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 trial data for Molnuparivir in cats with naturally occurring FIP. This information does not clearly state the amount of molnuparivir in one of their “50 mg tablets” and the actual dosing interval (q12h or q24h?). The dose used in this study also seemed too high. Fortunately, an estimated starting dose of molnuparivir in cats with FIP can be obtained from the published studies of 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.
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
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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.
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
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.
Pedersen NC. 2021. The neurological form of FIP and GS-441524 treatment. https://sockfip.org/the-neurological-form-of-fip-and-gs-441524-treatment/
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 /.
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.
Warren, TK et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531, 381–385.
FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
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.
Zdanowicz MM. The pharmacology of HIV drug resistance. Am J Pharm Educ. 2006; 70 (5): 100.doi: 10.5688 / aj7005100
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
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].
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FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
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).
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 formulations 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 the “pill-taking” difficulties some cats may have.
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 dosage of GS-441524 for cats with wet or dry FIP and no neurologic or ocular signs is 4-6 mg/kg SC q24h. The injectable dose for cats with ocular disease is 8 mg/kg SC q24h and for cats with neurological disease is 10 mg/kg SC q24h. If a cat is started on wet FIP and then develops eye disease, the dose is increased immediately to 8 mg/kg SC q24h and if neurological signs develop, the dose is increased to 10 mg/kg SC q24h. Failure to treat FIP at doses greater than 15 mg/kg SC q24h indicates drug resistance. The PO doses corresponding to 4-6, 8, and 10 mg/kg SC q24h are 10, 16, and 20 mg/kg PO q24h. (Note: some oral formulations are labeled SC equivalents but actually contain up to twice the mg GS listed.) The recommended duration of treatment is 12 weeks, with the dose increased as needed. However, it is known that some cats can be cured in 6 weeks with any form of GS-441524, many in 8-10 weeks, and almost all in 12 weeks. Young cats with abdominal wet FIP tend to respond most rapidly, cats with dry FIP more slowly, and cats with neurologic FIP the slowest. Therefore, the “universal” recommendation is to treat any cat with FIP, regardless of form, for a minimum of 12 weeks. The daily PO dose can be divided q12h, which may be advantageous when treating with higher doses to avoid an absorption ceiling. SC and PO treatments 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 well-known brand name for the oral form of GS-441524. It was sold in several different forms, including multiple tablet and capsule forms. In early 2021, Mutian switched to a tablet form, labeled as 200 mg or 50 mg “Mutian” or “Xraphconn” – these deliver an equivalent SC dose of 10 or 2.5 mg of GS-441524, respectively. The tablets are significantly larger (8.5 mm diameter) than tablets from other suppliers. Recently, a new capsule formulation has been available sporadically. As of September 2021, the Mutian website no longer offers a PO option. For all oral forms of Mutian, the supplier lists the dosage as: 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:
Marking
Injectable equivalent
Dosage instructions
Aura 12h-1kg
approx. 2.5 mg / kg
Wet / 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 kg
approx. 5 mg / kg
Wet / 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 kg
approx. 7.5 mg / kg
Wet / 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 kg
approx. 10 mg / kg
Wet / 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.
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
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.
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).
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
de Miranda, P., Krasny, HC, Page, DA, Elion, GB, 1981. The disposition of acyclovir indifferent species. J. Pharmacol. Exp. Ther. 219 (2), 309–315
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.
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
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 short
English expression
Slovak translation
PI
protease inhibitor
protease inhibitor
NPI
nucleoside polymerase inhibitor
nucleoside polymerase inhibitor
NNPI
non-nucleoside polymerase inhibitor
a non-nucleoside polymerase inhibitor
CPE
cytopathic effect
cytopathic effect
cACT
combined anti-coronaviral therapy
combined anticoronavirus therapy
cART
combined anti-retroviral therapy
combination antiretroviral therapy
CRFK cells
Crandell-Rees Feline Kidney cells
Crandell-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 compound
Drug category
EC50 (µM)
GC376
PI
0.04
EIDD 1931
NPI
0.09
Elbasvir
NS5A Inhibitor
0.16
EIDD 2801
NPI
0.4
GS-441524+
NPI
0.66
K777 / K11777
PI
0.67
Toremifene citrate
Other*
5
Amodiaquine
Other**
6.5
Lopinavir
PI
8.08
Ritonavir
PI
8.7
Grazoprevir
PI
12.13
Nelfinavir mesylate
PI
13.47
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.
Compound
Drug category
5μM
10μM
25μM
50μM
100μM
Elbasvir
NS5A Inhibitor
0.67
0.46
1.4
2
4.9
K777 / K11777
PI
0.61
0.29
2.3
9
16
Toremifene citrate
Other *
12
22
23
35
39
Amodiaquine
Other **
11
12
19
23
26
Lopinavir
PI
0.67
15
13
16
18
Ritonavir
PI
0.49
0.84
2
33
26
Grazoprevir
PI
0.55
0
0.81
3.9
19
EIDD1931
NPI
1.2
0.94
0.6
0.78
2.8
EIDD2801
NPI
0
0.95
0.63
1.7
0.8
GS-441524+
NPI
0.21
0
0
0
0
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.
Compound
Virus titer reduction fold
GC376 (20μM)
25000
GC376
7300
GS-441524 (NMPharmTech)
5280
EIDD-1931
3700
GS-441524 (MedChemExpress)
3500
EIDD-2801
2110
Lopinavir
309
Toremifene
10
K777
7
Grazoprevir
5
Amodiaquine
4
Elbasvir
2
Nelfinavir mesylate
1
Ritonavir
1
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 1
Compound 2
Compound 3
Virus titer reduction
Add
cACT / add
GC376 (20 μM)
Amodiaquine
—
1897000
25004
76
GC376 (20 μM)
Amodiaquine
Toremifene
256000
25014
10
GC376 (20 μM)
K777
—
248000
25007
10
GC376 (20 μM)
Toremifene
—
128000
25010
5.1
GC376 (20 μM)
Nelfinavir mesylate
—
91100
25001
3.6
Elbasvir (5 μM)
Lopinavir
—
14000
311
45
Elbasvir (5 μM)
GC376 (20 μM)
—
12600
25002
0.50
GC376 (10 μM)
Amodiaquine
—
11700
7304
1.6
GC376 (10 μM)
Grazoprevir
—
8290
7305
1.1
GC376 (20 μM)
GS-441524 (NMPharmTech)
8260
30280
0.27
GC376 (10 μM)
Amodiaquine
Elbasvir (5 μM)
7570
7306
1.0
GC376 (10 μM)
GS-441524 (MedChem)
—
6910
10800
0.64
GC376 (20 μM)
Ritonavir
—
6560
25001
0.26
K777
Lopinavir
—
5530
316
18
GC376 (10 μM)
GS-441524 (NMPharmTech)
—
4340
12580
0.34
GC376 (20 μM)
Lopinavir
—
3400
25309
0.13
Lopinavir
Ritonavir
—
3130
310
10
Lopinavir
Ritonavir
Toremifene
1740
320
5.4
GC376 (10 μM)
EIDD-2801
—
255
9410
0.03
K777
Toremifene
—
25
17
1.5
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.
Given the strong anti-FIPV activity of GC-376, as well as its potential availability for advancement 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 monotherapy and combination therapy (Fig. 7). Overall, GC376 demonstrated excellent anti-FIPV activity at 20μM both in monotherapy and combination therapy in vitro. The most significant reduction in FIPV RNA occurred when GC376 was combined 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 starting 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 then reverse transcribed using the High-Capacity RNA-to-CDNA Kit (Applied Biosystems) according to the manufacturers' protocols. Copy numbers of FIPV and feline GAPDH cDNA were determined using the Applied Biosystems' QuantStudio 3 Real-Time PCR System and PowerUp SYBR Green Master Mix according to the manufacturer's protocol for a 10 μL reaction. Each PCR reaction was performed in triplicate with aqueous template as a negative control and plasmid DNA as a positive control. A control reaction excluding reverse transcriptase was included in each set of real-time PCR assays. 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 housekeeping gene GAPDH was performed simultaneously using primers, 5'-GAPDH, 5'-AAATTCCACGGCACAGTCAAG, and 3'-GAPDH, 5'-TGATGGGCTTTCCATTGATGA. Cycling conditions for both FIPV and GAPDH amplicons were as follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 1 min. The final step included a dissociation curve to evaluate the specificity of primer binding. FIPV and GAPDH copy numbers were calculated based on standard curves generated in our laboratory. FIPV cDNA copies 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 Analogs
Nucleoside analog
NPI
GS-441524 (China-sourced)
Adenosine analog nucleoside
NPI
3-Deazaneplanocin A Hydrochloride
Adenosine analog nucleoside
NPI
Adefovir
Adenosine analog nucleoside
NPI
Galidesivir
Adenosine analog nucleoside
NPI
GS-441524 (Manufactured in China)
Adenosine analog nucleoside
NPI
MK-0608
Adenosine analog nucleoside
NPI
NITD008
Adenosine analog nucleoside
NPI
Didanosine
Adenosine analog nucleoside
NPI
Tenofovir alafenamide
Adenosine analog nucleoside
NPI
Tenofovir disoproxil fumarate
Adenosine analog nucleoside
NPI
EIDD 1931
Nucleoside analog Cytidine
EIDD 2801
Nucleoside analog Cytidine
2′-C-methylcytidine
Nucleoside analog Cytidine
NPI
Gemcitabine Hydrochloride
Nucleoside analog Cytidine
NPI
2-C-methylguanosine
Nucleoside analogue Guanosine
NPI
7-methylguanosine
Nucleoside analogue Guanosine
NPI
Entecavir
Nucleoside analogue Guanosine
NPI
Mizoribine
Nucleoside analogue Guanosine
NPI
Ribavirin
Nucleoside analogue Guanosine
NPI
PSI-6206
Nucleoside analog Uridine
NPI
6-Azauridine
Nucleoside analog Uridine
NPI
Balapiravir
Nucleoside analog Cytidine
NPI
Sofosbuvir
Nucleoside analog Uridine
NPI
Favipiravir
Nucleoside analog Purine
NPI
Total 36
Protease inhibitors
Grazoprevir
NS3 / 4A protease inhibitor
PI
Rupin trivir
Rhinoviral 3CP inhib
PI
Lopinavir
Antiretroviral PI
PI
Ritonavir
Antiretroviral PI
PI
Nelfinavir
Antiretroviral PI
PI
Disulfiram (tetraethyliuram disulfide)
Papain-like protease inhib
PI
K777 / K11777
Cysteine protease inhibitor
PI
Telaprevir
NS3 / 4A protease inhibitor
PI
Camostat mesylate
Serine protease inhibitor
PI
Paritaprevir
Serine protease inhibitor
PI
GC376
Coronavirus protease inhibitor
PI
Total 11
NS5A inhibitors
Velpatasvir
NS5A Inhibitor
NS5A Inhibitor
Ravidasvir / PPI-668
NS5A Inhibitor
NS5A Inhibitor
Ledipasvir
NS5A Inhibitor
NS5A Inhibitor
Ombitasvir
NS5A Inhibitor
NS5A Inhibitor
Pibrentasvir
NS5A Inhibitor
NS5A Inhibitor
Daclatasvir
NS5A Inhibitor
NS5A Inhibitor
Elbasvir
NS5A Inhibitor
NS5A Inhibitor
Total 7
NNPI
Dasabuvir
Non-nucleoside polymerase inhibitor
NNPI
Total 1
other
Monensin
Ionophore
other
Phenazopyridine hydrochloride
Crystalline solid
other
Pyrvinium pamoate hydrate
Androgen receptor inhibitor
other
Toremifene citrate
Selective estrogen receptor modulator
other
AM580
Retinobenzoic derivative
other
Homoharringtonine
Translation elongation inhib
other
Amodiaquine
4-aminoquinolone
other
Total 7
Midwestern Chemical Fragments
F0472-0017
Midwestern
F6190-0257
Midwestern
F6279-0675
Midwestern
F2167-1080
Midwestern
F6190-0740
Midwestern
F6438-2155
Midwestern
F3411-5663
Midwestern
F6233-0011
Midwestern
F9995-2543
Midwestern
F2124-0890
Midwestern
F2711-2577
Midwestern
F2130-0055
Midwestern
F2493-3358
Midwestern
F2459-0974
Midwestern
F2124-0465
Midwestern
F1899-2269
Midwestern
F2185-1982
Midwestern
F2189-0717
Midwestern
F2147-0158
Midwestern
F1371-0192
Midwestern
F2156-0057
Midwestern
F9995-2431
Midwestern
F3349-0218
Midwestern
F2156-0059
Midwestern
F2156-0070
Midwestern
F5856-0194
Midwestern
F2147-0975
Midwestern
Total 27
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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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Introduction - Pregnancy is an interesting immunological paradox. Although organs and tissues from individuals of the same (allograft) or other species (xenograft) are considered foreign and destroyed, this is not the case for a fetus that is an allograft. The prevention of fetal rejection is accompanied by events related to the forthcoming complex hormonal changes. 1-4 The effects of pregnancy affect many functions of the immune system. 1-4 However, pregnancy has the main effect on T cell immunity. T-cell immunity is mediated by a class of thymus-affected lymphocytes that are active in identifying infected host cells evaluated as foreign due to their interaction with pathogens. T-cell immunity also plays an important role in autoimmune diseases.
The modulation of immunity that occurs during pregnancy can variously affect maternal immunocompetence. Pregnancy is known to ameliorate or exacerbate autoimmune diseases, increase the postpartum incidence of autoimmune diseases, and increase susceptibility to many common bacterial, fungal, and viral diseases. 4-7 The incidence of these diseases is greatest in the third trimester, when estradiol and progesterone levels are highest in the blood.4
Although mothers may be more susceptible to certain infections during pregnancy, the fetus remains protected from maternal infections through a placental barrier that separates the mother's and fetus's bloodstream. Although this barrier is highly effective in retaining infectious agents, it is permeable to most drugs. Therefore, the role of the placental barrier in resisting mainly pathogens differs from the blood-brain barrier, which is a barrier for both drugs and pathogens.
FIP and pregnancy - Only limited information has been published on FIP in pregnant females. 8-10 Based on the author's further experience with the small number of cases observed during FIP treatment with GS-441524, the affected females appear to be in the subclinical or preclinical stage of FIP at the time of pregnancy. The immunosuppressive effect of pregnancy then allows the infection to progress to a clinical stage, usually in the last trimester. The most common clinical form of FIP in pregnant cats is abdominal and moist, and clinical signs usually appear in the late stages of pregnancy, at or shortly thereafter.
FIP in females also affects kittens, depending on the severity of the infection and its timing. Kittens either die at the beginning of pregnancy when they are resorbed, aborted at a later stage of pregnancy, or they may be born sick and die soon after birth. 8-10 Some may even survive for weeks before succumbing to FIP. However, some litters are born alive and remain healthy during foster care or bottle feeding. It is not clear what role infection plays in this mortality. Are early fetal resorptions and abortions due to fetal infection from maternal monocytes / macrophages? Are fetal deaths caused by the non-specific effect of the disease and the associated cytokine storm? It is likely to be a combination of both. However, there is no doubt that some kittens may be infected in late pregnancy, be relatively healthy and may develop confirmed FIP in the first weeks of life.
The advent of FIP treatment with GS-441524 significantly affected FIP during pregnancy, the fate of fetuses and newborn kittens. Females treated in the early stages of the disease often give birth to healthy kittens, and if they respond quickly to treatment, they can take care of them in the usual way. No studies have been performed to determine how much GS-441524 reaches the fetus from the mother's bloodstream, but it is a small molecule and should easily cross the placental barrier. GS-441524 has been shown to have no adverse effects on fetal or kitten development when administered to a female during the second or third trimester and / or during the neonatal period. GS-441524 is thought to pass easily from females to kittens in colostrum and milk.
The current recommendation is to treat pregnant females as if they were not pregnant and not to interfere with pregnancy or neonatal care unless necessary. Successive ultrasound examinations will provide an accurate picture of what is happening to the fetus. Some will be dead and absorbed in utero, others are weak and miscarried, others have a normal appearance and are healthy after birth. Females usually respond quickly to GS-441524 and most of them are healthy enough to care for and care for their kittens. In addition to the treatment itself, females should be allowed to interact normally with their kittens. Kittens do not need individual treatment, as a sufficient amount of GS-441524 is probably provided by breast milk. Kittens that are born healthy and not breastfed can be artificially fed and their weight monitored daily. GS-441524 treatment should only be used if a lack of weight gain and activity indicate that it may be necessary.
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Basic facts - Feline leukemia virus (FeLV) is a retrovirus related to the murine leukemia virus that existed among feral cats for tens of thousands of years before its discovery in 1964 (Jarrett et al., 1964). FeLV infection occurs mainly in cats less than 3-8 years of age (Pedersen, 1998, 1991). Cats in the asymptomatic stage of the infection are the main source of infection. The virus is secreted in all secretions and bodily secretions and spreads by close contact (Pedersen et al., 1977). FeLV infection usually occurs in nature after cats are old enough to socialize, and the primary phase of the infection is either asymptomatic or transient and ends with a long-lasting immune response in 95% or more cats. Only a small proportion of infections in nature lead to chronic viremia. FeLV-associated diseases occur predominantly in a small group of cats with persistent infection. FeLV disease in feral cats did not arouse increased interest prior to its discovery, and any associated mortality remained unnoticed among the spectrum of diseases that affect feral cats. What we know about the pathogenesis of FeLV infection in feral cats therefore originally came from studies conducted in the 1970s and 1980s on groups of domestic cats and laboratory infections (Review Pedersen, 1998, 1991).
Epizootiology - FeLV appears to have inadvertently migrated from the wild to the domestic cat population sometime before the 1960s, and the incidence has increased rapidly since then. The first indications that the virus may be behind the disease came in 1964 with the identification of intracellular particles resembling the murine leukemia virus in the cat's home with multiple cases of lymphosarcoma (Jarrett et al., 1964). Our understanding of the severity of FeLV infection and its relationship to diseases other than lymphosarcoma began in 1969 in research laboratories (Hardy et al., 1969). The main spectrum of FeLV-related diseases arose from the commercial application of the Indirect Fluorescence Antibody Test (IFA) for the detection of viremic cats, beginning in 1972 (Hardy, 1973; Hardy and Zuckerman, 1991). Rapid domestic ELISA-based FeLV assays followed (Lutz et al., 1979).
FeLV was retrospectively identified as the leading cause of domestic cat disease in the 1960s and prospectively in the 1970s and 1980s. What caused the panzootic of FeLV infection and diseases in domestic cats? It was later found that a human-controlled environment with a large population of cats, especially where young kittens were in contact with older infected kittens and cats, proved to be ideal for cat-to-cat transmission. The severity of these exposures, along with young age (Hoover et al., 1976) and other environmental stressors, has significantly increased the incidence of persistent infections compared to transient infections (Pedersen et al., 1977). While only a small percentage of cats in the wild become permanently viremic, one-third or more cats exposed in a controlled laboratory environment have developed persistent viremia.
The end of the FeLV panzootic came with extensive "testing and segregation" of viremic cats, as documented by Weijer et al. (1986). Testing and segregation were later supplemented by effective vaccines. Remarkably, FeLV infection is no longer the leading cause of disease in households with multiple cats. Again, it exists as a natural infection, with positivity only in 1-5% wild cats. However, several viremic cats, especially younger ones, continue to appear among cats moved from the wild to shelters and temporary homes.
Pathogenesis - primary FeLV infection is largely insignificant in nature and terminated by a strong immune response and lifelong immunity (Review Pedersen, 1991). However, if the extent of exposure is large enough and / or the feline's immunity is reduced in some way, primary and transient disease may occur. This stage can be manifested by fever, generalized lymphadenopathy, low platelet and WBC counts, and mild anemia. This stage is often followed by long-lasting and largely asymptomatic viremia lasting months and years. Cats with persistent FeLV infection eventually develop several primary and secondary diseases, which are usually fatal. The usual mortality estimate for viremic cats is around 50% per year (Pedersen, 1988), which means that only 12.5% of them will survive after three years. Because infection in nature occurs mainly in younger cats and most of them die within 3 years, few cases of FeLV infection are observed in nature in cats older than 5-8 years.
Primary FeLV diseases are associated with various mutants of the infecting strain and include aplastic anemia, various myeloproliferative disorders, and lymphoma, which is usually generalized, ocular, or neurological (Summary Pedersen 1988, 1991). FeLV-associated secondary disease is caused by several common feline infectious agents, which are usually not very pathogenic, but which are exacerbated by suppression of FeLV-associated T cell immunity.
Relationship between FeLV and FIP infection – It was found that one-third to one-half of cats with FIP during the 1970s and 1980s were infected with FeLV (Cotter et al., 1973; Pedersen et al., 1977). The association between the two infections was demonstrated when young laboratory cats infected with enzootic feline enteric coronavirus (FECV) were housed with FeLV carriers (Pedersen et al., 1977). When these young cats became persistently infected with FeLV, their coronavirus antibody titers began to rise and within weeks or months clinical signs of FIP appeared. FIP occurred in only one-third of cats with FeLV viremia, and two-thirds of cats that became immune to FeLV did not develop FIP. In a later study, cats chronically infected with a laboratory strain of feline immunodeficiency virus (FIV) were experimentally infected with FECV (Poland et al., 1996). Two of 19 cats in the chronic immunosuppressive stage of FIV infection developed FIP, whereas none of 20 FIV-uninfected “housemates” became ill. Studies similar to this one with immunosuppression of FeLV and FIV were important in concluding that FIP virus (FIPV) was a commonly occurring mutant of FECV and a minimal pathogen in healthy immunocompetent cats.
What does FeLV infection mean for the treatment of FIP? I admit that I am not in favor of FIP treatment in FeLV-positive cats. We know that FeLV infection induces the type of immunosuppression that leads to the development of FIP. I also suspect that successful treatment with FIP antivirals such as GS-441524 is based on restoring the protective immune response to FIPV. If true, FeLV-induced immunosuppression may interfere with FIPV immunity and reduce the cure rate in GS treatment, or interfere with any long-term protective immunity elicited by successful antiviral therapy. Two other problems with the treatment of such cats need to be considered. Breeding FeLV-infected cats causes financial and physical difficulties in terms of routine veterinary care and quarantine of susceptible cats. It is also known that only 10% cats infected with FeLV survive for more than three years. These facts raise questions about the best use of the resources of temporary / rescue groups and individuals - for cats with only FIP, cats with other treatable diseases or for healthy cats waiting for home?
Conclusion - Foster parents / rescuers are unlikely to choose not to treat FIP in FeLV positive cats. However, the decision to treat FIP in such cats must be based primarily on the accuracy of the initial FeLV test. About 5 of the 100 positive in-house FeLV ELISAs in a healthy cat population will be false positive. If FeLV is present in the 1% street cat population, false positive results will be five times more common than true positives. Therefore, it is important that positive ELISAs are confirmed by another test, such as PCR. Owners should, whenever possible, ensure that these cats do not have other FeLV-related diseases, such as aplastic anemia, lymphoma or myeloproliferative disorders. Cat owners who opt for GS treatment should also share the short-term and long-term results of their efforts in order to obtain a better prognosis in this FIP group.
References
Cotter SM, Gillmore CE, Rollins C. 1973. Multiple cases of feline leukemia and feline infectious peritonitis in a household. J Am Vet Med Assoc 162:1054-1058.
Hardy WD Jr., et al. Feline leukemia virus: occurrences of viral antigen in the tissues of cats with lymphosarcoma and other diseases. Science 1969, 166:1019-1021.
Hardy WD Jr. Horizontal transmission of feline leukemia virus in cats. Nature 1973, 244:266269.
Hardy WD Jr., Zuckerman EE. Ten-year study comparing enzyme-linked immunosorbent assay with the immunofluorescent antibody test for detection of feline leukemia virus infection in cats. J Am Vet Med Assoc. 1991, 199:1365-73.
Hoover EA, et al. Feline leukemia virus infection: Age-related variation in response of cats to experimental infection. J Natl Cancer Inst. 1976, 57:365-369.
Jarrett WFH, et al., Leukemia in the cat. A virus-like particle associated with leukemia (lymphosarcoma). Nature 1964, 202:567-56.
Lutz H, et al. The Demonstration of Antibody Specificity by a New Technique. The Gel
Electrophoresis-Derived Enzyme-Linked Immunosorbent Assay (GEDELISA) and its Application to Antibodies Specific for Feline Leukemia Virus. J Histochem Cytochem. 1979, 27: 1216-1218.
Pedersen NC. Feline Infectious Diseases. American Veterinary Publications, Inc., Goleta, CA, USA, 1988.
Pedersen NC. Feline leukemia virus infection. In: Feline Husbandry. Disease and management in the multiple-cat environments. American Veterinary Publications, Inc., Goleta, CA, USA, 1991, pp210-228.
Pedersen NC, Theilen G, Keane MA, Fairbanks L, Mason T, Orser B, Che CH, Allison C. 1977. Studies of naturally transmitted feline leukemia virus infection. Am J Vet Res 38: 1523–31.
Poland AM, Vennema H, Foley JE, Pedersen NC. 1996. Two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with feline enteric coronavirus. J Clin Micro 34: 3180–3844. Weijer K, de Haag U, Osterhaus A. 1986. Control of feline leukemia virus infection by a removal program. Vet Rec, 119, 555–556.
The use of supplements for dogs and cats is becoming more common, which reflects the trend of using supplements for humans. I have also noticed that many cat owners use numerous supplements to support the treatment of FIP with GS-441524. I strongly feel that these supplements have no effect and cost the owners a huge amount of money. Some owners decide to take the supplements themselves, but in some cases they use them on the advice of their veterinarians. People also rely on supplements in those parts of the world where there is a lack of veterinary care. In many cases, they are "prescribed" to prevent, slow down or reverse specific medical conditions. In fact, they are often used just to do something, and even if they don't work, at least they don't hurt. In some cases, it is sufficient to indicate that a particular authority "needs some form of assistance". A significant proportion of test panels performed even in healthy animals will show one or more suspicious values, especially in the blood, liver or kidneys. Such values should not be used as a reason for prescribing or selling supplements. As a person who believes in scientific methods and clinical trials aimed at ensuring safety and efficacy, I cannot in good conscience recommend owners to use untested over-the-counter supplements that they claim to prevent, alleviate or treat diseases.
I am aware of the many testimonies that exist on the web that prove the effectiveness of a wide range of products. However, there are also many articles from reputable sources that support my beliefs. I quote excerpts from such articles below.
"Much has been written about the nutritional supplements business, the billions of dollars-long industry with strong political ties, and the deplorable inadequacy of regulation, which allows for extensive marketing of supplements without a solid scientific basis or scientific evidence. …………. . The marketing used to promote these supplements, of course, goes beyond anything justified by real scientific evidence and is almost generally unreliable. Likewise, testimonies and anecdotes about their effects, whether from patients, pet owners, veterinarians or Nobel laureates, are just stories that have almost no probative value. And since most good medical ideas will not eventually become a real and effective clinical therapy, it is likely that many of these even more attractive products will prove ineffective or cover unknown risks. Without adequate supporting evidence and effective quality control, regulation and post-market surveillance, we can never be sure that by using them, we are helping and not harming our patients. "
"Claims about the effectiveness of many pet supplements and nutraceuticals are often based on subjective evaluation methods, including owner reviews, when they have not been rigorously tested in well-designed clinical trials and published in professional journals and should therefore be viewed with skepticism. Although the results extrapolated from studies performed in humans or rodent models are valuable for interspecies comparisons, they do not take into account the different pharmacokinetics and pharmacodynamics of different species. Furthermore, many pet supplements and nutraceuticals are consumed orally, and the bioavailability of orally administered drugs varies greatly between species. Consumers should also be advised to be skeptical of in vitro test-based marketing claims. For example, many of the declared benefits of joint supplements for pets are based on in vitro tests, which often rely on very high doses applied directly to cartilage explants or cultured chondrocytes. "
As far as animals are concerned, the Food and Drug Administration (FDA), the Center for Veterinary Medicine (CVM) in the United States, regulates two classes of products: food and medicine. Depending on the intended use, the animal nutritional supplement is considered to be a food or medicine. There is no separate category for "supplements" for animals.
The Health Supplements Act (DSHEA) of 1994 defined the term 'food supplement', but did not specify whether the definition applied to humans, animals or both. The main benefit of DSHEA has been the reclassification of certain regulated food ingredients into food additives that require approval before being placed on the market.
In 1996, CVM published a notice in the Federal Register explaining that DSHEA did not apply to animal products.
Federal laws and regulations simply do not know the category of animal products called "nutritional supplements." Depending on the intended use, the product is either a food or a drug regulated by the FDA.
Many owners buy products for their pets for human use. It is important to know that manufacturers of human nutritional supplements may not provide the FDA with evidence that their supplements are effective or safe. However, they are not allowed (knowingly) to place dangerous or ineffective products on the market.
As soon as a nutritional supplement is placed on the market, the FDA must prove that the product is not safe in order to limit or withdraw it from the market.
On the contrary, before manufacturers can get a drug on the market, they must obtain FDA approval by providing convincing evidence that it is safe and effective.
Some supplements that are safe for humans can be toxic to dogs or cats. Therefore, it is essential that pet owners consult their veterinarians before administering the supplement to their pet.
Original article: 2021 - FIP IN AGED CATS Niels C. Pedersen, DVM, PhD, Professor Emeritus, University of California School of Veterinary Medicine, Davis; 2/11/2021
Facts: The relationship between age and the incidence of FIP has already been discussed in the literature and has even been the subject of research [1]. 29% cases of FIP occur in kittens up to 0.5 years, 50% up to 1 year, 80% up to 3 years of age and 96% up to 8 years of age (Table 1). The incidence of FIP between the ages of 7 and 11 is extremely low. At least two studies, one from the United States in 1976 [2] and the other from Europe in 2021 [3] [Table 1], confirm a real but less dramatic increase in the incidence of 3% in older cats. The purpose of this article is to discuss the causes of this increase and its implications for the diagnosis and successful treatment of FIP in old cats.
Table 1. Age of cats at the time of FIP diagnosis based on data obtained from cats treated with GS-441524 from Europe 2019-2021.
Source of FIP exposure: FIP is the result of FECV exposure [4] and the same applies to older cats. However, there are several unique situations associated with such exposure. The first scenario is analogous to that which occurs in younger cats, ie mass exposure of younger cats. However, it is common for older cats to mate as kittens and live their lives together in relative isolation. This leads to another interesting scenario in which one cat in a pair is likely to die sooner than the other, leaving her without a companion. You will then get a new companion, most often a kitten, from a rescue organization, shelter or kennel. The chances of the kitten excluding FECV from these sources are high. There is also a second source of FECV exposure that does not require cat-to-cat contact. It is known that FECV can be transmitted from one cat to another through human clothing. FECV is present in high concentrations in litter dust, especially in young cats and kittens, and can survive for many days in the environment. Therefore, the owner's contact with younger cats away from home is another source of FECV infection.
Unique aspects of FIP in old cats: Experience with the treatment of old cats GS-441524 leads to three possible factors that should interest us: 1) misdiagnosed FIP, 2) the existence of FIP in connection with one or more other disorders common in the aging cat and 3) treatment of FIP in connection with weakened immune system.
Incorrect FIP diagnosis: Observations made in cats treated with GS-441524 suggest that FIP in older cats is more prone to misdiagnosis and more difficult to treat. The misdiagnosis of FIP in old cats can best be explained by a simple probability. For example, one study found that FIP is the most common cause of spinal disease in cats less than two years old, while cancer was the most common single disorder in the 2-8 age group [5]. Therefore, the first consideration for a spinal cord disorder in a young cat should be FIP, while the first consideration for an old cat should be cancer. The incidence of many non-retroviral cancers is also on the rise, starting at around 7-9 years of age, accounting for about one-third of deaths in old cats. Chronic kidney disease also begins to manifest clinically at about the same age and accounts for about one-third of deaths in old cats. Remaining deaths in old cats include diabetes and hyperthyroidism and numerous musculoskeletal, cardiovascular, neurological and gastrointestinal disorders. Older cats, like older people, tend to have a decrease in immune function, which is often manifested by increased serum immunoglobulin levels and diseases associated with relative immunodeficiency and autoimmunity. The clinical and laboratory symptoms of these aging disorders often mimic the symptoms of FIP, but the probability that aging cats will have FIP is much lower than in these other conditions. In contrast, a young cat with FIP-compatible clinical and laboratory features that has FIP is much higher than an old cat with similar findings.
FIP as a secondary disease: The second scenario is more common in many old cats with FIP suffering from other serious medical conditions. Chronic renal failure is the most common of these underlying conditions, with cancers such as lymphoma being uncommon. Older cats also suffer from an aging immune system, leading to a state of relative immunodeficiency, which is another predisposing factor for FIP.
FIP and immunodeficiency due to aging: The immune system is sensitive to the adverse effects of aging in all animal species, including cats [6, 7]. Decreased immune function in aging cats is associated with changes in B and T cell populations and increased levels of non-specific immunoglobulin. Therefore, older cats often have higher levels of serum protein and globulin than usual. Increases in total serum protein and globulin levels in younger cats are often considered symptoms of FIP, while their diagnostic value in older cats is less significant.
The relative immunodeficiency caused by aging makes it difficult to fight new infections and older infections that remain hidden or latent in the body for decades. The best studied examples of the impact of aging on resistance to new and latent infections come from humans. COVID-19 deaths in the elderly with complicating diseases such as diabetes and chronic lung disease are the best examples of the impact of aging and chronic degenerative diseases on resistance to infectious agents. It is well known that tuberculosis remains latent in the pulmonary lymph nodes for decades before reactivation. Therefore, TB is a particular problem in humans in facilities for the elderly and in individuals treated for autoimmune disorders with cytokine inhibitors. There are also indications that FIP in some cats may exist in a subclinical state for months to years before it becomes clinically evident. There are also cases of cats that live their entire lives in residential isolation until they develop FIP in old age.
Conclusion: Fortunately, FIP in old cats is unusual, but it has unique properties that affect diagnosis and treatment. Particular attention should be paid to verifying the diagnosis of FIP and identifying other health factors that either predispose to FIP or complicate its successful treatment. This is a major diagnostic challenge in older cats and other degenerative disorders common to this age group make treatment difficult. The FIP cure rate, which is higher than 80% in young cats, is not as high in old cats.
References
Pedersen NC, Liu H, Gandolfi B, Lyons LA. The influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis. Vet Immunol Immunopathol. 2014, 162(1-2):33-40.
Pedersen NC. Virologic and immunologic aspects of feline infectious peritonitis virus infection. Adv Exp Med Biol. 1987, 218: 529-50.
Marioni-Henry K, Vite CH, Newton AL, Van Winkle TJ. Prevalence of diseases of the spinal cord of cats. Journal Veterinary Internal Medicine, 2004, 18, 851-58.
Day MJ. Aging, immunosenescence and inflammageing in the dog and cat. J Comp Pathol. 2010, 142 Suppl 1: S60-9.
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