevirPI0.5500.813.919
EIDD1931NPI1.20.940.60.782.8
EIDD2801NPI00.950.631.70.8
GS-441524+NPI0.210000
Table 2
Percent cytotoxicity by compound and concentration.
PI = Protease inhibitor; NPI = Nucleoside polymerase inhibitor
* Selective estrogen receptor modulator
** 4-Aminoquinoline
+ NMPharmTech
Figure 4
Representative cytotoxicity profiles.
Bar graphs of percent cytotoxicity +/- standard deviation (SD) for four compounds with anti-FIPV activity. Percent cytotoxicity values were determined by normalizing cytotoxicity for control wells with positive toxicity (set to 100% cytotoxicity) and untreated CRFK cells (set to 0% baseline cytotoxicity).

Quantification of inhibition of viral RNA production in monotherapy

A real-time RT PCR assay was used to measure the ability of each antiviral agent to inhibit coronavirus replication in monotherapy (Viral RNA knock-down assay). The compounds demonstrating the greatest inhibition of FIPV RNA production were GC376, a 3C-like coronavirus protease inhibitor, GS-441524, EIDD-1931 and EIDD-2801, the last three being nucleoside analogs (Fig. 5, Table 3). Substances with the least inhibitory effect on viral RNA production include elbasvir, nelfinavir and ritonavir. Ritonavir, a protease inhibitor, is used in combination with lopinavir to treat HIV-1 infection (Kaletra, AbbVie). Lopinavir monotherapy has unsatisfactory oral bioavailability in humans, but when used in combination, ritonavir has been shown to significantly improve lopinavir plasma concentrations [33]. Therefore, despite the relatively minimal inhibition of FIPV identified with ritonavir as monotherapy, this compound has been further tested, including combined anti-cancer evaluation.

CompoundVirus titer reduction fold
GC376 (20μM)25000
GC3767300
GS-441524 (NMPharmTech)5280
EIDD-19313700
GS-441524 (MedChemExpress)3500
EIDD-28012110
Lopinavir309
Toremifene10
K7777
Grazoprevir5
Amodiaquine4
Elbasvir2
Nelfinavir mesylate1
Ritonavir1
Table 3
Multiple reduction in viral RNA copy number for anti-FIPV compounds on monotherapy
* Unless otherwise indicated, all compounds were used at 10 μM.
Figure 5
Multiple reduction in FIPV RNA copy number using antiviral compounds as monotherapy.

FIPV-infected CRFK cells were incubated for 24 hours with compounds with detected anti-FIPV activity. The viral copy number was then determined by RT-qPCR and normalized to feline GAPDH copy number to determine the fold reduction effect for each compound. All compounds were tested at 10μM unless otherwise noted. All experimental treatments were performed in triplicate wells, and the fold decrease was calculated by dividing the average experimental normalized FIPV copy number by the average normalized FIPV copy number determined for untreated FIPV-infected wells.
1GS-441524 - NMPharmTech (China).
2GS-441524 - MedChemExpress (China).

Quantification of inhibition of viral RNA production in cACT

To identify drug combinations with synergistic antiviral activity versus monotherapy, combinations of two or more compounds were selected based on (i) established combinations used in other viral infections such as HIV-1 and HCV, (ii) drugs with different mechanisms of action, (iii) potential changes in the systemic distribution of the compound (eg ability to cross the blood-brain or blood-eye barrier according to chemical classification) and (iv) minimal cytotoxicity (based on CSP). For each cACT, any resulting reduction in FIPV copy number in excess of the calculated additive effect for each drug used in the monotherapy regimen was considered synergistic (Table 4). The combination of GC376 and amodiaquin achieved the greatest synergistic effect with the highest overall fold reduction in viral RNA with a 76-fold reduction in viral RNA compared to the additive effect (Fig. 6). This particular synergistic combination was one of the surprising results, given that amodiaquin alone showed only limited inhibition of FIPV viral RNA copies as determined by qRT-PCR.

Compound 1Compound 2Compound 3Virus titer reductionAddcACT / add
GC376 (20 μM)Amodiaquine18970002500476
GC376 (20 μM)AmodiaquineToremifene2560002501410
GC376 (20 μM)K7772480002500710
GC376 (20 μM)Toremifene128000250105.1
GC376 (20 μM)Nelfinavir mesylate91100250013.6
Elbasvir (5 μM)Lopinavir1400031145
Elbasvir (5 μM)GC376 (20 μM)12600250020.50
GC376 (10 μM)Amodiaquine1170073041.6
GC376 (10 μM)Grazoprevir829073051.1
GC376 (20 μM)GS-441524 (NMPharmTech)8260302800.27
GC376 (10 μM)AmodiaquineElbasvir (5 μM)757073061.0
GC376 (10 μM)GS-441524 (MedChem)6910108000.64
GC376 (20 μM)Ritonavir6560250010.26
K777Lopinavir553031618
GC376 (10 μM)GS-441524 (NMPharmTech)4340125800.34
GC376 (20 μM)Lopinavir3400253090.13
LopinavirRitonavir313031010
LopinavirRitonavirToremifene17403205.4
GC376 (10 μM)EIDD-280125594100.03
K777Toremifene25171.5
Table 4
Multiple reduction in FIPV viral RNA copy number in combination therapy (cACT).
The expected additive effect reflects the sum of the fold reductions in viral RNA of each compound used in monotherapy (Table 3).
* Unless otherwise indicated, all compounds were used at 10μM.
cACT / add - ratio of FIPV titer reduction in combination therapy versus the sum of fold reduction titers in monotherapy
Add - the sum of fold reduction titers in monotherapy
Figure 6
Selected examples of fold FIPV RNA copy reduction using combination therapy (cACT).
The bars represent the mean fold decrease in three wells of treated CRFK cells compared to the mean fold decrease in three untreated wells infected with FIPV. All compounds were tested at 10μM unless otherwise noted.

Due to the strong anti-FIPV activity of GC-376, as well as its potential availability for advances in in vivo pharmacokinetic studies, clinical trials, and promising use in cACT, this compound was selected for a series of "viral RNA knock-down" assays in mono and combination therapy (Fig. 7). Overall, GC376 demonstrated excellent anti-FIPV activity at 20 μM in both monotherapy and in vitro combination therapy. The most significant reduction in FIPV RNA occurred in combination with GC376 at 20μM with amodiaquine at 10μM. The experiment combining GC376 with amodiaquine was repeated and both results are shown for comparison in FIG. 7C.

Figure 7
GC376 antiviral activity in monotherapy and in combination therapy at 10 and 20 μM

(A) FIPV RNA reduction quantified by RT-qPCR using GC376 as monotherapy at 10 and 20 μM. There is a significant difference between the two concentrations, with 20μM being better than 10μM. (unpaired t-test; p <0.0001).
(B) Combination in vitro therapy using GC376 at 10 μM.
(C) Combination in vitro anti-FIPV therapy using GC376 at 20 μM.

Discussion

Because there is currently no effective vaccine against FIP, there is a strong clinical and worldwide need for effective antiviral treatment options for FIPV-infected cats. We tested 89 compounds, which resulted in the identification of 25 antiviral agents with antiviral activity and strong safety profiles against feline coronavirus, FIPV. We also identified combinations of antiviral agents (cACT) that resulted in greater efficacy or synergism over monotherapy alone. Of particular interest was the finding regarding the use of elbasvirus, which repeatedly demonstrated excellent protection of CRFK against CPE-induced FIPV at concentrations below 1 μM based on multiple assays (EC50 0.16 μM). In principle, however, no difference in viral RNA copy number was found between infected cells treated with or without elbasvirus. Further visual analysis of FIPV-infected CRFK cells treated with elbasvirus revealed an atypical cell morphology relative to uninfected cells, which was characterized by variable enlargement, cell rounding, and partial cell detachment (cytopathic effect). These "atypical cells" were rarely detached from the culture plate, and as a result, absorbance values were comparable to uninfected control wells. This dichotomous result between platelet analysis and viral RNA knock-down assay suggests that the antiviral effect of elbasvir occurs after viral replication and, as a result, elbasvir may not protect cells from viral RNA accumulation. Elbasvir is used to treat patients infected with hepatitis C virus (HCV) and is thought to target the HCV NS5A protein, which prevents replication and also to complete virions [34]. Although no NS5A homolog has been identified for FIPV, it is possible that elbasvir exhibits a similar antiviral effect by preventing the assembly of FIPV virions without blocking viral RNA synthesis in CRFK cells. Additional evaluation of treated FIPV-infected CRFK cells by transmission electron microscopy may shed light on the effect of elbasvirus on protecting CRFK from FIPV-related damage and death.

Co-administration of ritonavir with lopinavir has been shown to significantly increase lopinavir plasma concentrations in rats, dogs and humans [33]. Ritonavir is a potent inhibitor of CYP3A, which is the primary enzyme responsible for protease inhibitor metabolism, and therefore its co-administration with other protease inhibitors results in increased systemic concentrations of co-administered protease inhibitors such as lopinavir [38, 39]. The increase in the antiviral effect of lopinavir associated with ritonavir was relatively minimal in the viral RNA elimination assays with only a 10-fold inhibition of FIPV over the additive effect. This may be the result of an in vitro testing artifact on a feline kidney cell line (ie CRFK cells) that lacks the enzyme CYP3, an enzyme that typically occurs at sites of high protease inhibitor metabolism at the first pass effect (ie enterocytes). and hepatocytes) [39]. These results suggest that in vitro tests alone may not fully predict the effect of antiviral agents in FIPV-infected cats in vivo.

Grazoprevir, a serine protease inhibitor NS3 / 4, has been used in combination with elbasvirus, an NS5A inhibitor, to treat HCV-infected patients (Zepatier, Merck) [40]. Here, we demonstrated that grazoprevir has anti-FIPV activity when used as monotherapy. The cysteine protease inhibitor K777 / K11777 has been investigated for its ability to block coronavirus (MERS-CoV and SARS-CoV-1) and ebolavirus entry and has been found to completely inhibit coronavirus infection, but only in target cell lines without virus-activating serine proteases. [41]. For other cell lines, K777 inhibited coronavirus cell entry in combination with a serine protease inhibitor [41]. It is possible that limited inhibition of FIPV RNA K777 production could be increased if combined with a serine protease inhibitor.

Amodiaquine is an antimalarial drug and belongs to the class of 4-aminoquinoline drugs. Amodiaquine, along with related 4-aminoquinolines such as chloroquine and hydroxychloroquine, was originally developed to treat malaria [42], and like chloroquine and hydroxychloroquine, it has a wide range of anatomical distributions, including the eyes and brain [43-49]. Penetration of antiviral agents into the CNS and / or ocular compartments is particularly important in FIPV-infected cats with neurological and / or ocular disorders. While several studies have defined the antiviral properties of chloroquine and hydroxychloroquine [28, 50, 51], the antiviral activity of amodiaquin has also been investigated with the identification of antiviral activity against dengue virus, Ebola virus and severe fever with viral thrombocytopenia syndrome (SFTS) [52–55]. The mechanism of action of amodiaquine may involve an increase in cytoplasmic lysosomal and / or endosomal pH, which prevents the release of viable virions into the cytoplasm [56]. Due to its unique drug class status and presumed ability to cross the blood-brain barrier [57], amodiaquine is a promising candidate for the combined treatment of neurological and / or ocular forms of FIP.

Toremifene citrate, a selective estrogen receptor modulator (SERM), is used to treat metastatic breast cancer in human patients. Recently, toremifene has been evaluated for its antiviral properties and has demonstrated anticorrosive activity against zoonotic coronaviruses, Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1 coronaviruses [58]. Toremifene has also been shown to be active against Ebola virus (EBOV) [59, 60]. Although the exact mechanism of antiviral action is not defined, the antiviral effect of toremifene against EBOV appears to be to destabilize the EBOV glycoprotein [59].

Interestingly, GC376 demonstrated confusing differences between 10 μM and 20 μM in combination therapy. When used at 10 μM with other compounds, synergism and in some cases a decrease in antiviral effect compared to additive values ranging from 0.03 to 1.6 were absent (Table 4). When used at 20 μM, there were still cases where combination with another compound resulted in a reduced antiviral effect compared to GC376 used as monotherapy at 20 μM. However, many more variations in the 20 μM combinations showed a fold effect compared to additive values ranging from 0.13 to 76 (Table 4). A specific example is the contrast between GC376 at 20 μM compared to GC376 at 10 μM combined with amodiaquine at 10 μM. The first caused the greatest inhibition of viral RNA as well as the greatest multiple of the additive (synergistic) effect, while the second caused almost a loss of synergism with a value of the additive multiple of 1.6.

The identification of effective antiviral strategies for the treatment of FIPV-infected cats has translational implications for the ongoing SARS-CoV-2 pandemic. FIPV infection in cats resembles coronavirus infection in ferrets [61, 62] and is compared with the pathogenesis of other chronic macrophage-dependent diseases such as tuberculosis [63]. Because the clinical and pathogenic details of SARS-CoV-2 infection in humans are still emerging, there appears to be some overlap with FIPV in anatomical distribution, clinical manifestations, and likely response to certain antiviral therapies. In cats, the feline enteric coronavirus biotype (FECV) is restricted to the gastrointestinal tract due to enterocyte tropism. Clinical symptoms in FECV-infected cats range from mild gastrointestinal disease (diarrhea) to the absence of symptoms. The mutated FIPV coronavirus feline biotype acquires macrophage tropism and preferentially targets serous abdominal and thoracic surfaces with a subset of cats that demonstrate CNS or eye involvement [7]. Similarly, in patients with COVID-19, there are reports of diarrhea and a subset of patients with CNS involvement [14]. Although the cellular receptor for SARS-CoV-2 has been identified as ACE2 [64], the cellular receptor for FIPV serotype I has yet to be determined. The cellular receptor for less clinically relevant FIPV serotype II has been identified as feline aminopeptidase peptidase (fAPN) [4]. A study using RNAseq to evaluate gene expression profiles of ascites cells obtained from cats with FIP did not identify ACE2 expression, suggesting that ACE2 is unlikely to be a FIPV serotype I receptor [63]. A more detailed examination of the identity of the FIPV serotype I receptor is needed.

Clinical successes with GS-441524 or GC-376 in cats with experimental and naturally occurring FIPs indicate that FIP can be effectively treated, but treatment of dry (granulomatous), neurological and ocular FIPs remains a challenge. The protease inhibitor 3C-like protease, GC-376, appears to be relatively effective in the treatment of FIPV effusion infection limited to body cavities, but may be less effective in the treatment of neurological or ocular forms of the disease [24]. These different results may be the result of ineffective penetration of the blood-brain and blood-eye barriers, making GC-376 a promising candidate for combination therapy with a CNS-penetrating antiviral drug.

Materials and methods

FIPV inoculation for in vitro experiments

Crandell-Reese cat kidney cells (CRFK, ATCC) were cultured in T150 flasks (Corning), seeded with FIPV serotype II (WSU-79-1146, GenBank DQ010921) and propagated in 50 ml of Dulbecco's modified Eagle's medium (DMEM) with 4 , 5 g / l glucose (Corning) and 10% fetal bovine serum (Gemini Biotec). After 72 hours of incubation at 37 ° C, extensive cytopathic effect (CPE) and large cell clearing / separation areas were noted. The flasks were then flash frozen at -70 ° C for 8 minutes, thawed briefly at room temperature, and the cells and supernatant were then centrifuged at 1500 g for 5 minutes, followed by a second centrifugation step at 4000 g for 5 minutes to isolate cell - free viral volumes. The supernatant containing the virus base was divided into 0.5 and 1.0 ml aliquots in 1.5 ml cryotubes (Nalgene) and archived at -70 ° C. After freezing, one tube was allowed to thaw and the virus titer was determined using biological assays (TCID50) and real-time RT PCR methods (below).

The tissue culture dose-50 infectious dose (TCID50) was determined using a viral plaque assay. CRFK cells were grown in a 96-well tissue culture plate (Genesee Scientific) until CRFK cells reached approximately 75-85% confluence. Serial 10-fold dilutions were prepared from FIPV stock solution and 200 μl samples from each dilution were added to 10-well replicates. 72 hours after infection, cells were fixed with methanol and stained with crystal violet (Sigma-Aldrich). Individual wells were visually evaluated for virus-induced CPE, evaluated as CPE positive or negative, and TCID50 was determined based on the log equation.10TCID50 = [total number of # wells CPE positive / # replicates] + 0.5 to reflect infectious virions per milliliter of supernatant [68].

Quantification of FIPV by qRT-PCR

Cell-free viral RNA was isolated from the parent virus using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The isolated RNA was treated with DNase (Turbo DNase, Invitrogen) and subsequently reverse transcribed using a High-Capacity RNA-to-CDNA kit (Applied Biosystems) according to the manufacturers' protocols. Copy number of FIPV and feline GAPDH cDNA was determined using an Applied Biosystems' QuantStudio 3 Real-Time PCR System and a PowerUp SYBR Green Master Mix according to the manufacturer's protocol for a 10 μL reaction. Each PCR reaction was performed in triplicate with an aqueous template as a negative control and plasmid DNA as a positive control. A reverse transcriptase-secreting control reaction was included in each set of real-time PCR assays. The cDNA templates were amplified using the FIPV forward primer, 5'-GGAAGTTTAGATTTGATTTGGCAATGCTAG and the FIP reverse primer, 5'-AACAATCACTAGATCCAGACGTTAGCT (terminal part of the FIPV 7b gene) [25]. Real-time PCR for the feline domestic GAPDH gene was performed simultaneously using primers, 5 GAPDH, 5'-AAATTCCACGGCACAGTCAAG and 3 GAPDH, 5'-TGATGGGCTTTCCATTGATGA. The cycling conditions for both FIPV and GAPDH amplicons were as follows: 50 ° C for 2 minutes, 95 ° C for 2 minutes, followed by 40 cycles of 95 ° C for 15s, 58 ° C for 30s, 72 ° C for 1 minute. The last step included a dissociation curve to evaluate the specificity of the primer binding. The copy number of FIPV and GAPDH was calculated on the basis of standard curves generated in our laboratory. Copies of FIPV cIPNA determined by real-time RT PCR were normalized to 106 copies of feline GAPDH cDNA.

Development of anti-helicase chemical fragments

The drugs studied and described in this study were already known antiviral agents. In contrast, the helicase enzyme FIPV was cloned, expressed and used as a target for coronavirus and enzyme-specific viral discovery. The AviTag-FIP Helicase-HisTag target DNA sequence was optimized and synthesized. The synthesized sequence was cloned (Adeyemi Adedeji) into the Avi-His tagged pET30a vector to express the protein in E. coli. E. coli strain BL21 (DE3) was transformed with a recombinant plasmid. One colony was inoculated into 1 liter of auto-induced medium containing the antibiotic and the culture was incubated at 37 ° C at 200 rpm.

When the OD600 reached about 3, the cell culture temperature was changed to 15 ° C for 16 hours. Cells were harvested by centrifugation. The cell pellets were resuspended in lysis buffer followed by sonication. The centrifuge precipitate was dissolved with a denaturing agent. The target protein was obtained by one-step purification on a Ni column. The target protein was sterilized with a 0.22 μm filter. The yield was 7.2 mg at 0.90 mg / ml and was stored in PBS, 10% glycerol, 0.5 mM L-arginine, pH 7.4. The concentration was determined by the Bradford protein assay with BSA as a standard. Protein purity and molecular weight were determined by SDS-PAGE with Western blot confirmation.

Surface plasmon resonance (SPR) fragments were screened on a ForteBio Pioneer FE SPR platform. A HisCap sensor chip containing an NTA surface matrix was used. Channels 1 and 3 were filled with 100 μM NiCl 2, followed by injection of 50 μg / ml FIP protein. Channel 2 was left protein-free as well as NiCl 2 as a reference. Channel 1 was immobilized to a density of 88000 RU, while channel 3 contained approximately 12000 RU. Channel 1 was used. The buffer used for immobilization was 10 mM HEPES, pH 7.4, 150 mM NaCl and 0.1% Tween-20. DMSO was added to a final concentration of 4% for this assay. The proprietary compound library was diluted in the same DMSO-free buffer to a final DMSO concentration of 4% DMSO. Compounds from the library were tested at a concentration of 100 μM using the OneStep gradient injection method. The findings were selected based on RU and kinetics and used for cell screening.

Viral plaque test

To screen for antiviral activity of compounds, infected CRFK cells were treated with compounds in six-well replicates and compared to positive control wells (infected cells), negative controls (uninfected cells) and treatment controls (infected cells treated with a known active antiviral compound) simultaneously on each tissue plate. culture. CRFK cells were grown in 96-well tissue culture plates (Genesee Scientific) containing 200 μl of culture medium. At 7575-85% cell confluence, the medium in uninfected control wells was aspirated and replaced with 200 μl of fresh medium. The medium in the infected wells was aspirated and replaced with FIPV inoculated medium at a multiplicity of infection (MOI) of 0.004 infectious virion per cell. The tissue culture plate was incubated for 1 hour with periodic gentle agitation ("number eight" manipulations) every 15 minutes to facilitate virus-cell interaction. One hour after infection, each putative antiviral compound was added to six wells infected with FIPV (to determine the antiviral activity of the compound) and six uninfected control wells (to screen for cytotoxicity of the compound in CRFK cells). All compounds were initially screened at 10 μM, except for the "chemical fragment" compounds supplied by M. Olsen (Midwestern University), which were evaluated at 50 μM. Tissue culture plates were incubated at 37 ° C for 72 hours and then fixed with methanol and stained with crystal violet. Plates were scanned for absorbance at 620 nm using an ELISA plate reader (FilterMax F3, Molecular Devices; Softmax Pro, Molecular Devices). For each treatment condition, individual well absorbance values ​​were recorded along with the mean absorbance value and mean error of the mean for 6-well experimental replicates.

For substances that demonstrated antiviral efficacy at initial screening at 10 or 50μM (protected from CPE-associated virus), the EC50 was determined by performing a series of progressive 2-fold dilutions of the compounds in a viral plaque assay. To determine the EC50, CRFK cells were grown in 96-well tissue culture plates similar to the antiviral screening assay. Except for uninfected control wells, all remaining wells were infected with FIPV as described above. A two-fold dilution series ranged from 20μM to 0μM and each concentration was performed in six well replicates. The number of dilution steps ranged from 6 to 14 and was compound dependent. Six well replicates of uninfected CRFK cells served as a control for normal CRFK cells; six FIPV-infected CRFK cell replicates served as untreated FIPV-infected control wells; and six well replicates of FIPV-infected CRFK cells treated with GS-441524 served as control wells for protection against virus-induced cell death based on published data on the efficacy of using GS-441524 in vitro in CRFK cells [26].

Tissue culture plates were incubated for 72 hours and then fixed with methanol, stained with crystal violet, and the absorbance at 620 nm was scanned using an ELISA plate reader. Individual absorbance values along with the mean absorbance value and standard deviation for 6-well experimental replicates were recorded for each treatment condition. The EC50 was calculated by plotting a non-linear regression equation (dose-response curve) using Prism 8 software (GraphPad).

Viral RNA knock-down test

Real-time RT-PCR assays were used to quantify inhibition of viral RNA production by the compound. CRFK cells were cultured in a 6-well tissue culture plate (Genesee Biotek). At approximately 75-85% cell fusion, the culture medium was replaced with fresh medium and the cells were infected with FIPV serotype II at an MOI of 0.2 (MOI based on TCID50 bioassay / pfu). The plates were incubated for one hour with periodic gentle shaking every 15 minutes. Wells infected with FIPV were treated with one (monotherapy), two or three (combined anti-cancer therapy) antiviral compounds; each experimental treatment was performed three times. The compound dose was based on the EC50 of the compounds and ranged from 0.001 to 20 μM. For each experimental set, three culture wells with FIPV-infected and untreated CRFK cells served as virus-infected controls. Infected cell cultures were then incubated for 24 hours and total RNA associated with the cells was isolated using a PureLink-RNA mini kit (Invitrogen). RNA was treated with DNAse (TurboDNAse, Ambion), reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), and FIPV cIPNA and feline GAPDH cDNA were measured by real-time qRT-PCR as described above. . The fold reduction in viral titer was determined by dividing the normalized mean FIPV RNA copy number for untreated FIPV-infected CRFK cells into the normalized mean FIPV RNA copy number for CRFK treated cells with the desired compound (s). The expected additive effect was determined by adding a fold reduction for each monotherapy used in combination. The composite additive effect was determined by dividing the predicted additive effect by the combined multiple reduction value for a particular combination therapy.

Determination of cytotoxicity safety profiles (CSP).

The cytotoxicity of the compound in feline cells was assessed using a commercially available kit (CellTox Green Cytotoxicity Assay, Promega) according to the manufacturer's instructions. Untreated CRFK cells were used as negative controls and the cells were treated with a cytotoxic solution provided by the manufacturer as positive toxicity controls. Briefly, in addition to control wells, CRFK cells were plated in 96-well tissue culture plates (Genesee Scientific) in four well replicates with 5, 10, 25, 50 or 100 μM concentrations of the desired compound and incubated for 72 hours. After 72 hours, all wells were stained with the DNA kit, incubated at 37 ° C protected from light for 15 minutes, and the fluorescence intensity at 485-500 nm Ex / 520-530 nm EM was subsequently determined using a plate reader (FilterMax F3, Molecular Devices; Softmax Pro, Molecular Devices). The cytotoxicity of a compound at a particular concentration was thought to be proportional to the fluorescence intensity based on the selective penetration and binding of the dye to the DNA of degenerated, apoptotic or necrotic cells. The extent of cytotoxicity was determined by adjusting the fluorescence value for cells treated with the positive control reagent to 100% and untreated feline cells as 0% cytotoxicity. The average fluorescence value for the four wells containing each compound concentration was then interpolated as a percentage (percent cytotoxicity) ranging from 0 to 100%.

Conflict of interests

The authors declare that there has been no conflict of interest.

We appreciate funding provided by the Winn Feline Foundation (MTW 17-020; MTW 19-026) and the University of California, Davis, Center for Companion Animal Health (CCAH; 2018-92-F; 2018-94-FE) through multi-FIP research donations individual donors and organizations (SOCK FIP, Davis, CA) and foundations (Philip Raskin Fund, Kansas City, KS).

Additional information

Complete list of compounds tested in vitro for FIPV activity

Nucleoside polymerase inhibitors

12x GS Nuc AnalogsNucleoside analogNPI
GS-441524 (China-sourced)Adenosine analog nucleosideNPI
3-Deazaneplanocin A HydrochlorideAdenosine analog nucleosideNPI
AdefovirAdenosine analog nucleosideNPI
GalidesivirAdenosine analog nucleosideNPI
GS-441524 (Manufactured in China)Adenosine analog nucleosideNPI
MK-0608Adenosine analog nucleosideNPI
NITD008Adenosine analog nucleosideNPI
DidanosineAdenosine analog nucleosideNPI
Tenofovir alafenamideAdenosine analog nucleosideNPI
Tenofovir disoproxil fumarateAdenosine analog nucleosideNPI
EIDD 1931Nucleoside analog Cytidine 
EIDD 2801Nucleoside analog Cytidine 
2′-C-methylcytidineNucleoside analog CytidineNPI
Gemcitabine HydrochlorideNucleoside analog CytidineNPI
2-C-methylguanosineNucleoside analogue GuanosineNPI
7-methylguanosineNucleoside analogue GuanosineNPI
EntecavirNucleoside analogue GuanosineNPI
MizoribineNucleoside analogue GuanosineNPI
RibavirinNucleoside analogue GuanosineNPI
PSI-6206Nucleoside analog UridineNPI
6-AzauridineNucleoside analog UridineNPI
BalapiravirNucleoside analog CytidineNPI
SofosbuvirNucleoside analog UridineNPI
FavipiravirNucleoside analog PurineNPI
Total 36

Protease inhibitors

GrazoprevirNS3 / 4A protease inhibitorPI
Rupin trivirRhinoviral 3CP inhibPI
LopinavirAntiretroviral PIPI
RitonavirAntiretroviral PIPI
NelfinavirAntiretroviral PIPI
Disulfiram (tetraethyliuram disulfide)Papain-like protease inhibPI
K777 / K11777Cysteine protease inhibitorPI
TelaprevirNS3 / 4A protease inhibitorPI
Camostat mesylateSerine protease inhibitorPI
ParitaprevirSerine protease inhibitorPI
GC376Coronavirus protease inhibitorPI
Total 11

NS5A inhibitors

VelpatasvirNS5A InhibitorNS5A Inhibitor
Ravidasvir / PPI-668NS5A InhibitorNS5A Inhibitor
LedipasvirNS5A InhibitorNS5A Inhibitor
OmbitasvirNS5A InhibitorNS5A Inhibitor
PibrentasvirNS5A InhibitorNS5A Inhibitor
DaclatasvirNS5A InhibitorNS5A Inhibitor
ElbasvirNS5A InhibitorNS5A Inhibitor
Total 7

NNPI

DasabuvirNon-nucleoside polymerase inhibitorNNPI
Total 1

other

MonensinIonophoreother
Phenazopyridine hydrochlorideCrystalline solidother
Pyrvinium pamoate hydrateAndrogen receptor inhibitorother
Toremifene citrateSelective estrogen receptor modulator other
AM580Retinobenzoic derivativeother
HomoharringtonineTranslation elongation inhibother
Amodiaquine4-aminoquinoloneother
Total 7

Midwestern Chemical Fragments

F0472-0017Midwestern
F6190-0257Midwestern
F6279-0675Midwestern
F2167-1080Midwestern
F6190-0740Midwestern
F6438-2155Midwestern
F3411-5663Midwestern
F6233-0011Midwestern
F9995-2543Midwestern
F2124-0890Midwestern
F2711-2577Midwestern
F2130-0055Midwestern
F2493-3358Midwestern
F2459-0974Midwestern
F2124-0465Midwestern
F1899-2269Midwestern
F2185-1982Midwestern
F2189-0717Midwestern
F2147-0158Midwestern
F1371-0192Midwestern
F2156-0057Midwestern
F9995-2431Midwestern
F3349-0218Midwestern
F2156-0059Midwestern
F2156-0070Midwestern
F5856-0194Midwestern
F2147-0975Midwestern
Total 27

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Read "A rational approach to the identification of effective combined anti-coronavirus therapies against feline coronavirus"

The Neurological Form of Feline Infectious Peritonitis and GS-441524 treatment

1.6.2021
Niels C. Pedersen, DVM PhD
Original article: The Neurological Form of Feline Infectious Peritonitis and GS-441524 treatment

Introduction

Neurological impairment occurs in about 5–10% cases of FIP. This may vary from region to region, as the author's experience suggests that Turkish street cats are more prevalent. The age of onset is similar to other forms of FIP, with most cases occurring within 3 years.

Neurological FIP is considered a form of dry FIP, and typical dry FIP lesions in the abdomen, chest, or eyes occur in about half of the neurological cases of FIP. Neurological symptoms are only visible in about 5% cats with manifestations of wet FIP.1 However, in cats, there was a significant increase in the incidence of neurological FIP either during treatment with GS-441524 or in the form of post-treatment relapse periods in cats that were originally treated for non-neurological FIP.

Clinical signs

Neurological FIP occurs in two forms, primary and secondary. Abnormal neurological symptoms are present in cats with primary disease. However, general symptoms of ill health are also common, such as failure to thrive, weight loss, lethargy and anorexia. The fever may be overt or covert. About half of cats with primary neurological FIP will also have identifiable lesions outside the CNS and typical blood test results. However, cats with no obvious signs of CNS damage will often have normal or near-normal levels on the CBC and in their serum.

Early neurological symptoms, recognizable prospectively or retrospectively, include licking the floor or walls, sporadic muscle twitching, and indeterminate behavior and cognitive abnormalities. Anisocoria is another early sign. Suspicion of neurological FIP increases as clinical symptoms become more functional. The earliest sign is usually a gradual loss of coordination and balance (ataxia). The reluctance to jump up or down from high places is one of the first signs of incoordination. Incoordination is initially most noticeable on the hind legs, but quickly becomes general. In some cases, seizures of the grand mal type or psychomotor type may also occur. Grand mal seizures are manifested by a brief loss of consciousness, strong rhythmic muscle cramps affecting the whole body. Psychomotor epilepsy is associated with varying degrees of consciousness and uncontrolled or partially controlled body movements.

Cats with secondary neurological FIP show signs of extra-intestinal disease and CNS involvement occurs at a later stage of the disease. It often occurs during antiviral treatment of other forms of FIP and is a common cause of relapse in cats treated with other forms of FIP. These relapses usually occur within the first 1-4 weeks after successful treatment.

Spinal cord involvement is often overlooked in neurological FIP, although more than 50% cats with inflammatory spinal cord disease have FIP.2 Spinal cord involvement leads to fecal and / or urinary incontinence of varying severity. Paralysis of the tail or hind limbs are also symptoms of spinal cord disease. Spinal cord involvement is likely to lead to permanent neurological deficits and then to brain disease.

Diagnosis

The sudden onset of neurological abnormalities in cats less than 5-7 years of age is strong evidence of FIP on the basis of probability alone, as few other diseases will have similar symptoms in this age group. However, there is a tendency among veterinarians to include systemic toxoplasmosis on their diagnostic list above, especially when ocular or CNS symptoms are observed. Systemic toxoplasmosis in cats is a rare disease compared to FIP and often occurs in immunocompromised hosts, including hosts with FIP. 15-17 This is understandable because cats are the definitive host of Toxoplasma gondii in nature and have developed a state of facultative symbiosis. In addition, the main clinical manifestation of systemic toxoplasmosis is characteristic pneumonia, sometimes associated with hepatitis, pancreatic necrosis, myositis, myocarditis, and dermatitis.3-8 FIP-like uvitis occurs in approximately 10% cats with systemic toxoplasmosis, 4 and encephalitis is even less common.7,17 The diagnostic test for systemic toxoplasmosis is based on a comparison of IgG and IgM antibody titers using the indirect fluorescent antibody (IFA) procedure. 3 High IgG titers in the absence of IgM antibodies indicate previous toxoplasma exposure, which can reach up to 50% in feral cat populations.4 The presence of high titers of IgM antibodies is an indication of the systemic spread of the organism from the intestine to other tissues and is one of the requirements for the diagnosis of systemic disease. However, many cats with ocular and neurological signs are inappropriately treated for systemic toxoplasmosis only on the basis of high IgG titers.

The diagnosis of typical forms of FIP is usually made by combining information on the age and origin of the cat, historical and physical signs (eg ill health, fever, abdominal or thoracic effusions, palpable abdominal mass) with certain laboratory abnormalities in the complete blood count (anemia; high white blood cell count, low lymphocyte count and high neutrophil count), serum biochemical panel (high total protein, high globulin, low albumin and low A: G ratio), effusion tests, if present (exudate or modified exudate, yellow tint) and determining the likelihood that these findings can best be explained by the FIP. A definitive diagnosis can be obtained by identifying coronavirus proteins or RNA in effusions or tissue samples by PCR or immunohistochemistry. However, it is possible that cats that develop neurological FIP during or after treatment with a non-neurological form will lack many or all of these clinical signs.

Diagnosis of neurological FIP, especially in the primary form, is usually made in three ways: 1) consider all historical, clinical, and laboratory findings and estimate the likelihood of FIP, 2) examine the brain for FIP by magnetic resonance imaging (MRI), and / or cerebrospinal fluid (CSF) analysis, 8,9 and 3) treat on the assumption that it is a neurological FIP, and hope for a positive response to antiviral therapy.

Contrast-enhanced magnetic resonance imaging is increasingly being used in the diagnosis of neurological FIP. Dilation (hydrocephalus) of one or more ventricles is a common lesion in the brain.8,9 Similar dilatations in the form of syringomyelia can be observed in the spinal cord. Dilatations are secondary to inflammation of the surrounding ependyma. The ependyma ensures the excretion, circulation and maintenance of CSF homeostasis. Therefore, the severity of FIP secondary obstructive hydrocephalus is proportional to the degree of ependymal inflammation and the associated increase in contrast. Discrete lesions of the parenchyma are not identified. MRI significantly increases the cost of diagnosis, anesthesia increases the risk of death in seriously ill cats, and expertise and equipment are not always available. Therefore, the final diagnosis often falls in response to a specific antiviral treatment. The drug of choice for FIP neurological cases is GS-441524.9,12

CSF analysis is an alternative way to quantify the nature and severity of inflammation in the ependymus and meninges. CSF protein levels and cell numbers are elevated in cats with FIP, and it is often possible to obtain suitable samples for the detection of infected macrophages by IHC or PCR.10,11 CSF analysis is associated with a low risk of anesthesia and needle puncture into the magna tank.

Treatment

Neurological FIP can be cured if a sufficient amount of antiviral drug crosses the blood-brain barrier and the virus does not acquire drug resistance.9,12 Field tests with the GS376 viral protease inhibitor were the first to show that neurological symptoms could be significantly suppressed, but the infection could not be cured. The reason was considered the inability to reach sufficiently high levels of GC376 in the CNS. Greater success in treating cats with neurological FIP has been achieved with the nucleoside analog and viral RNA transcription inhibitor GS-441524.9,12 GS-441524 was shown to enter cerebrospinal fluid (CSF) at concentrations from 7-21% blood, depending on the cat tested. 13 These differences in the blood-brain barrier between cats are likely to explain the variable doses of GS-441524 from 4 to 10 mg / kg per day required for the treatment of naturally occurring cases of neurological FIP.9,12

The current starting dose for GS-441524 was based on recent findings7 set at 10 mg / kg daily by the subcutaneous route. Although it is possible to treat some cats at lower doses, 9,12 There is no easy way to measure the strength of the blood-brain barrier, so use the lowest dosage that will have a healing effect for most cats. Treatment success is measured by both improvement in clinical symptoms and improvement in critical blood test abnormalities. Weight gain and coat quality are also important quality traits that need to be observed. Sequence analyzes of MRI and CSF will provide more direct evidence of response to treatment,9 but in most cases they are impractical.

Improvement in general health and neurological symptoms usually appear within 24-48 hours, and most cats destined for complete recovery will return to normal within 4-6 weeks. However, a significant proportion of cats will respond more slowly and require a reassessment of their clinical condition and blood test status every 4 weeks. Slowing down the course of treatment, either clinically or in the form of a reversal in the initial abnormalities of the blood test, will require an increase in the dose from +2 to +5 mg / kg per day.9,12

Discontinuation of treatment, which is usually after 84 days, is not always easy to confirm. Typical blood test abnormalities used in most other forms of FIP either do not occur at the time of diagnosis or return to normal before treatment is stopped. Persistent neurological abnormalities may persist after the infection has healed, making clinical evaluation difficult. Without magnetic resonance and / or cerebrospinal fluid analysis to confirm that the disease has passed, the only option left is to stop treatment and hope that there will be no relapse.

Complications of neurological FIP

Relapses in cats treated for neurological FIP usually occur within a few days of stopping treatment and are caused by either inappropriate dosing and / or the acquisition of drug resistance. The incidence of relapses appears to be slightly higher than after treatment of forms of FIP without CNS involvement. Underdosing may be the result of a stronger blood-brain barrier in some cats compared to others, a poor quality antiviral drug, or incorrect dose calculation. However, it is common for cats to recover from re-treatment until drug resistance has occurred.

The acquisition of drug resistance is well known in antiviral drugs used in humans for diseases such as HIV / AIDS. It has also been reported with GC37611,14 also GS-441524 in cats.12 Drug resistance can occur by mutations in either the native FECV or its wild-type FIP biotype14, and manifested by an insufficient initial response to treatment, but this is not a common phenomenon.12 Resistance is more likely to occur during treatment and is due to both chronic drug exposure and lower sub-inhibitory drug levels. Drug resistance is usually partial and can often be overcome by increasing the dose. Drug resistance may worsen over time, and further dose increases will have no effect.

Cats with neurological FIP may show residual brain and / or spinal cord damage and permanent consequences after cessation of treatment. Disabilities include varying degrees of incoordination, behavioral changes, and dementia. The most problematic consequences are associated with spinal cord injury. The spinal cord is enclosed in a bone tube that does not allow for large expansion in the event of inflammation or some form of syringomyelia. Spinal cord involvement in FIP is often manifested by varying degrees of fecal and / or urinary incontinence. Paralysis of the hind limbs and tail is also observed, but is less common. Unfortunately, these clinical abnormalities are often permanent, especially if the neurological disease is not treated for a long time.

One of the most common negative antiviral treatment outcomes in cats with neurological FIP is failure to cure, although continuing high-dose treatment still allows for a sustainable quality of life (ie, management of disease symptoms without cure). This situation suggests that inhibition of virus replication by antiviral drugs may not be sufficient to cure the infection, and that an effective immune response is also required. This phenomenon of "treatment without cure" has in many cases led many owners to continue treatment at all costs for more than a year. It has also led to many experiments with ultra-high doses of GS441524 (> 15 mg / kg daily), divided doses, switching from injections to oral therapy, concomitant oral and injectable therapy, combination antiviral therapy (eg GS-441424 plus GC376) and antiviral support. treatment with high doses of corticosteroids and other immunosuppressants. Treatment with such treatments is occasionally required, but the result has been unfavorable for most of these cats.

There is circumstantial evidence that the host's immunity to FIP is shared between the CNS and other parts of the body. The incidence of CNS involvement appears to be increased when GS-441524 inhibits infection outside the CNS. Therefore, active disease outside the CNS appears to have an inhibitory effect on CNS disease. Cats with pure neurological disease often do not show abnormal blood test values on the CBC panel or in the serum, even with significant inflammatory changes in the cerebrospinal fluid.8 Compared to other forms of FIP, cats with neurological FIP often have the highest serum, ie the highest CSF antibody titers.8 These are all evidence of "compartmentalization" of the infection on either side of the blood-brain barrier.

References

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