FIP treatment with subcutaneous remdesivir followed by GS-441524 oral tablets

Richard Malik DVSc PhD FACVS FASM Center for Veterinary Education, University of Sydney
Original article: Treatment of FIP in cats with subcutaneous remdesivir followed by oral GS-441524 tablets

Translator's note: The article contains information about the real content of GS-441524 in tablets. However, this content may not correspond to the "equivalent" amount of GS-441524 in tablets from other manufacturers, where the actual content of GS-441524 is always slightly higher due to the known reduced oral bioavailability of the drug. Therefore, it is not possible to easily and unambiguously compare the recommended dosage of GS-441524 from BOVA in Australia and in our country.

Introduction

Infectious feline peritonitis (FIP) is an infectious disease, especially of young cats. It occurs when a feline enteric coronavirus that multiplies in the gut undergoes a critical mutation that changes its tissue tropism from enterocytes to macrophages. The FIP virus then circulates in the body in macrophages - this is the ultimate mechanism of the Trojan horse. This leads to disseminated infection and the development of fibrinoid necrotizing vasculitis and serositis due to the deposition of immune complexes consisting of feline antibodies and FIP viral antigens.

In general, there are two forms of FIP - effusive ("wet") FIP and non-effusive ("dry") FIP. The disease process itself can occur in the abdomen, thoracic cavity, pericardium, eyes or central nervous system. Combinations of dry and wet FIP with various tissues are not uncommon.

Until recently, the diagnosis of feline infectious peritonitis (FIP) was a death judgment for a feline patient. In recent years, however, this vision has been turned upside down as a result of the pioneering work of Professor Niels C. Pedersen and colleagues at UC Davis.

Over the last 12 months, many veterinarians in Australia have also successfully managed many cases of FIP using remdesivir and GS-441524.

Omega-interferon (Virbagen) and polyprenyl immunostimulant (PPI) were the first drugs used to treat FIP, and both had some effects in some patients. Omega interferon has been useful in cases of effusive ("wet") FIP, often combined with low-dose prednisolone according to the Ishid protocol, while PPI, pioneered by Al Legendre, has been more useful in cases of non-fusible FIP. In some cases, both drugs were used at the same time. The problem was that both forms of therapy were often expensive, especially when both drugs were used, so that although patients improved and could have transient clinical remissions during treatment, permanent clinical cures were rare. As a result, most veterinarians still considered the diagnosis of FIP a prelude to euthanasia.

That all changed a few years ago thanks to the culmination of FIP's lifelong research. Niels Pedersen. Niels is an amazing North American veterinarian of Danish descent. He grew up on a chicken farm and originally wanted to be a clinician for large animals, but with great foresight he decided on a scientific career. Shortly after graduating, he traveled to Canberry to the John Curtin School of Medical Research at ANU, where in the late 1960s he received a PhD in kidney transplant rejection immunology from Professor Bede Morris, using sheep as an experimental model to study lymphocyte kinetics.

When Niels returned to UC Davis, he focused on studying infections and immunity. Although he has contributed to a large number of topics in internal medicine and the genomics of dogs and cats, FIP has become his favorite disease due to its commonness and current complexity. His studies date from the 1980s, when he specialized in diagnostics, virology and pathogenesis, to the present, with an increasing focus on therapy.

Niels, in collaboration with colleagues from Kansas State University, has shown that a purposefully designed protease inhibitor GC-376 could prevent and cure experimentally induced FIP in laboratory cats.1,2 Field clinical trials with cats with naturally occurring disease have been disappointing, especially when cats have had an ocular form of FIP or CNS disease. He did not give up, so he switched to another drug - GS-4415243,4 - a nucleoside analogue developed by the North American pharmaceutical company Gilead. This molecule has been shown to be much more effective than GC-376 in the treatment of FIP, both in experimental infections and in spontaneous cases of FIP. Starting with pharmacokinetics and dose escalation studies using a wide range of clinical cases, Niels and colleagues found that the required dose depended on whether the patient had dry or wet FIP and whether the eye or central nervous system (CNS) was affected.5

Surprisingly, Gilead, the manufacturer who developed GS441524, has not yet shown interest in developing this molecule for the treatment of cats. To fill the gap for effective FIP therapy around the world, various laboratories in China and Eastern Europe have begun producing GS-441524 and selling it on the black market.

The wide availability of the GS-441524, often of high quality and initially very high price, provided dedicated owners with a way to save their cats with FIP. Studies by clinical pathologist Samantha Evans of Ohio State University have indicated a cure rate of approximately 80 % in the field. Until recently, the procurement of the drug was complicated and full of problems, which at some level were circumvented by various "FIP Warriors" groups on Facebook. Unfortunately for Australian cat lovers, APVMA and Vet Boards finally understood what was going on and the Border Force made it much more difficult to obtain GS-441524 and its safe import for veterinary use. Regulatory and Veterinary Committees' warnings against prosecutors were directed against veterinarians who allowed cats with FIP to be treated with black market drugs.

Ironically, the COVID 19 pandemic provided a new solution to this problem. Gilead developed remdesivir (GS-5734) as a drug for the treatment of hepatitis C, Ebola and human coronavirus disease. Remdesivir is a prodrug of GS-441524, which contains an additional chemical side chain (including a phosphate group) to improve intracellular penetration (Figure 1B). Remdesivir (as a product of Veklura) obtained a temporary marketing authorization (for two years) from TGA in July 2020 for the treatment of SARS-CoV-2 infections in human patients with COVID-19. This registration process would normally take several years, but the severity of the pandemic has accelerated this process, taking into account preliminary data from clinical trials. As remdesivir became a licensed human drug and Gilead licensed production worldwide, it meant more access to quality raw material. This circumvented the problems with the use of the drug purchased on the black market, as well as the problems of unknown purity and consistency of the product over time.

In 2020, the veterinary compounding company BOVA Australia provided reliable supplies of remdesivir in a suitable format for IV and subcutaneous application. Studies in Australia have determined that the shelf life after reconstitution exceeds 12 days and have confirmed in vitro efficacy against coronaviruses in tissue cultures. The analytical purity of the drug is regularly checked by HPLC. Over the past year, veterinarians in every Australian state have used remdesivir to treat cats with FIP. There have been a number of effusive and non-fusive cases, including some cats with ocular disorders (uveitis) and others with multifocal CNS disease. Based on treatment of approximately 500 cats treated between October 2020 and November 2021, remdesivir has been shown to be highly effective in managing FIP infections. It allows for a slightly simpler subcutaneous administration and the injection appears to be slightly less painful compared to GS-441524 and does not cause the local injection site reactions observed with GS-441524 injection. Remdesivir was originally used exclusively in Australia, although it has also been available in the UK from BOVA UK for the last 2 months.

The molecular weight of remdesivir is 603 g / mol, while the molecular weight of GS-441524 is 291 g / mol. This could suggest that treatment of cats with remdesivir requires approximately twice the dose of GS-441524, although this does not take into account the possible improvement in intracellular penetration of remdesivir into certain tissues compared to GS-441524. The proposed dose of remdesivir in human patients with COVID19 is 200 mg intravenously (IV), followed by 100 mg IV daily. For a 70 kg human patient, this represents a daily dose of 1.3 mg / kg, so using allometric scaling, a dose of 5-10 mg / kg per day was considered correct for a cat. However, our experience with the first 500 cases was that many cats eventually needed a higher dose of remdesivir for permanent cure, so we adjusted our recommended dosage upwards (see below). Remdesivir provides BOVA as a sterile 10 mg / ml solution ready for use in a 10 ml vial.

Figure 1. (A) BOVA Remdesivir reconstituted and ready for treatment. After reconstitution, the contents of the vial are stable for at least 120 days at 5 ° C - and the vial is usually consumed in 3-7 days. It is best to store the vial in the refrigerator. (B) The pathway that remdesivir travels intracellularly to activate as GS-441524.

At present, Australia and the United Kingdom are the only countries where remdesivir is readily available by prescription for veterinary use. However, veterinarians in India, New Zealand, South Africa and parts of Europe have also started using human medicine suppliers to access the medicine.

Diagnosis

Figure 2: Amazingly comprehensive and practical overview of FIP diagnostics by Severine Tasker.

A complete differential diagnosis of FIP is beyond the scope of this article, but readers are strongly encouraged to read the excellent article by Séverine Tasker in the Journal of Feline Medicine & Surgery. 6

Although FIP can occur in cats of any age, most cases occur in kittens and cats less than 3 years of age. Persistent and often high fever that does not respond to antibiotic therapy (and often NSAIDs) is a common finding, as is increased plasma total protein levels due to elevated globulin concentrations (diffuse gammopathy in serum electrophoresis). In effusive or "wet" FIP, the albumin to globulin ratio may drop to <0.45. Acute phase reactants such as serum amyloid A and α1-acid glycoprotein tend to be markedly elevated. Many cats with FIP also exhibit secondary immune-mediated hemolytic anemia, increased AST and ALT activities, and jaundice.

Diagnostic imaging is crucial for early diagnosis, which has been greatly facilitated by the introduction of digital radiology and the widespread availability of diagnostic ultrasound in small animal practice. Pleural effusion is readily recognizable from chest X-rays, while abdominal effusion is best detected by ultrasound (Figure 3), especially if high frequency probes are available. It is worth noting that in some cases, the fluid pockets may be focal and localized. Often there is some fluid around the kidney under the kidney sheath, kittens may have scrotal edema, while in rare cases the discharge is limited to the pericardial sac. But the key is - to look for (i) effusion in any body cavity, (ii) granulomas in the kidneys, liver or lungs, (iii) enlarged intra-abdominal and mesenteric lymph nodes (Figure 5) or marked thickening of the iliac-ecological area (f focal FIP ’) ( Figure 5). Chest X-rays after drainage of pleural effusion may show changes corresponding to viral pneumonia.

Figure 2: (A) Ultrasound of the abdomen showing abundant highly echogenic fluid (fibrin fibers) in cats with high protein ascites due to effusive FIP. (B) The fusion contains a viscous yellow to straw-colored liquid. (C) An X-ray of the abdomen with the appearance of cut glass indicating fluid in the abdomen.

If you see an effusion - puncture - because fluid is the best diagnostic sample.

Figure 3: Marked mesenteric lymphadenomegaly in a cat with dry FIP.

A fluid with a high protein content, often yellow to straw in color, is characteristic (Figure 3B). If you see granuloma in the organ or if the lymph nodes are clearly enlarged - do FNA (thin needle aspiration biopsy), apply a smear, use RapidDiff staining and look for neutrophils and macrophages (pyogranulomatous inflammation) without visible infectious agents (Figure 4). The two diseases most commonly confused with FIP in adult cats are lymphoma and some types of lymphocytic cholangitis (associated with high protein ascites).

Figure 4: RapidDiff stained aspirate with a thin needle from the mesenteric lymph nodes of a 4-year-old oriental cat with dry FIP. Distinctive macrophages are the key to cytological diagnosis. Photo courtesy of Trish Martin.

Of course, effusive disease is much easier to diagnose because ascitic, pericardial or pleural fluid provides a suitable sample that can be examined cytologically, by fluid analysis and immunofluorescence (IFA) for FIP antigen, or reverse transcriptase PCR to detect FIP nucleic acid. IFA is performed at VPDS, B14, University of Sydney (via Vetnostics, QML, ASAP, VetPath, Gribbles or IDEXX). However, it is usually the cheapest way to send the sample directly to the university laboratory.

Dry FIP is more problematic because it usually requires a thin-needle aspiration biopsy of pyogranulomatous lesions in the liver, kidneys, or abdominal lymph nodes. Occasionally, cases of wet FIP may show fluid samples that are negative for IFA and / or PCR testing, but the patient is still likely to have FIP, which is reflected in a favorable response to remdesivir or GS-441524 treatment.

Treatment

Since October 2020, we have been treating cats with FIP with remdesivir (IV and SCI) and more recently with GS-441524 (oral), so our protocols are constantly evolving with experience. About 500 cats have been treated so far. We try to avoid being too prescriptive in our recommendations, as we suspect that there is no one-size-fits-all protocol and that each case presents unique circumstances, including patient size, whether the cat is still "happy" and reasonably , or is depressed and dehydrated. An important factor is the emotional and financial commitment of the owner. A key feature that needs to be mentioned is that both drugs are very safe, even in sick cats and kittens.

Note that the following recommended doses are higher than those originally recommended a year ago. Although lower doses worked in many patients, we found that this was often the wrong economic consideration, as disease recurrence at the end of treatment and the development of viral resistance during treatment appear to be related to insufficient initial dosing. So we have learned to be more aggressive from the beginning, which is cheaper in the long run (ie 2nd therapy is not required)

Our greatest experience is with remdesivir. This drug is expensive and the owner has to commit to a costly treatment process that takes 3 months. For most clients, this represents an emotional and financial burden. My view is that in many cases it is better to spend money on antiviral therapy as such than on extensive diagnostics and monitoring.

Figure 5: Significant thickening of the ilico-ecological area of the Devon Rex cat with the so-called "focal FIP", a common form of non-fusive FIP. Photo courtesy of Penny Tisdall.

One of the approaches in newly diagnosed cats with severe disease is hospitalization of cats during the first 3-4 days of treatment. Patients begin treatment with remdesivir when receiving IV fluid therapy (typically 2-4 ml / kg / hr; first day Hartmann's solution or Plasmalyte followed by 0.45 % NaCl and 2.5 % dextrose containing 20 mmol KCl / l). On the 1st day of hospitalization, remdesivir is administered in a high dose intravenously (10-15 mg / kg diluted in 10 ml with saline and is given SLOWLY for 20-30 minutes or longer, manually or by infusion pump; in human patients, administration lasts 2 hours. ) to achieve an increased starting dose of drug distribution volume. This achieves fast antiviral efficacy. In cases with CNS disease, we recommend a daily IV dose of 20 mg / kg. Many cats may appear slightly depressed several hours after IV remdesivir infusion. In human patients, remdesivir may cause infusion-related reactions, including low blood pressure, nausea, vomiting, sweating or chills, but we have not observed these events in our feline patients.

The advantage of starting treatment intravenously is that dehydration, if present, is corrected and you have IV access if you need to take other medicines (eg anticonvulsants, corticosteroids). Importantly, once an IV catheter is inserted, daily injections of remdesivir do not cause any pain or discomfort. However, if the cat eats and is diagnosed in the early stages of the disease, then IV therapy is not required and the same doses can be given subcutaneously, saving a lot of money.

FIP cats treated with remdesivir typically improve significantly during the first 2-3 days. However, we found that cases of effusion, and especially those that resulted in pleural effusion prior to treatment, should be closely monitored, as the combination of the antiviral effect of remdesivir and a higher than maintenance dose of crystalloids may lead to transient worsening of pleural effusion. This requires drainage twice a day using a 19G butterfly needle (1.1 mm - cream color) and a 3-way stopcock (ideally using an ultrasonic guide to find the best place to insert the needle). These "secondary" pleural effusions can be fatal if not detected in time and appear to occur in approximately 1 in 10 cases of effusive FIPs treated with remdesivir.

Another problem that occasionally occurs at this time is the development of neurological symptoms, including seizures. Our view is that this is not the effect of the drug as such, but rather the unmasking of the subclinical CNS FIP. Such cats require careful monitoring, while the development of seizures requires the use of anticonvulsant drugs such as midazolam (0.3 mg / kg IV), alfaxane or propofol (administered IV to be effective), followed by levetiracetam (Keppra) (10- 20 mg / kg, PO every 8 hours). Phenobarbitone is a reliable anticonvulsant, but it tends to increase the metabolism of many drugs, and levetiracetam is probably safer until we better understand the pharmacokinetics and metabolism of remdesivir and GS-441524. Some doctors also administer dexamethasone or prednisolone as a single treatment to relieve CNS inflammation.

Although advocating initial IV therapy for the most severe cases of FIP, cats and kittens that are still "happy" and eating do not require IV therapy at first and may instead begin subcutaneous injections at 10-12 mg / kg / day (20 mg / kg in CNS diseases). This is, of course, much cheaper because cats or kittens do not have to be placed in an infusion pump and hospitalized in a stressful environment. For clients who have financial limits, this may be a more appropriate way to start therapy. Some skilled colleagues, such as Jim Euclid, have developed a hybrid approach where kittens receive subcutaneous fluids daily as a bolus with injected remdesivir.

The cats were then given continuous subcutaneous injections of remdesivir. It originally took 84 days, and such cases accounted for most of the cases we have dealt with so far. Recently, we have been using aggressive IV / SCI remdesivir for initial therapy, and then cats are switching to oral GS-441524 for 10 weeks of consolidation therapy.

After the initial use of lower doses, which were not successful in every patient, we now use the following treatment protocols:

  • for cats with wet FIP: 10-12 mg / kg once daily (SID) for 2 weeks
  • for cats with severe eye impairment: 15 mg / kg SID by subcutaneous injection (SCI) for 2 weeks; Cats with severe uveitis should also be given topical corticosteroids (Before Forte or Maxidex) for 2-3 days (no longer!) and atropine eye ointment.
  • for cats with neurological FIP with CNS symptoms: administer 20 mg / kg SID SCI for 2-4 weeks. 5

It is important that owners are properly instructed on how to optimally administer daily injections. Cats will perceive the injection as less painful if the remdesivir solution in the syringe is allowed to warm to room temperature instead of being refrigerated. In addition, if you teach them simple tasks such as using a new needle when injecting (ie use a needle other than the one used to draw the medicine from the vial) and using 21G (0.8mm - green) or 23G diameter needles (0.6mm - blue), injections will be more tolerable. Although 21G needles are larger, some cats may have the advantage of injecting faster. Alternatively, for simplicity, veterinarians can prepare injections for the whole week, which they will keep in the refrigerator, and will give a new injection every day.

For cats that continue to perceive SC injections as painful, we used gabapentin orally (50 to 100 mg per cat) and / or transmucosally or SC administered buprenorphine 30-60 minutes before sedation / analgesia injection. The area to be injected can also be trimmed so that a topical EMLA cream can be applied 30 minutes before the injection. BOVA produces a faster-acting local anesthetic gel that may be useful in some patients. In exceptional cases, we inserted a cephalic catheter every 4-5 days so that owners could administer IV therapy instead of SC injections. Injection site reactions reported with GS-441524 injected abroad do not appear to occur with remdesivir.

After 2-4 weeks of taking remdesivir and after the abdominal fluid has disappeared and the ocular and CNS symptoms have improved or disappeared, we are now proposing a switch to GS-441524 tablets. This is done for 3 reasons: (i) it reduces costs (ii) eliminates the pain problem of SC injections (iii) in some patients it is more effective. Remdesivir injections are probably more reliable than oral GS-441524, and in the worst cases, you might choose to give them for 4 weeks, but for most cats, 2 weeks and comfort and lower oral formulation costs outperform everything else.

The use of GS-441524 tablets is relatively new in Australia, but is widely used overseas. The recommended oral dose of GS441524 is usually the same as the SCI / IV remdesivir dose: wet cases of FIP receive 10-12 mg / kg PO SID, ocular cases 15 mg / kg PO SID and CNS cases 20 mg / kg (or higher). GS-441524 is more economical and even safer than remdesivir. In CNS cases where high doses are administered, it is probably best to administer 10 mg / kg PO every 12 hours (BID) to circumvent the "ceiling" effect referred to in the limited absorption of high doses.

Figure 6. Focal dry FIP with pyogranulomatous inflammation of intra-abdominal lymph nodes. Instead of exploratory laparotomy, lymph node biopsy, histology, and immunohistology, 3 days of remdesivir IV treatment may be more cost-effective if FIP is highly suspected. Enlarged lymph node FNA is probably an ideal diagnostic option for physicians with this set of skills.

Why are the dosages about the same? At mg / kg, GS441524 has twice as many active molecules as remdesivir (due to the difference in their molecular weight), but the bioavailability of GS-441524 is only 50 % (only half of what is given is absorbed, and this is affected by feeding and also the effect of the ceiling dose) - so these two factors cancel each other out.

We recommend that GS-441524 tablets be given with a small treat to mask the tablet, with the main meal being served 1 hour later. The tablets provided by BOVA are 50 mg tuna-flavored tablets, with four score lines, so they can be divided into halves or even quarters.

In situations where owners cannot afford full treatment, we use mefloquine (Lariam; 5 mg / kg orally once daily in capsules or 62.5 mg twice a week) after initial treatment with remdesivir / GS-441524.

Phillip McDonagh, Jacqui Norris, Merran Govendir and colleagues at the Sydney School of Veterinary Science have shown that mefloquine has an antiviral effect. 7 This is probably due to the fact that mefloquine usurps the biochemical intracellular pathways used by the FIP virus, a mechanism that has recently been demonstrated with clofazimine. 8 (anti-leprosy medicine), and several other medicines. In several cats, where owners could not afford a complete treatment with remdesivir, mefloquine proved to be effective in reaching the limit of clinical cure.

The main advantage of buying remdesivir and GS-441524 from BOVA for the treatment of FIP cases is that the products we use are subject to quality control. It's just a prescription with the client's name and address, the patient's name and the dose to be given, and the compounder can usually deliver the vials or tablets to any veterinarian in Australia within 24-48 hours.

At present, the price is 100 mg of vials of remdesivir 250$ plus GST and postage (the total price is usually about 280$). GS-441524 is sold in packs of 10 tablets for 600$ plus shipping and handling. By purchasing more vials and tablets at the same time, of course, postage and handling fees will be reduced. We believe that most owners will feel much more comfortable getting a product from a well-known Australian company than sending money overseas and hoping that drugs of unknown quality on the black market will reach Australia safely without being detained by customs.

There is no reason why a well-motivated veterinarian would not be able to handle these cases in his own practice. This is often more convenient for the owner, especially if they struggle with daily injections and need a practice near them.

Figure 7: Gs-441524 tablets from BOVA Australia. They are tuna flavored. They can be divided into halves or even quarters. MUCH EASIER than injections for most cats. Less stress and less cost.

Veterinarians who wish to explore this option or have general questions about FIP case management may email Sally Coggins (dr.sallyc@gmail.com), Richard Malik (richard.malik@sydney.edu.au), David Hughes (concordvets@concordvets.com.au), Grette Howard (drgretta@gmail.com) or Professor Jacqui Norris (jacqui.norris@sydney.edu.au), for advice on diagnosis or treatment. Many Australian veterinarians interested in FIP have gained considerable expertise in the management of these cases. For example, Andrew Spanner in Adelaide treated more than 20 cases with excellent results. Thus, there are already many feline medicine physicians and internal medicine specialists with experience in the treatment of FIP, and so veterinarians who are hesitant to treat their own cases have the opportunity to recommend these specialists to their clients.

Physicians who accept FIP cases from GPs include: QLD Rhett Marshall, Marcus Gunew, Alison Jukes, Rachel Korman; NSW Katherine Briscoe, Michael Linton, Randolph Baral, Melissa Catt; VIC - Carolyn O'Brien, Keshuan Chow, Amy Lingard; WA-Martine Van Boeijen and Murdoch University Veterinary Hospital; TAS Moira van Dorsselaer.

All of these doctors (and probably even more we don't know about) are happy to accept cases for diagnosis and therapy. Everyone is probably happy to discuss case management with you.

Figure 8: Bengal kitten with CNS and ocular FIP (A: before) and (B: after) after Remdesivira. This cat also had pulmonary granulomas.

Sally Coggins, working with Lara Boland, Emily Pritchard, Associate Professor Mary Thompson and Professor Jacqui Norris at the Sydney School of Veterinary Science, is interested in treating cases with comprehensive diagnosis and free monitoring. It will be part of Sally's doctoral program, so you will help her advance in her studies by sending her cases. We hope that through these studies, we will get a better idea of how quickly cats respond and when exactly treatment can be safely stopped. Owners will only have to pay for remdesivir and GS-441524 for therapy. This group is also interested in treating cases with interferon-omega and mefloquine.

In most cases, FIP is doing very well with GS-441524 or remdesivir. Niels Pedersen has gathered an amazing resource for veterinarians interested in FIP case management - https://sockfip.org/dr - pedersen - research / . The site also provides some recommendations on how to monitor cats during treatment. I'm not very protocol-oriented, so the key things for me to keep track of are appetite, attitude, activity levels, and changes in body weight and fitness over time. Most physicians like to monitor serum hematology and biochemistry every month to ensure that all measurable abnormalities improve, although this can be stressful for the patient and increase treatment costs. The trade-off is taking a few drops of blood to monitor PCV, total plasma protein (TPP) using refractometry, and plasma color to determine if anemia is improving, jaundice is subsiding, and gamma globulin levels are lowering, resulting in lower TPP.

Do not worry about transient increases in globulin levels at the start of treatment; when high protein effusions are absorbed, a lot of immunoglobulins enter the patient's plasma. This phenomenon may be common until the 8th week of treatment, but disappears by the 12th week.

Figure 9: Transverse plane MRI image in contrast to T1 weighting. Note: dilatation of the lateral ventricles with very slight emphasis on the ependymal lining (orange arrows). Image courtesy of Christine Thomas.

And what about a kitten with multifocal CNS disease, where FIP CNS is the most likely cause of clinical symptoms? The traditional approach is serology (to rule out cryptococcosis and toxoplasmosis), a good history and thiamine test to rule out vitamin B1 deficiency, followed by MRI scans (Figure 9) and CSF collection for fluid analysis and multiplex neuro-qPCR analysis). This approach is very expensive and there is also a certain risk of anesthesia and especially CSF collection. We found that a 3-5 day intravenous or sc. Remdesivir therapy can be used as a therapeutic test in cats with probable CNS FIP and is a cost-effective alternative to complete diagnostic processing, which can cost 3-5000$ or more.

Similarly, if exploratory laparotomy, abnormal tissue biopsy, histology, and immunohistochemistry for FIP antigen are used to diagnose dry intra-abdominal FIP versus 3-5 days of treatment with remdesivir or GS-441524, a drug test may be considered, which is a better choice from in terms of patient well-being and reduced costs. Most cats with non-fusive FIP experience rapid improvement with antiviral therapy, with normalized fever, improved appetite, and better overall attitude within 2 to 3 days. If the patient does not respond to antiviral therapy, then exploratory laparotomy and representative organ biopsy are reasonable, as the main differential diagnoses are lymphoma and lymphocytic cholangitis.

This is a matter of personal approach for each doctor. FNA for cytological and sometimes immunohistochemical examination or PCR is a convincing non-invasive option where this expertise is available, but sometimes it does not give a definitive answer. Some veterinarians insist on tissue diagnosis and positive immunohistology or PCR in each patient, while others would like to "treat treatable" with a 3-5-day remdesivir / GS-441524 application and proceed to exploratory laparotomy only when there is no clear response to therapy.

It is incredibly satisfying to see the transformation of cats and kittens, which are not well, into normal and happy cats. It's really something that will lift your spirits as a doctor. It's good science and good veterinary medicine!

Conclusions

In the past, the diagnosis of FIP was an intellectual exercise so that we could end the suffering of a cat or kitten with the certainty of an accurate diagnosis. Now, thanks to FIP's lifelong study, Dr. Niels Pedersen, we are able to successfully treat perhaps 80 % or more cats with FIP if the client has sufficient funds. It is too early to predict whether or how many will be repeated later.

There is a need for intensive study in diagnosis and case management, but with the necessary effort, a good veterinarian should be able to work with a determined owner to achieve a clinical cure. The most important thing is not to put too many obstacles in the way of the dedicated owner and support him during the 12-week marathon treatment course by helping him find the best way to treat his patient. This may include sedative / analgesic treatment to help the cat improve controllability and prevent discomfort when the client brings their cat to the clinic daily for remdesivir injections or switching to GS-441524 tablets when the stress from the injections is too great for the owner. It is important to go a long way and a payment plan can be provided that will allow determined clients to improve the affordability of treatment.

Finally, the impact of COVID-19 on coronavirus research has been indeed profound, and several very promising drugs are under development, such as molnupiravir from Merck and another oral drug from Pfizer.

OVERALL SUMMARY

2-step approach to therapy

Phase 1 - INDUCTION

IV / SC injections of Remdesivir

  • For cats with wet FIP: 10-12 mg / kg remdesivir by subcutaneous injection (SCI) once daily (SID) for 2 weeks
  • For cats with eye: 15 mg / kg SID remdesivir SCI for 2 weeks
  • For cats with neurological symptoms of FIP and CNS: remdesivir 20 mg / kg SID for 2 weeks

Phase 2 - CONSOLIDATION

Switch to GS-441524 tablets after 2 weeks of remdesivir injection

  • For cats with wet FIP: 10-12 mg / kg GS-441524 oral SID for 10 weeks
  • For cats with eye impairment: 15 mg / kg SID GS-441524 oral SID for 10 weeks
  • For cats with neurological symptoms of FIP and CNS: GS-441524 10 mg / kg oral BID (20 mg / kg / day) for 10 weeks

References

  1. Kim, Y .; Liu, H .; Galasiti Kankanamalage, AC; Weerasekara, S .; Hua, DH; Groutas, WC; Chang, KO; Pedersen, NC Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog. 2016, 12, e1005531.
  2. Pedersen, NC; Kim, Y .; Liu, H .; Galasiti Kankanamalage, AC; Eckstrand, C .; Groutas, WC; Bannasch, M .; Meadows, JM; Chang, KO Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J. Feline Med. Surg. 2018, 20, 378–392.
  3. Murphy, BG; Perron, M .; Murakami, E .; Bauer, K .; Park, Y .; Eckstrand, C .; Liepnieks, M .; Pedersen, NC The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet. Microbiol. 2018, 219, 226–233.
  4. Pedersen, NC; Perron, M .; Bannasch, M .; Montgomery, E .; Murakami,
    E .; Liepnieks, M .; Liu, H. Efficacy, and safety of the nucleoside analog GS441524 for treatment of cats with naturally occurring feline infectious peritonitis. J. Feline Med. Surg. 2019, 21, 271–281.
  5. Dickinson PJ, Bannasch M, Thomasy SM, et al. Antiviral treatment using the adenosine nucleoside analogue GS-441524 in cats with clinically diagnosed neurological feline infectious peritonitis. Journal of Veterinary Internal Medicine. 2020. doi: 10.1111 / jvim.15780.
  6. Tasker S. Diagnosis of feline infectious peritonitis: Update on evidence supporting available tests. Journal of Feline Medicine and Surgery.
    2018; 20 (3): 228-243. doi: 10.1177 / 1098612X18758592
  7. McDonagh, P .; Sheehy, PA; Norris, JM Identification, and characterization of small molecule inhibitors of feline coronavirus replication. Vet. Microbiol. 2014, 174, 438–447.
  8. Yuan, S., Yin, X., Meng, X. et al. Clofazimine broadly inhibits coronaviruses including SARS-CoV-2. Nature (2021).
    https://doi.org/10.1038/s41586-021-03431-4
  9. https://sockfip.org/ - THE BEST resource on the internet or anywhere for FIP.
Figure 10: Two cats with dry FIP after successful therapy. As an avid young veterinarian wrote to me not so long ago - "that's why I did science!"

COSTS:

2 kg kitten with wet FIP
4 × 100 mg remdesivir vials - 1000$
35 × 50 mg tablets GS-441524 - 2100$
Manipulation and GST - 30$ plus 310$ = 340$
A total of 3440$, approximately 290$ per week for 12 weeks

4 kg cat with dry FIP
7 × 100 mg remdesivir vials - 1750$
70 × 50 mg tablets GS-441524 - 4200$
Handling and GST 30$ plus 600$
A total of 6550$, about 545$ per week for 12 weeks

Figure 11: Two siblings who developed FIP and were successfully treated with remdesivir and GS441524.
Read "FIP treatment with subcutaneous remdesivir followed by oral tablets GS-441524"

Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis

31.3.2020, Translation 25.4.2021
Tomoyoshi Doki, Masahiro Toda, Nobuhisa Hasegawa, Tsutomu Hohdatsu & Tomomi Takano
Original article: Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis

Abstract

Infectious feline peritonitis (FIP) is a deadly disease of wild and domestic species of cats. Although some drugs have been shown to be effective in the treatment of FIP, they are still not available in clinical practice. In this study, we evaluated the therapeutic effect of the combined use of adalimumab (anti-human TNF-alpha monoclonal antibody, ADA) and itraconazole (ICZ), which is currently available to veterinarians. ADA neutralizing activity against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. Ten specific pathogen-free (SPF) cats were inoculated intraperitoneally with FIPV KU-2 type I. Cats that developed FIP were administered ADA (10 mg / animal) twice between day 0 and day 4 after the start of treatment. ICZ (50 mg / head, SID) was administered orally every day from day 0 after the start of treatment. ADA demonstrated dose-dependent neutralizing activity against rfTNF-alpha. In an animal experiment, 2 of 3 cats showed an improvement in FIP clinical symptoms and biochemistry results, an increase in peripheral blood lymphocytes, and a decrease in plasma alpha 1-AGP levels after initiation of treatment. One of the cats did not respond to treatment and was euthanized, although the level of the viral ascites gene temporarily decreased after treatment. ADA was found to have neutralizing activity against rfTNF-alpha. The combined use of ADA and ICZ has shown a therapeutic effect on experimentally induced FIP. We consider these drugs to be a treatment option until effective anti-FIPV drugs are legally available.

Introduction

Feline Infectious Peritonitis Virus (FIPV), Feline Coronavirus (FCoV) Coronaviridae, causes a deadly disease in wild and domestic cat species called feline infectious peritonitis (FIP). In cats that develop FIP, several organs are affected, including the liver, lungs, spleen, serosis, kidneys, eyes, and central nervous system, and the formation of lesions in these organs is accompanied by necrosis and pyogenic granulomatous inflammation. Accumulation of pleural effusion and ascites fluid has been reported in some cats [1].

The FCoV virion consists mainly of nucleocapsid (N), envelope (E), membrane (M) and peplomer spike (S) proteins. FCoVs are classified into two serotypes, FCoV types I and II, based on differences in the amino acid sequence of the S protein. [2, 3]. FCoV type II was generated by genomic recombination between FCoV type I and canine coronavirus II. type (CCoV) [4,5,6]. Several serological and genetic surveys have shown that type I FCoV is dominant and that most cases of FIP are caused by FIPV type I infection [7,8,9].

We have previously shown that TNF (tumor necrosis factor) -alpha plays a crucial role in the progression of FIP. TNF-alpha is overproduced by FIPV-infected macrophages. TNF-alpha is involved in lymphopenia and elevated levels of the cellular receptor FIPV serotype II, aminopeptidase N (APN) [10, 11]. Neutrophil apoptosis in cats with FIP is also reported to be inhibited by TNF-alpha [12]. This finding suggests that neutrophilia in cats with FIP is due to TNF-alpha-induced neutrophil survival. We have found that monoclonal antibody (mAb) 2-4 with high neutralizing activity against feline TNF-alpha (fTNF-alpha) is useful as a treatment for FIP [13, 14]. However, the anti-fTNF-alpha mAb has never been marketed and cannot be used clinically in animal hospitals. In human medicine, the anti-human TNF-alpha mAb is used to treat inflammatory diseases such as rheumatoid arthritis and Crohn's disease. [15]. It is commercially available and can also be used in veterinary practice. However, it is not known whether this human antibody is effective against FIP, feline disease.

Several antiviral agents have been described for the treatment of FIPV [16,17,18,19,20]. These drugs have been shown to reduce FIPV proliferation, but are not commercially distributed as veterinary drugs. We have previously found that itraconazole (ICZ), which is used in veterinary medicine, reduces the multiplication of FIPV type I [21,22,23]. However, the therapeutic effects of ICZ in cats with FIP remain unclear.

In this study, we evaluated whether adalimumab (ADA), an anti-human TNF-alpha mAb, has neutralizing activity against fTNF-alpha. We further evaluated the therapeutic effects of ADA and ICZ on FIP by administering them to cats with experimentally induced FIP.

Materials and methods

Cell cultures and viruses

WEHI-164 mouse sarcoma cells were maintained in RPMI 1640 growth medium supplemented with 10% FCS, 600 U potassium benzylpenicillin per ml, 240 μg streptomycin sulfate per ml and 50 μM 2-mercaptoethanol. WEHI-164 mouse sarcoma cells were from the American Type Culture Collection (ATCC CRL1751).

FIPV KU-2 type I was grown in whole Felis catus fetal (fcwf) -4 cells at 37 ° C. FIPV type I KU-2 type I was isolated in our laboratory.

Monoclonal antibody

Anti-fTNF-alpha mAbs 2-4 have been described in [13] and have been shown to have neutralizing activity for recombinant fTNF-alpha (rfTNF-alpha) and native fTNF-alpha. mAbs 2-4 were purified from the hybridoma culture supernatant using Protein G Sepharose (GE Healthcare Life Sciences, Marlborough, MA, USA). The anti-human TNF-alpha mAb, adalimumab (ADA, Humira®), was purchased from Eisai Co., Ltd. (Tokyo, Japan) as a 20 mg / 0.2 ml solution. ADA was diluted to a concentration of 2 mg / ml in saline.

Itraconazole

Itraconazole (ICZ) was purchased from NICHI-IKO (Toyama, Japan) and provided as a tablet (100 mg). The cats were given half a tablet, ground to a powder and mixed with food.

Neutralization of feline TNF-alpha by ADA in WEHI-164 cells

TNF-alpha neutralization in WEHI-164 cells was assayed as described by Doki et al. [13]. Briefly, WEHI-164 cells were suspended at a density of 1 × 106 cells / ml in dilution medium containing 1 μg actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) per ml and preincubated at 37 ° C for 3 hours. Serially diluted mAbs were mixed with 40ng rfTNF-alpha per ml. The mixture was incubated at 37 ° C for 1 hour. Preincubated cells were seeded in a volume of 50 μl in wells of a 96-well plate. Then 50 microliters of the mixture was added to each well. After incubation at 37 ° C for 24 hours, 10 μl of WST-8 solution (WST-8 Cell Proliferation Assay Kit; Kishida Chemical Co., Ltd., Osaka, Japan) was added, and the absorbance of the formazan produced was measured at 450 nm. The percent neutralization was calculated using the following formula: Neutralization (%) = (OD wells containing mAb and rfTNF-alpha - OD wells containing rfTNF-alpha without mAb) / OD wells without mAb and rfTNF-alpha × 100.

Experiment on animals

All relevant national and institutional guidelines for the care and use of animals have been complied with. The protocol on animal experimentation was approved by the President of Kitasato University based on the decision of the Institutional Committee on the Care and Use of Animals at Kitasato University (Approval No. 18-152). Specific pathogen-free (SPF) cats were bred in our own laboratory and maintained in an isolated temperature-controlled facility.

Experimental schedule

According to the experimental schedule shown in FIG. 1, 10 SPF cats (9- or 10-month-old domestic shorthair cats) were inoculated intraperitoneally with FIPV KU-2 type I (103.6 TCID50 / ml / animal). The cats were then examined daily for clinical signs and their body temperature and weight were measured. Cats that developed FIP were administered ADA (10 mg / animal) twice intravenously between day 0 and day 4 after the start of treatment. ICZ (50 mg / animal, SID) was administered orally every day from day 0 after the start of treatment. Blood was collected from cats at weekly intervals after virus inoculation and at the time of ADA administration. Heparinized blood was used to measure complete and differential cell numbers and to isolate plasma. Plasma samples were stored at -30 ° C until the day of analysis. Cats were euthanized after reaching the human endpoint or 60 days after infection. The diagnoses of FIP were confirmed by post-mortem examination with the detection of peritoneal and pleural effusions and pyogranuloma in the main organs.

Figure 1
Experimental schedule of treatment and vaccination of FIPV cats

Measurement of plasma alpha1-glycoprotein (AGP)

AGP plasma concentrations were determined using an alpha1 AG cat plate (The Institute for Metabolic Ecosystem Lab., Osaki, Japan) according to the manufacturer's protocol.

Measurement of plasma vascular endothelial growth factor (VEGF) concentration

Plasma VEGF concentrations were determined using a human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. The ELISA kit primarily detects feline VEGF isoform 164 [24].

Real-time quantitative RT-PCR (qRT-PCR)

To quantify viral RNA in supernatant and ascites cells, qRT-PCR was performed using a primer targeting the 3'-UTR [17]. One milliliter of ascites was collected from cat no. 6 and after centrifugation, the supernatant and cells were stored separately. Total RNA was isolated from the supernatant and ascites cells using a high purity RNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. RNA was reverse transcribed and amplified using RNA-direct Realtime PCR Master Mix (TOYOBO, Osaka, Japan) with specific primers 3′-UTR-F (5′-GGAGGTACAAGCAACCCTATT-3 ') and 3′-UTR-R (5' -GATCCAGACGTTAGCTCTTCC-3 ') and probe (FAM-5′-AGATCCGCTATGACGAGCCAACAA-3'-BHQ1). The reaction was performed in a total volume of 20 μl / well in 48-well PCR plates using the StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) at 90 ° C for 30 s, 60 ° C for 20 minutes and at 95 ° C for 1 minute, followed by 45 cycles at 90 ° C for 15s and 60 ° C for 1 minute. To obtain control RNA for quantification, cDNA fragments amplified with primers 3'-UTR-F and 3'-UTR-R were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). The linearized and purified plasmid was transcribed using the RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, WI, USA) according to the manufacturer's instructions. The concentration of transcribed RNA was measured using a spectrophotometer. Ten-fold dilutions of RNA transcript ranging from 1 × 10 were prepared1 up to 1 × 109 copies / μl with 10ng MS2 RNA per ml. RNA copy number was calculated according to the procedure described in Fronhoffs et al. [25]. In vitro stock solutions of transcribed RNA were stored at -80 ° C and diluted working solutions were stored at -30 ° C.

Protein sequence

The deduced amino acid sequences of feline and human TNF-alpha were obtained from RefSeq (accession numbers NP_001009835.1 and NP_000585.2). Comparison of the deduced amino acid sequences of feline and human TNF-alpha was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

The results

Neutralizing activity of ADA against rfTNF-alpha

A comparison of the deduced amino acid sequences of feline and human TNF-alpha is shown in FIG. 2. The sequences were found to be identical to 89.7%.

Figure 2.
Amino acid sequence comparison of feline and human TNF-alpha. *: residues that interact with adalimumab

The neutralizing activity of ADA against rfTNF-alpha was measured using the WEHI-164 cytotoxicity assay. ADA neutralized rfTNF-alpha activity in a dose-dependent manner, similar to anti-fTNF-alpha mAb 2-4 (Fig. 3). The dose of mAb2-4 and ADA required to inhibit 50% cell death (IC50) was 18.1 ± 4.9 ng / ml and 36.9 ± 23.4 ng / ml, respectively.

Figure 3.
Neutralization dose-response curve against recombinant fTNF-alpha. ADA neutralizing activity against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. WEHI-164 cells were treated with mixtures of serial dilutions of ADA and fTNF-alpha and the level of TNF-alpha-induced cytotoxicity was measured after 24 hours.

Changes in body temperature and body weight

Ten SPF cats were inoculated intraperitoneally with 103.6 TCID50 FIPV KU-2 type I. Three cats (2, 3 and 6) showed fever, anorexia, lethargy and jaundice. Abdominal distension caused by ascites was observed in cat no. 6. These 3 cats were used in the following experiments as FIP cats. Treatment for cats 2, 3 and 6 was started on days 34, 32 and 21 after virus vaccination.

The changes in body temperature and body weight of the three cats are shown in FIG. 4. All developed fever ≥ 39 ° C several days before treatment (Fig. 4A). In cats 2 and 3, body temperature decreased to a pre-FIP range on day 7 after the start of treatment. Then the body temperature of cat no. 3 increased to 38.7 ° C on day 31 after the start of treatment. Cat body temperature no. 6 remained at ≥ 39.0 ° C even after the start of treatment.

Figure 4.
Changes in body temperature and weight of cats. †: The cat was euthanized because its clinical condition reached a human endpoint

In cats 2 and 3, no weight loss was observed compared to body weight on day 0 of treatment (Fig. 4B). Body weight of cat no. 6 continued to decrease from day 0 to day 15, resulting in a 8% loss compared to day 0.

Changes in WBC and lymphocyte counts

Total white blood cell and lymphocyte counts were measured in FIPV-infected cats. Cats 2 and 3 showed a small change in total white blood cell count throughout the experiment (Fig. 5A). In cat no. 6, the total white blood cell count increased after the start of treatment and remained at ≥ 20,000 cells / μl after day 6.

Figure 5.
Changes in WBC and lymphocyte counts in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

In cats 2 and 3, the number of lymphocytes increased slightly after the start of treatment (Fig. 5B). In cat no. 6, the lymphocyte count remained low throughout the experiment. It decreased to approximately 800 cells / μl on day 6 after the start of treatment, but recovered to near pre-FIP levels on day 13 and did not show a significant change up to the human endpoint.

Changes in alpha1-acid glycoprotein (AGP) plasma concentrations

Plasma alpha 1-AGP levels were measured sequentially in three cats. In all three cats, it increased to a high level ≥ 750 μg / ml at the start of treatment (Fig. 6). In cats 2 and 3, plasma alpha 1-AGP levels decreased to near pre-FIP levels on days 14 and 16 after treatment. However, in cat no. 6, the increase in plasma alpha 1-AGP levels was suppressed until day 6 after the start of treatment, but increased to approximately 1,900 μg / ml on day 13.

Figure 6.
Changes in plasma alpha1-acid glycoprotein (AGP) levels in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

Changes in plasma vascular endothelial growth factor (VEGF) concentration

Plasma VEGF levels were measured sequentially in three cats. In cat no. 2 remained low (FIG. 7). In cat no. 3, increased temporarily to approximately 200 pg / ml on day 5 before treatment, but decreased to near pre-FIP levels on day 0 of treatment and showed no increase after treatment. In cat no. 6, plasma VEGF levels increased to approximately 170 pg / ml after FIPV inoculation on day 0 of treatment. It then decreased from day 6 of treatment and was similar to the level before the onset of FIP on day 13.

Figure 7.
Changes in plasma vascular endothelial growth factor (VEGF) levels in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

Levels of viral RNA in supernatant and ascites cells in cat no. 6

Levels of viral RNA in the supernatant and ascites cells collected from cat no. 6 were quantified by qRT-PCR. The level of viral RNA temporarily decreased on day 4 after the start of treatment in the supernatant and in the ascites cells (Fig. 8). However, on day 20 after the start of treatment, viral RNA levels in the supernatant and ascites cells doubled on day 0 of treatment.

Figure 8.
Levels of viral RNA in the supernatant and ascites cells collected from cat no. 6 were quantified by qRT-PCR. The level of viral RNA temporarily decreased on day 4 after the start of treatment in the supernatant and in the ascites cells (Fig. 8). However, on day 20 after the start of treatment, viral RNA levels in the supernatant and ascites cells doubled on day 0 of treatment.

Biochemical panel

After treatment, TP and A / G ratio, which are diagnostic indices of FIP, and AST, LDH, and TB, which are related to liver function, were measured (Fig. 9). TP increased slightly in cat no. 3, but remained within the normal range for all three cats. On the other hand, the A / G ratio decreased after FIPV exposure in all three cats and was ≤ 0.8 on day 0 of treatment. In cat no. 2, the A / G ratio remained similar to that after the start of treatment until the end of the experiment. In cat no. 3, decreased until day 16 of treatment, but then showed no further decrease. In cat no. 6 the decrease continued even after the start of treatment.

Figure 9.
Biochemical panel. The white areas of the graphs represent normal ranges. †: The cat was euthanized because its clinical condition reached a human endpoint

AST, LDH and TB were higher than the normal range on day 0 or 3 of treatment in all three cats. In cats 2 and 3, AST, LDH and TB decreased to near pre-FIP levels on day 7 or 9 after treatment. In cat no. 6, AST, LDH and TB were higher than the normal range on day 0 or 4 after the start of treatment and continued to increase until the end of the experiment.

Discussion

A strong therapeutic effect can be achieved when cats with FIP are administered an anti-fTNF-alpha mAb, which suppresses TNF-alpha activity, a FIP exacerbator, in combination with ICZ, which reduces FIPV proliferation. However, approving an anti-fTNF-alpha mAb as a veterinary drug in each country is costly and time consuming. Therefore, we focused on the anti-human TNF-alpha mAb, which is already used to treat human inflammatory diseases.

Prior to animal experiments, we investigated whether the anti-human TNF-alpha mAb could neutralize fTNF-alpha. ADA, an anti-human TNF-alpha mAb, demonstrated dose-dependent neutralizing activity against rfTNF-alpha, similar to anti-fTNF-alpha mAb 2-4. The nine previously reported anti-fTNF-alpha mAbs had IC50s ranging from 5 to 700 ng / ml against rfTNF-alpha at the same concentration [13]. The IC50 of ADA (36.9 ± 23.4 ng / ml) indicates that it has a high neutralizing activity against fTNF-alpha compared to these anti-fTNF-alpha mAbs. As a rule, anti-human cytokine mAbs do not show any cross-reactive neutralizing activity against animal cytokines. However, Aguirre et al. reported that human and feline TNF-alpha have similar neutralizing epitopes [26]. In addition, the deduced amino acid sequence of human TNF-alpha at 89.7% is identical to that of feline TNF-alpha, and both have 10 of the 12 residues that interact with ADA. [27]. This appears to be the reason for the strong neutralizing activity of adalimumab against feline TNF-alpha.

We administered ADA and ICZ to cats with experimentally induced FIP and investigated their therapeutic effect. Of the 10 SPF cats that received intraperitoneally administered FIPV KU-2 type I, three developed FIP. Of these three cats, cat no. 6 had apparent ascites. These three cats were considered to have developed FIP for the following reasons: (i) all three cats were infected with FIPV, (ii) clinical signs associated with FIP were observed, (iii) elevated plasma alpha-1 levels of AGP and VEGF, and (iv) as a reference data from our previous animal experiments were used. ADA (10 mg / animal) was administered intravenously to three cats on day 0 and within 4 days after the start of treatment. In addition, ICZ (50 mg / animal) was administered orally every day, starting on day 0 of treatment. In cats 2 and 3, an improvement in clinical signs and blood test results, an increase in peripheral blood lymphocytes and a decrease in plasma alpha 1-AGP levels were observed after initiation of treatment. Symptomatic improvements were observed between weeks 1 and 2 after the start of treatment. Furthermore, no ascites or pyogenic granulomas were pathologically observed in cats 2 and 3 at the end of the experiment, and no changes suggestive of recurrence of FIP were observed by the end of the treatment period. Cat no. 6 did not respond to treatment and was euthanized, although the level of the viral ascites gene temporarily decreased after the start of treatment. These results strongly suggest that anti-human TNF-alpha mAbs and ICZs are effective in the treatment of FIP.

VEGF is produced by FIPV-infected macrophages and improves feline endothelial cell permeability [28]. In cats with FIP, plasma VEGF levels correlate with ascites. Cat no. 6, in whom ascites was evident on both preliminary and palpation examinations (160 ml of ascites was obtained by euthanasia), had a high plasma VEGF level ≥100 pg / ml for 3 weeks. However, in cats 2 and 3, which showed no signs of ascites, plasma VEGF levels remained low or increased only temporarily. Thus, plasma VEGF levels were confirmed to correlate with the amount of ascites in cats with FIP.

Azole antifungal agents, including ICZ, are known to have hepatotoxicity as a side effect and are likely to cause cholestasis and jaundice. [29,30,31,32]. Because an increase in liver enzymes is observed in cats with FIP, the development of side effects after ICZ administration would be worrying. However, in cats 2 and 3, liver marker markers, which increased during treatment, returned to normal and the plasma jaundice disappeared. Therefore, no adverse effects of ICZ have been reported. Therefore, in terms of side effects, ICZ administration is considered safe for FIP cats.

Recently, GC-376, a 3C-like protease inhibitor, and GS-441524, a nucleoside analog, have been developed as drugs to inhibit FIPV proliferation. [16,17,18,19,20]. GC-376 and GS-441524 are effective drugs that have therapeutic effects in 30-80% cats with FIP. Although these drugs are expected to be useful in the treatment of FIP, they have not yet been approved. Therefore, no drugs are available in clinical practice to treat FIP. In this study, we demonstrated that the combined use of ADA and ICZ, which is currently available to veterinarians, is effective in the treatment of FIP. We consider these drugs to be a treatment option until antiviral drugs such as GC-376 and GS-441524 become available. In addition, these antiviral drugs have therapeutic effects on FIP by mechanisms different from ADA and ICZ [16, 18, 21, 23]. In the future, evaluating the therapeutic effects of their current use may help to develop a more effective treatment for FIP.

Conclusions

The therapeutic effects of adalimumab, anti-human TNF-alpha mAb and itraconazole on FIP were investigated. Adalimumab was found to have neutralizing activity against rfTNF-alpha, similar to anti-fTNF-alpha mAbs. FIP was successfully treated in 2 of 3 cats with experimentally induced FIP by adalimumab and itraconazole.

References

  1. Pedersen NC (2014) An update on feline infectious peritonitis: diagnostics and therapeutics. Vet J 201: 133–141. https://doi.org/10.1016/j.tvjl.2014.04.016Article PubMed Google Scholar 
  2. Tekes G, Thiel HJ (2016) Feline coronaviruses: pathogenesis of feline infectious peritonitis. In: Ziebuhr J (ed) Advances in virus research, vol. 96, Academic Press, pp 193–218
  3. Jaimes JA, Whittaker GR (2018) Feline coronavirus: insights into viral pathogenesis based on the spike protein structure and function. Virology 517: 108–121. https://doi.org/10.1016/j.virol.2017.12.027TIME Article PubMed Google Scholar 
  4. Decaro N, Mari V, Campolo M et al (2009) Recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs. J Virol 83: 1532–1537. https://doi.org/10.1128/jvi.01937-08TIME Article PubMed Google Scholar 
  5. Herrewegh AA, Smeenk I, Horzinek MC et al (1998) Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus. J Virol 72: 4508–4514TIME Article PubMed Google Scholar 
  6. Terada Y, Shiozaki Y, Shimoda H et al (2012) Feline infectious peritonitis virus with a large deletion in the 59-terminal region of the spike gene retains its virulence for cats. J Gen Virol 93: 1930–1934. https://doi.org/10.1099/vir.0.043992-0TIME Article PubMed Google Scholar 
  7. Addie DD, Schaap IAT, Nicolson L, Jarrett O (2003) Persistence and transmission of natural type I feline coronavirus infection. J Gen Virol 84: 2735–2744. https://doi.org/10.1099/vir.0.19129-0TIME Article PubMed Google Scholar 
  8. Kummrow M, Meli ML, Haessig M et al (2005) Feline coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland. Clin Diagn Lab Immunol 12: 1209–1215. https://doi.org/10.1128/CDLI.12.10.1209-1215.2005TIME Article PubMed Google Scholar 
  9. Hohdatsu T, Okada S, Ishizuka Y et al (1992) The prevalence of types I and II feline coronavirus infections in cats. J Vet Med Sci 54: 557–562. https://doi.org/10.1292/jvms.54.557TIME Article PubMed Google Scholar 
  10. Takano T, Hohdatsu T, Hashida Y et al (2007) A “possible” involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis. Vet Microbiol 119: 121–131. https://doi.org/10.1016/j.vetmic.2006.08.033TIME Article PubMed Google Scholar 
  11. Takano T, Hohdatsu T, Toda A et al (2007) TNF-alpha, produced by feline infectious peritonitis virus (FIPV) -infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages. Virology 364: 64–72. https://doi.org/10.1016/j.virol.2007.02.006TIME Article PubMed Google Scholar 
  12. Takano T, Azuma N, Satoh M et al (2009) Neutrophil survival factors (TNF-alpha, GM-CSF, and G-CSF) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions. Arch Virol 154: 775–781. https://doi.org/10.1007/s00705-009-0371-3TIME Article PubMed Google Scholar 
  13. Doki T, Takano T, Nishiyama Y et al (2013) Generation, characterization and therapeutic potential of anti-feline TNF-alpha MAbs for feline infectious peritonitis. Res Vet Sci 95: 1248–1254. https://doi.org/10.1016/j.rvsc.2013.09.005TIME Article PubMed Google Scholar 
  14. Doki T, Takano T, Kawagoe K et al (2016) Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis. Res Vet Sci 104: 17–23. https://doi.org/10.1016/j.rvsc.2015.11.005TIME Article PubMed Google Scholar 
  15. Tracey D, Klareskog L, Sasso EH et al (2008) Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther 117: 244–279TIME Article PubMed Google Scholar 
  16. Kim Y, Lovell S, Tiew KC et al (2012) Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses. J Virol 86: 11754–11762. https://doi.org/10.1128/JVI.01348-12TIME Article PubMed Google Scholar 
  17. Kim Y, Liu H, Galasiti Kankanamalage AC et al (2016) Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog 12: e1005531. https://doi.org/10.1371/journal.ppat.1005531TIME Article PubMed Google Scholar 
  18. Murphy BG, Perron M, Murakami E et al (2018) The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet Microbiol 219: 226–233. https://doi.org/10.1016/j.vetmic.2018.04.026TIME Article PubMed Google Scholar 
  19. Pedersen NC, Kim Y, Liu H et al (2018) Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg 20: 378–392. https://doi.org/10.1177/1098612X17729626Article PubMed Google Scholar 
  20. Pedersen NC, Perron M, Bannasch M et al (2019) Efficacy and safety of the nucleoside analog GS-441524 for the treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg 21: 271–281. https://doi.org/10.1177/1098612X19825701Article PubMed Google Scholar 
  21. Takano T, Akiyama M, Doki T, Hohdatsu T (2019) Antiviral activity of itraconazole against type I feline coronavirus infection. Vet Res 50: 5. https://doi.org/10.1186/s13567-019-0625-3Article PubMed Google Scholar 
  22. Takano T, Endoh M, Fukatsu H et al (2017) The cholesterol transport inhibitor U18666A inhibits type I feline coronavirus infection. Antivir Res 145: 96–102. https://doi.org/10.1016/j.antiviral.2017.07.022TIME Article PubMed Google Scholar 
  23. Takano T, Wakayama Y, Doki T (2019) Endocytic pathway of feline coronavirus for cell entry: differences in serotype-dependent viral entry pathway. Pathogens 8: 300. https://doi.org/10.3390/pathogens8040300TIME Article Google Scholar 
  24. Koga L, Kobayashi Y, Yazawa M et al (2002) Nucleotide sequence and expression of the feline vascular endothelial growth factor. J Vet Med Sci 64: 453–456. https://doi.org/10.1292/jvms.64.453TIME Article PubMed Google Scholar 
  25. Fronhoffs S, Totzke G, Stier S et al (2002) A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol Cell Probes 16: 99–110. https://doi.org/10.1006/mcpr.2002.0405TIME Article PubMed Google Scholar 
  26. Aguirre A, Escobar A, Ferreira V et al (2003) An anti-human recombinant tumor necrosis factor alpha (TNFα) monoclonal antibody recognizes an epitope in the TNFα feline. Vet Res 34: 177–184. https://doi.org/10.1051/vetres:2002064TIME Article PubMed Google Scholar 
  27. Hu S, Liang S, Guo H et al (2013) Comparison of the inhibition mechanisms of Adalimumab and Infliximab in treating tumor necrosis factor α-associated diseases from a molecular view. J Biol Chem 288: 27059–27067. https://doi.org/10.1074/jbc.M113.491530TIME Article PubMed Google Scholar 
  28. Takano T, Ohyama T, Kokumoto A et al (2011) Vascular endothelial growth factor (VEGF), produced by feline infectious peritonitis (FIP) virus-infected monocytes and macrophages, vascular permeability induces and effusion in cats with FIP. Virus Res 158: 161–168. https://doi.org/10.1016/j.virusres.2011.03.027TIME Article PubMed Google Scholar 
  29. García Rodrǐguez LA, Duque A, Castellsague J et al (1999) A cohort study on the risk of acute liver injury among users of ketoconazole and other antifungal drugs. Br J Clin Pharmacol 48: 847–852. https://doi.org/10.1046/j.1365-2125.1999.00095.xArticle PubMed Google Scholar 
  30. Kao WY, Su CW, Huang YS et al (2014) Risk of oral antifungal agent-induced liver injury in Taiwanese. Br J Clin Pharmacol 77: 180–189. https://doi.org/10.1111/bcp.12178TIME Article PubMed Google Scholar 
  31. Susan M, Grant SPC (1989) Itraconazole. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in superficial and systemic mycoses. Drugs 37: 310–344Article Google Scholar 
  32. Khoza S, Moyo I, Ncube D (2017) Comparative hepatotoxicity of fluconazole, ketoconazole, itraconazole, terbinafine, and griseofulvin in rats. J Toxicol. https://doi.org/10.1155/2017/6746989Article PubMed Google Scholar 

Thanks

We thank the members of our laboratory for supporting animal experiments. This work was partially supported by KAKENHI (Grants-in-Aid for Scientific Research (B), No. 16H05039) from the Ministry of Education, Culture, Sports, Science and Technology in Japan and the Kitasato University Research Grants for Kitasato University Young Scientists.

Ethical statements

Conflict of interests

The authors are not aware of any potential conflicts of interest in connection with the research, authorship or publication of this article.

Ethical approval

All relevant national and institutional guidelines for the care and use of animals have been complied with. The protocol on animal experimentation was approved by the President of Kitasato University based on the decision of the Institutional Committee on the Care and Use of Animals at Kitasato University (Approval No. 18-152). SPF cats were bred in our own laboratory and maintained in an isolated temperature-controlled facility. Sample sizes were determined based on our experience with FIPV infection models and a minimum number of cats were used.

Informed consent

Informed consent was obtained from the legal guardian of all experimental animals described in this work for the procedures performed. No animals or humans are identifiable in this publication and therefore no further informed consent to the publication was required.

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Editor: Sheela Ramamoorthy. Read "Therapeutic effect of anti-human TNF-alpha antibody and itraconazole in the treatment of FIP"

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

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

Abstract

Goals

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

Methods

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

The results

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

Conclusion and meaning

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

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

Introduction

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

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

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

Materials and methods

Preparation of the drug

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

Study concept

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

Case selection and diagnosis confirmation

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

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

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

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

Treatment regimen

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

Monitoring during the initial treatment period

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

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

Monitoring of initial and long-term response to treatment

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

The results

Disease signaling and presentation

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

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

Treatment results

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

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

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

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

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

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

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

Indicators of favorable response to treatment

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

CBCs (Hematology)

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

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

Serum protein changes

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

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

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

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

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

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

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

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

Side effects observed during and after treatment

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

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

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

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

Autopsy findings

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

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

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

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

Discussion

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

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

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

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

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

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

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

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

Conclusions

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

Additional material

Owner consent form

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

Thanks

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

Notes

Received: December 28, 2018

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

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

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

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

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

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

References

1.De Clercq, E, Li, G. Approved antiviral drugs over the past 50 years. Clin Microbiol Rev 2016; 29: 695–747.
Google Scholar | Crossref | Medline
2.Hartmann, K. Efficacy of antiviral chemotherapy for retrovirus-infected cats: what does the current literature tell us? J Feline Med Surg 2015; 17: 925–939.
Google Scholar | SAGE Journals | ISI
3.Thomasy, SM, Shull, O, Outerbridge, CA. Oral administration of famciclovir for treatment of spontaneous ocular, respiratory, or dermatologic disease attributed to feline herpesvirus type 1: 59 cases (2006–2013). J Am Vet Med Assoc 2016; 249: 526–538.
Google Scholar | Crossref | Medline
4.Pedersen, NC, Elliott, JB, Glasgow, A. An isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus. Vet Microbiol 2000; 73: 281–300.
Google Scholar | Crossref | Medline | ISI
5.Kim, Y, Shivanna, V, Narayanan, S. Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses. J Virol 2015; 89: 4942–4950.
Google Scholar | Crossref | Medline
6.Pedersen, NC, Kim, Y, Liu, H. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg 2018; 20: 378–392.
Google Scholar | SAGE Journals | ISI
7.Pesteanu-Somogyi, LD, Radzai, C, Pressler, BM. Prevalence of feline infectious peritonitis in specific cat breeds. J Feline Med Surg 2006; 8: 1–5.
Google Scholar | SAGE Journals | ISI
8.Riemer, F, Kuehner, KA, Ritz, S. Clinical and laboratory features of cats with feline infectious peritonitis - a retrospective study of 231 confirmed cases (2000–2010). J Feline Med Surg 2016; 18: 348–356.
Google Scholar | SAGE Journals | ISI
9.Rohrbach, BW, Legendre, AM, Baldwin, CA. Epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals. J Am Vet Med Assoc 2001; 218: 1111–1115.
Google Scholar | Crossref | Medline | ISI
10.Warren, TK, Jordan, R, Lo, MK. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531: 381–385.
Google Scholar | Crossref | Medline | ISI
11.Sheahan, TP, Sims, AC, Graham, RL. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 2017; 9. DOI: 10.1126 / scitranslmed.aal3653.
Google Scholar | Crossref
12.Murphy, BG, Perron, M, Murakami, E. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet Microbiol 2018; 219: 226–233.
Google Scholar | Crossref | Medline
13.Takano, T, Endoh, M, Fukatsu, H. The cholesterol transport inhibitor U18666A inhibits type I feline coronavirus infection. Antiviral Res 2017; 145: 96–102.
Google Scholar | Crossref | Medline
14.Takano, T, Nakano, K, Doki, T. Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II. Arch Virol 2015; 160: 1163–1170.
Google Scholar | Crossref | Medline
15.Kim, Y, Liu, H, Galasiti Kankanamalage, AC. Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog 2016; 12: DOI: 10.1371 / journal.ppat.1005531.
Google Scholar | Crossref
16.Gut, M, Leutenegger, CM, Huder, JB. One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses. J Virol Methods 1999; 77: 37–46.
Google Scholar | Crossref | Medline | ISI
17.Agostini, ML, Andres, EL, Sims, AC. Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI: 10.1128 / mBio.00221-18.
Google Scholar
18.Weiss, G, Goodnough, LT. Anemia of chronic disease. N Engl J Med 2005; 352: 1011–1023.
Google Scholar | Crossref | Medline | ISI
19.Kurnick, JE, Ward, HP, Pickett, JC. Mechanism of the anemia of chronic disorders: correlation of hematocrit value with albumin, vitamin B12, transferrin, and iron stores. Arch Intern Med 1972; 130: 323–326.
Google Scholar | Crossref | Medline
20.Taylor, SS, Tappin, SW, Dodkin, SJ. Serum protein electrophoresis in 155 cats. J Feline Med Surg 2010; 12: 643–653.
Google Scholar | SAGE Journals | ISI
21.Hirschberger, J, Hartmann, K, Wilhelm, N. Clinical symptoms and diagnosis of feline infectious peritonitis. Tierarztl Prax 1995; 23: 92–99.
Google Scholar | Medline
22.Pedersen, NC, Liu, H, Gandolfi, B. The influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis. Vet Immunol Immunopathol 2014; 28: 152–154.
Google Scholar
23.Jain, N, Reddy, DH, Verma, SP. Cardiac abnormalities in HIV-positive patients: results from an observational study in India. J Int Assoc Provid AIDS Care 2014; 13: 40–46.
Google Scholar | SAGE Journals
24.Payne, JR, Brodbelt, DC, Luis Fuentes, V. Cardiomyopathy prevalence in 780 apparently healthy cats in rehoming centers (the CatScan study). J Vet Cardiol 2015; 17 Suppl 1: S244 – S257.
Google Scholar | Crossref | Medline
Read "Efficacy and safety of nucleoside analogue GS-441524 in the treatment of cats with naturally occurring feline infectious peritonitis"

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

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

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

Abstract

The goal

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

Methods

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

The results

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

Conclusions and significance

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

Introduction

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

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

Materials and methods

Official protocols

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

Organization of a clinical trial

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

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

Test group description

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

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

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

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

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

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

Confirmation of the disease

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

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

Treatment regimen

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

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

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

The results

Determination of treatment duration

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

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

Response to initial treatment and indicators of favorable response

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Viral resistance testing

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

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

Occurrence of permanent clinical remissions

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

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

Side effects observed during and after treatment

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

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

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

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

Autopsy findings

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

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

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

Discussion

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

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

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

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

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

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

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

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

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

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

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

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

Conclusions

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

Footnotes

Received: 3.8.2017

Additional material: Informed consent form of the owner.

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

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

References

1.Prokofiev, MM, Kochetkov, SN, Prassolov, VS. Therapy of HIV infection: current approaches and prospects. Acta Naturae 2016; 8: 23–32.
Google Scholar | Crossref | Medline
2.Carter, W., Connelly, S., Struble, K. Reinventing HCV treatment: past and future perspectives. J Clin Pharmacol 2017; 57: 287–296.
Google Scholar | Crossref | Medline
3.Kim, Y, Lovell, S, Tiew, KC. Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses. J Virol 2012; 86: 11754–11762.
Google Scholar | Crossref | Medline
4.Kim, Y, Mandadapu, SR, Groutas, WC. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease. Antiviral Res 2013; 97: 161–168.
Google Scholar | Crossref | Medline
5.Kim, Y, Shivanna, V, Narayanan, S. Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses. J Virol 2015; 89: 4942–4950.
Google Scholar | Crossref | Medline
6.Kim, Y, Liu, H, Galasiti Kankanamalage, AC. Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog 2016; 12: e1005531.
Google Scholar | Crossref | Medline | ISI
7.Miller, FG, Brody, H. What makes placebo-controlled trials unethical? Am J Bioethics 2002; 2: 3–9.
Google Scholar | Crossref | Medline
8.Chiodo, GT, Tolle, SW, Bevan, L. Placebo-controlled trials good science or medical neglect? West J Med 2000; 172: 271–273.
Google Scholar | Crossref | Medline
9.Van Kruiningen, HJ, Ryan, MJ, Shindel, NM. The classification of feline colitis. J Comp Pathol 1983: 93: 275–294.
Google Scholar | Crossref | Medline | ISI
10.Pedersen, NC, Eckstrand, C, Liu, H. Levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection. Vet Microbiol 2015; 175: 157–166.
Google Scholar | Crossref | Medline | ISI
11.Pellerin, M, Lopez-Aquirre, Y, Penin, F. Hepatitis C virus quasispecies variability modulates nonstructural protein 5A transcriptional activation, pointing to cellular compartmentalization of virus-host interactions. J Virol 2004; 78: 4617–4627.
Google Scholar | Crossref | Medline
12.Pedersen, NC, Allen, CE, Lyons, LA. Pathogenesis of feline enteric coronavirus infection. J Feline Med Surg 2008; 10: 529–541.
Google Scholar | SAGE Journals | ISI
13.Pedersen, NC. A review of feline infectious peritonitis virus infection: 1963–2008. J Feline Med Surg 2009; 11: 225–258.
Google Scholar
14.Pedersen, NC. An update on feline infectious peritonitis: virology and immunopathogenesis. Vet J 2014; 201: 123–132.
Google Scholar | Crossref | Medline
15.Pedersen, NC, Liu, H, Durden, M. Natural resistance to experimental feline infectious peritonitis virus infection is decreased rather than increased by positive genetic selection. Vet Immunol Immunopathol 2016; 171: 17–20.
Google Scholar | Crossref | Medline | ISI
16.Pedersen, NC, Liu, H, Gandolfi, B. The influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis. Vet Immunol Immunopathol 2014; 162: 33–40.
Google Scholar | Crossref | Medline
17.Clifford, DB. HIV-associated neurocognitive disease continues in the antiretroviral era. Top HIV Med 2008; 16: 94–98.
Google Scholar | Medline
18.Foley, JE, Lapointe, JM, Koblik, P. Diagnostic features of clinical neurologic feline infectious peritonitis. J Vet Intern Med 1998; 12: 415–423.
Google Scholar | Crossref | Medline | ISI
19.Mesquita, LP, Hora, AS, de Siqueira, A. Glial response in the central nervous system of cats with feline infectious peritonitis. J Feline Med Surg 2016; 18: 1023–1030.
Google Scholar | SAGE Journals | ISI
20.Pedersen, NC. Feline infectious peritonitis: something old, something new. Feline Pract 1976; 6: 42–51.
Google Scholar
21.Valcour, V, Sithinamsuwan, P, Letendre, S. Pathogenesis of HIV in the central nervous system. Curr HIV / AIDS Rep 2011; 8: 54–61.
Google Scholar | Crossref | Medline
22.Joseph, SB, Arrildt, KT, Sturdevant, CB. HIV-1 target cells in the CNS. J Neurovirol 2015; 21: 276–289.
Google Scholar | Crossref | Medline
23.Spudich, S, Gonzalez-Scarano, F. HIV-1-related central nervous system disase: current issues in pathogenesis, diagnosis and treatment. Cold Spring Harb Perspect Med 2012; 2: a007120
Google Scholar | Crossref | Medline
24.Cavalcante, LN, Lyra, AC. Predictive factors associated with hepatitis C antiviral therapy response. World J Hepatology 2015; 7: 1617–1631.
Google Scholar | Crossref | Medline
25.Saunders, BM, Cooper, AM. Restraining mycobacteria: role of granulomas in mycobacterial infections. Immunol Cell Biol 2000; 78: 334–334.
Google Scholar | Crossref | Medline
26.Smyk-Pearson, S, Tester, IA, Klarquist, J. Spontaneous recovery in acute human hepatitis C virus infection: functional T-cell thresholds and relative importance of CD4 help. J Virol 2008; 82: 1827–1837.
Google Scholar | Crossref | Medline
27.Legendre, AM, Bartges, JW. Effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis. J Feline Med Surg 2009; 11: 624–626.
Google Scholar | SAGE Journals | ISI
28.Legendre, AM, Kuritz, T, Galyon, G. Polyprenyl immunostimulant treatment of cats with presumptive non-effusive feline infectious peritonitis in a field study. Front Vet Sci 2017; 4: 7.
Google Scholar | Crossref | Medline
Read "Efficacy of a 3C-Like protease inhibitor in the treatment of various forms of acquired feline infectious peritonitis"

Pharmacokinetic profile of oral mefloquine in clinically normal cats: Preliminary in vivo study of the potential treatment of feline infectious peritonitis (FIP).

Jane Yu, Benjamin Kimble, Jacqueline M. Norris and Merran Govendir
Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia; jane.yu@sydney.edu.au (JY); benjamin.kimble@sydney.edu.au (BK); jacqui.norris@sydney.edu.au (JN); merran.govendir@sydney.edu.au
Original article: Pharmacokinetic Profile of Oral Administration of Mefloquine to Clinically Normal Cats: A Preliminary In ‐ Vivo Study of a Potential Treatment for Feline Infectious Peritonitis (FIP)
8.6.2020

Brief summary:

In the search for antiviral agents against feline coronaviruses and feline caliciviruses, mefloquine, a human antimalarial drug, has been shown to reduce the viral load of feline coronaviruses and feline calicivirus in infected cells. In this study, mefloquine was administered orally to seven clinically healthy cats twice a week in four doses, and blood mefloquine concentrations were measured to determine the pharmacokinetic profile - drug movement in the body. The maximum blood concentration of mefloquine was 2.71 ug / ml and was reached 15 hours after a single oral dose. Side effects of mefloquine in some cats included vomiting after feeding without food and a slight increase in symmetric dimethylarginine (SDMA), an early renal biomarker. This study provides valuable information on the profile of mefloquine in cats as an initial step to its research as a possible treatment for feline coronavirus and feline calicivirus infection in cats.

Abstract: The pharmacokinetic profile of mefloquine was investigated to preliminary study the potential treatment of feline coronavirus infections (such as feline infectious peritonitis) or feline calicivirus infections. Mefloquine was administered orally at a dose of 62.5 mg to seven clinically healthy cats twice a week in four doses, and plasma mefloquine concentrations over 336 hours were measured by high performance liquid chromatography (HPLC). The maximum plasma concentration (Cmax) after a single oral dose of mefloquine was 2.71 ug / ml and the time to Cmax (Tmax) was 15 h. The elimination half-life was 224 h. Plasma concentrations reached a higher level of 4.06 ug / ml when mefloquine was fed. Dosage side effects in some cats included vomiting after administration without food. A slight increase in symmetric dimethylarginine (SDMA) but not creatinine serum concentrations was observed. Mefloquine can provide a safe and effective treatment for coronavirus and calicivirus infections in cats. 

Keywords: mefloquine; feline infectious peritonitis; pharmacokinetics; coronavirus; calicivirus 

Introduction

Feline coronavirus (FCoV) is an alpha coronavirus that occurs in two distinct pathotypes that can be distinguished by biological behavior but not by morphology [1]. Although the two pathotypes belong to the same type of virus, different names are used - feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV is highly contagious by the fecal-oral route. The infection is usually asymptomatic or can cause mild diarrhea [1,2]. FIP is a deadly, immune-mediated disease caused by virulent FCoV biotypes known as feline infectious peritonitis virus (FIPV) in domestic and wild cats. When cats with FIP show clinical signs, life expectancy ranges from a few days to a few weeks for the effusive form and from a few weeks to a few months for the non-fusive form [3-6]. However, there are a small number of cats that can survive for several years [7,8]. Treatment options are traditionally limited, but recent experimental treatments using protease inhibitors and nucleoside analogs have yielded promising results [9-13], although these treatment options have not yet been registered for the treatment of cats. The lack of available treatment options exacerbates the need to explore other available antiviral treatments. In addition, the search for treatment targeting various aspects of FCoV replication is of therapeutic value, as combination therapy for other viral infections is associated with a higher rate of pathogen suppression and minimizing the development of antiviral resistance [14,15]. 

Feline calicivirus is an important and common cause of upper respiratory tract disease and oral ulceration in cats, with more virulent forms of the virus emerging recently, leading to an outbreak of systemic disease, which is often fatal and, like FIP, has no effective antiviral treatment. [16] 

In the search for antiviral agents against feline coronavirus and feline calicivirus, mefloquine, a human antimalarial, has been shown to inhibit FCoV viral load in infected Crandell feline kidney cells without cytotoxic effects [17]. Its inhibition of cytopathic effects and viral replication at low concentrations supports further investigation of this drug as a potential antiviral therapeutic. In our previous project, we developed an in vitro model to determine the extent and rate of hepatic clearance (Cl) of mefloquine [18]. In cats, mefloquine undergoes phase I hepatic metabolism but not phase II conjugative metabolism [18]. There is no evidence that mefloquine delays elimination in clinically healthy cats. Plasma protein binding of mefloquine is approximately 99 % in the plasma of clinically normal cats as well as in the plasma of cats with FIP [19].

As mefloquine is currently used for malaria prophylaxis, information on its availability is available, including absorption, distribution and rate and extent of elimination of the drug in adults and infants, with some information also available in dogs [20], but not at this stage. no information is available on the pharmacokinetic profile (PK) of mefloquine in cats other than plasma protein binding [19]. Therefore, in order to assess whether the administration of mefloquine as an antiviral has any therapeutic advantage, it is necessary to determine the pharmacokinetic profile of mefloquine in clinically normal cats in order to develop the dosage and frequency of doses. Recognition of the pharmacokinetic profile of mefloquine in a clinically normal cat is a transitional step that bridges preclinical observations of mefloquine to feline medication in the future. The aim of this study was to examine the pharmacokinetic profile of mefloquine when administered 62.5 mg (10-12 mg / kg) orally twice weekly. The second objective was to document any changes in hematological and / or biochemical analytes and physiological responses to mefloquine in this dosing regimen.

Materials and methods 

The animals 

Eight adult cats (4 females, 4 males) were acquired by Invetus Pty Ltd. (Casino, New South Wales, Australia), an animal research facility, from its clinically normal animals. Body weight ranged from 5.0 to 5.8 kg (average 5.4 kg). The cats were 3 to 7 years old (average 5.5 years). Cats were selected based on clinically normal physical examination, normal fitness score, and body weight ≥ 5 kg. Exclusion criteria included cats with an abnormal physical examination, underweight cats, or cats that took medication. Seven cats were originally selected for the study. The cats were housed individually in boxes at the kennel and supplied with food and water ad libitum. The selection of clinically normal cats, their dosing with mefloquine, blood collection and shelter was carried out by the company Invetus. This study was approved by the Wongaburra Research Center's Animal Ethics Committee as a USY F 18120 W project on August 29, 2019 and by the University of Sydney Animal Ethics Committee as Protocol 2019/1662.

Drug administration and sampling

The cats were given a mask for blood collection and anesthesia with isoflurane in 100 % oxygen, and 2-4 ml of blood was collected from the jugular veins using a 22 mm diameter needle. Blood was collected into lithium heparin tubes, ethylenediaminetetraacetic acid (EDTA) tubes and serum tubes for mefloquine quantification, hematology and biochemistry. A 250 mg tablet of mefloquine (Lariam, Roche, Millers Point, New South Wales, Australia) was quartered along the line on the tablet. For each cat, the dose was a quarter of a tablet or 62.5 mg of mefloquine. The weight of each quarter of the tablet was recorded for consistency of dosing. Mefloquine (62.5 mg) was administered orally to each cat on days 0, 4 (corresponding to 96 h), 7 (168 h) and 10 (240 h). The cats were then monitored for any adverse reactions 2 hours after dosing. To determine mefloquine plasma concentrations, serial blood samples were taken into lithium heparin tubes at time 0 (before treatment), 1, 2, 4, 8, 12, 24, 48, 96, 168, 240 and 336 h after drug administration. On mefloquine administration days other than the first administration day (t = 96, 168 and 240 h), blood samples were taken before the mefloquine dose, followed by wet or dry food for several minutes. Blood was also collected into EDTA and serum tubes for hematological and biochemical examination at 168 and 336 hours. The samples were centrifuged within 1 h after blood collection. Plasma and serum were immediately stored in a freezer (-20 ° C) within 90 minutes of blood collection. EDTA tubes were immediately shipped to Idexx East Brisbane, Qld. Australia, for hematological analysis. Serum tubes were sent to the Veterinary Pathology Diagnostic Services, The University of Sydney and the Idexx Reference Laboratory for biochemical analysis. Serum was also sent to the Idexx Reference Laboratory for analysis of serum symmetric dimethylarginine (SDMA) and creatinine. To determine plasma mefloquine concentrations, samples were analyzed at the Sydney School of Veterinary Science, The University of Sydney, within two months of blood collection. 

Drug analysis method and sample processing

The concentration of mefloquine in the samples was quantified by high pressure liquid chromatography (HPLC), and the plasma samples were adjusted according to a validated method [19]. 

Chemicals

Mefloquine, verapamil (as internal standard [IS]), sodium phosphate, trimethylamine and phosphoric acid were purchased from Sigma-Aldrich (Castle Hill, Sydney, New South Wales, Australia). Acetonitrile and HPLC grade methanol were purchased from Thermo Fisher Scientific (Macquarie Park, NSW, Australia).

HPLC conditions

The HPLC system consisted of a Shimadzu LC-20AT feed unit, a DGU-20A3 HT feed unit for solvent degassing, an SIL-20A automatic injector, a SPD-20A UV detector and a CTO-20A column furnace. Shimadzu LC Solution software (Kyoto, Japan) was used for chromatographic control, data collection and processing. Chromatographic separation was performed using a Polaris C18-A column (5 μm, 150 × 4.6 mm) with a 1.0 mm diameter Optic-guard C 18 precolumn (Choice Analytical, Thornleigh, NSW, Australia) while adjusting the column temperature. at 35 ° C. The isocratic mobile phase contained a mixture of 25 mM sodium phosphate with 0.5 L of TP 2 T triethylamine adjusted to pH 6.0 with phosphoric acid, acetonitrile and methanol (50:25:25) at a flow rate of 0.8 ml / min. For each sample, the injection volume was 10 μl and the total duration was 15 min. The diode array detector was set to a wavelength of 220 nm. 

Plasma mefloquine concentrations of 0.156, 0.313, 0.625, 1.25, 2.50 and 10.0 μg / ml were prepared by serial dilution for sample preparation. Solution IS was prepared in 100 % acetonitrile at a final concentration of 10 ug / ml. Feline plasma samples were used to prepare the standard curve. 

To extract proteins from plasma samples, 100 μl of acetonitrile containing 10 μg / ml IS was added to 100 μl of feline plasma samples. The samples were then vortexed and centrifuged at 14,000 for 10 minutes. Ten microliters of supernatant was injected into the HPLC system for analysis. 

Pharmacokinetic analysis 

The data were evaluated by non-compartmental analysis, as the elimination phase was evident at only two time points, ie 48 and 96 h. The mean maximum plasma concentration (C max) and the time to C max (T max) of the first dose were determined by visual inspection of the plasma concentration-time curve of each cat over 96 hours. The difference in the natural logarithm of the plasma concentrations at 24 and 96 hours, ie the slope of the curve from 24 to 96 hours, gave. The elimination half-life was estimated using ln 2 / ke. Area under the curve (AUC0 ‐ t) at 96 h was calculated to the last measurable concentration using the linear trapezoidal method. Apparent volume of distribution was calculated as: 

V / F = (Dose / AUC) × (1 / ke), (1)

where F is the oral bioavailability that cannot be determined because intravascular (intravenous) administration of mefloquine to cats has not occurred. Apparent clearance was calculated as: 

Cl / F = V × ke. (2)

Area under the torque curve (AUMC00-96h ) was calculated as the sum of the AUC when each of the concentration data was multiplied by time. The average residence time was calculated as 1 / ke.   

Statistical analysis

Two cats were excluded from the statistical analysis due to vomiting. Mefloquine plasma concentration data for five cats (including cat E) at 24, 96, 168, 240 and 336 h were subjected to the Shapiro-Wilk normality test and all distributions were normal. However, SDMA concentrations were not normal at t = 0 h, but were normal at 168 and 336 h. Creatinine concentrations were normal at 0, 168 and 336 h. Mean plasma mefloquine concentrations were compared at 24, 96, 168, 240 and 336 h and were subjected to repeated on-way ANOVA measurements, as were mean SDMA and creatinine concentrations at 0, 168 and 336 h. Tukey's multiple comparison test was used to demonstrate whether the mean values at each time point differed significantly. Statistical analysis was accepted if <0.05. Statistical analysis was performed using Graphpad Prism 8 (San Diego, CA, CA). 

The results

Following administration of 62.5 mg of mefloquine per cat, the mean dose was 11.8 mg / kg (median 12.3, range 10.8-12.5). The change in mefloquine plasma concentrations over 336 h (14 days) in seven cats is shown in Figure 1 and the actual mefloquine plasma concentrations of each cat at each time point are shown in Table 1. Figure 2 shows mefloquine plasma concentrations (ug / ml). during the first 24 hours. A single oral dose of mefloquine results in a Cmax of 2.71 μg / ml after the first dose, averaging 15 h (Tmax). Increases in mefloquine plasma concentrations were observed after 168, 240 and 336 h (Figure 1), after the second dose given just after 96 h, the third dose given just after 168 h and the fourth dose given just after 240 h, respectively when mefloquine was administered with food, with peak plasma concentrations reaching 4.06 μg / ml (average) after 240 h. One cat (cat C) vomited 15 minutes after dosing on day 0 (treatment 1). Mefloquine was re-administered to this cat on day 4 (treatment 2), but vomited approximately one hour after dosing and was therefore excluded from the study. Another cat (cat F) vomited 5 minutes after dosing on day 1 (treatment 1). This cat was successfully given a dose of mefloquine after feeding on the following day of treatment (96 h) and was re-enrolled in the study. This time, no vomiting was observed after dosing. Blood samples were taken from cat F at 168, 240 and 336 h as shown in Table 1. 

Figure 1. Mefloquine plasma concentrations (ug / ml) in seven cats over 336 hours (14 days after the first dose). Cats were treated with a dose of 62.5 mg per cat (10-12 mg / kg) after t = 0, 96, 168 and 240 h. Blood was collected just prior to treatment, as indicated by vertical dotted lines.
Figure 2. Plasma mefloquine concentrations (ug / ml) during the first 24 hours after administration to cats 62.5 mg (10-12 mg / kg) mefloquine at t = 0.
Cat DCat ACat BCat GCat ECat CCat F
HCastrated male Castrated maleCastrated female Castrated female Castrated male Castrated female Castrated female
00.00 0.00 0.00 0.00 0.00 0.00 0.00 
10.54 1.580.31 1.970.22 0.86 
20.95 1.950.89 2.790.40 2.19
41.132.101.843.230.49 
81.592.232.202.890.64 
121.772.972.143.220.72 
242.93.352.93.120.56 
481.863.341.972.880.56 
961.512.761.752.500.57 
1682.754.153.363.733.581.39
2401.856.512.934.944.201.54
3363.74.193.604.562.92.11
Table 1. Plasma mefloquine concentrations in individual cats (ug / ml). Cats C and F were excluded from the pharmacokinetic analysis (PK) due to incomplete data. Cat C was excluded from the study due to vomiting, while cat F vomited after the first dose, but was successfully re-enrolled in the study on the second day of treatment. Cat E was excluded from the PK analysis because the profile of this cat was remote and distorted the data.

Pharmacokinetic (PK) parameters are shown in Table 2. Cats C and F were excluded from the PK analysis due to incomplete data. Because the PK profile of cat E distorts the data, indices for four cats (cat A, B, D, and G) with more consistent profiles were used for analysis. 

PK indices Units Average SD Median MinMax
ke (48-96 h)1 / h0.0030.0010.0030.0030.005
t1 / 2h224.1851.60233.94153.24275.60
Tmaxh15.0010.5216.004.0024.00
Cmaxμg / ml2.710.662.712.093.35
AUC 0-96 hμg / ml × h228.3062.23228.18166.59290.25
AUMC 0-96 hμg / ml × h 2107372971.7105767826.013968
MRT 0-96 hh326.5013.60337.46221.17397.47
V / Fobs (calculated 0-96 h)L / kg17.414.0815.7414.7323.41
Cl / Fobs (calculated 0-96 h)L / h / kg0.060.020.0520.040.085
Table 2. Pharmacokinetic parameters of cats A, B, D and G during the first 96 hours.
SD - standard deviation; ke - elimination rate constant; t1 / 2 - elimination half-life; Tmax - time to maximum plasma concentration; Cmax - maximum plasma concentration; AUC 0 –96 h- area under the curve for 96 h; AUMC 0-96 h - area under the torque curve during 96 h; MRT 0 –96 h- average residence time; V / Fobs - apparent volume of distribution; Cl / Fobs - Apparent clearance.

Hematology and biochemistry of the serum of six cats (cats A, B, D, E, F and G) were performed before treatment (0 h), after 168 and 336 h. Cat C was excluded from the study after the first two checkpoints and therefore blood collection was not continued. Haematological results were unambiguous at all six time points. The results of biochemistry are shown in Table 3.

0h0h168h168h336h336h
Biochemical analyteUnitsAverageRangeAverageRangeAverageRangeReference interval (Idexx reference laboratory)
Glucosemmol / l5.103.90-6.304.503.40-5.403.20-7.50
SDMAug / dl6.701.00-8.0011.08.00-13.013.510.0-16.00.00-14.0
Creatinineumol / l11590.0-140.12280.0-160.120.100.-140.80.0-200.
Ureammol / l8.006.80-10.27.706.90-9.108.086.90-9.205.00-15.0
Phosphorusmmol / l1.701.40-2.001.401.17-1.631.321.20-1.600.00-2.30
Calciummmol / l2.402.30-2.502.402.40-2.602.302.20-2.402.10-2.80
Sodiummmol / l152149-153154152-156151148-153144-158
Potassiummmol / l5.104.50-5.204.504.10-5.204.404.10-4.703.70-5.40
Chloridemmol / l115111-117123120-125118116-120106-123
Bicarbonatemmol / l16.015.0-18.016.3 (4 cats)15.0-18.012.0-24.0
Anion gapmmol / l25.825.2-27.120.6 (4 cats)20.1-21.315.0-31.0
Total proteing / L75.367.0-86.071.065.2-80.771.365.0-83.760.0- 84.0
Albuming / l31.729.0-36.028.927.8-30.030.028.0-32.025.0-38.0
Globuling / L43.735.0-37.042.335.3-52.941.233.0-55.731.0-52.0
ALTU / L79.243.0-16147.326.0-11658.722.0-16619.0-100.
ASTU / L47.825.0-83.029.722.0-53.029.820.0-42.00.00-62.0
ALPU / L39.821.0-75.037.024.0-72.041.827.0-87.05.00-50.0
GGTU / L0.700.00-1.000.500.00-2.000.00-5.00
Total bilirubinumol / l3.003.002.100.40-2.702.602.00-3.000.00-7.00
Cholesterolmmol / l3.852.60-4.903.903.00-4.803.100.00-4.702.20-5.50
CKU / L299133-73517897.0-39814817.0-24864.0-400
TT4nmol / l32.521.0-39.032.628.6-36.7 32.4-35.410.0-60.0
Table 3. Biochemical results (mean and range) at 0, 168 and 336 h. Numbers outside the reference intervals are highlighted in bold. Not all biochemical analytes were listed due to the availability of different biochemical analytes in different laboratories, so the fields are blank.
SDMA-symmetric dimethylarginine; ALT-alanine transaminase; AST-aspartate transaminase; ALP-alkaline phosphatase; GGT-gamma-glutamyl transpeptidase; CK-creatine kinase; TT4-total thyroxine. Numbers outside the reference intervals are highlighted in bold.

Biochemical examination showed a trend of increasing SDMA concentrations at 168 and 336 hours in all six cats. One-way repeated measures ANOVA comparing SDMA at all time points was statistically significant <0.002. Tukey's multiple comparison tests show that the average SDMA value at each time point was statistically different: 0 vs 168 h = 0.002; 168 vs 336 h = 0.005. Figure 3 shows the median SDMA concentrations at each time point with upper and lower ranges from six cats. The median SDMA at 0 h was 8.0 g / dl (range 1.0 - 8.0), the median SDMA at 168 h was 11.5 g / dl (range 8.0 - 13.0) and at 336 h was 14 .0 g / dl (range 10.0 - 16.0). Creatinine concentrations did not differ significantly at 0, 168, and 336 h in the six cats. Three cats had elevated liver parameters before treatment, including ALT, AST and ALP. Of these three cats, one cat (Cat G) had an ALT of up to 161 U / L and ALP was 75.0 U / L prior to treatment, and ALT and ALP remained elevated to 166 U / L and 87.0 U / L, respectively. at 336 h. This cat had no side effects during the study. One cat with slightly elevated ALT (142 U / L) was excluded from the study due to vomiting (cat C). Another cat with slightly elevated ALT (144 U / L) and AST (83.0 U / L) before treatment remained clinically healthy throughout the study and both liver parameters returned to normal after 168 and 336 h. Other biochemical analytical changes were unnoticed. Blood glucose was not provided at 0 h because artificial hypoglycemia was observed in all samples. This was suspected to be due to a problematic blood sampling method for glucose determination. 

Figure 3. SDMA results (ug / dl) of six cats at 0, 168 and 336 h. The median SDMA concentration at each time point is displayed with an upper and lower range. The dashed line represents the reference interval (0.00 - 14.0).

Although cats C and F vomited on the first administration of mefloquine, all other cats tolerated the drug well on the next administration when mefloquine was administered with food. No other side effects were observed in any cat during the two weeks of treatment.  

Discussion

This is the first pharmacokinetic study of mefloquine in cats. The only reported use of mefloquine in animals is as an antimalarial for predators and penguins [21,22]. In addition to its use as an antimalarial, successful treatment with this drug has been reported in people with progressive multifocal leukoencephalopathy caused by John Cunningham virus (JCV) [23,24]. Its antiviral activity has been demonstrated in vitro with FCoV [17], feline calicivirus [25], dengue virus type 2 and Zika virus in humans [26] and more recently with pangolin coronavirus GX_P2X, which is a model for SARS-CoV-2, the causative agent COVID-19 in humans [27]. The exact mechanisms of its action as an antimalarial or antiviral agent are not known [17,28,29]. 

As mefloquine is an antimalarial prophylaxis and treatment of humans, its pharmacokinetic profile has been documented. In healthy volunteers, the oral half-life of mefloquine is 1-4 h (mean 2.1 h) [30]. The oral bioavailability of mefloquine in cats is unknown, as the required IV AUC required for calculation has not been performed in cats. However, the oral bioavailability of mefloquine in dogs has been found to be approximately 67-90 % (mean 78 %) [20]. Mefloquine reaches maximum plasma concentrations after approximately 6-24 h (median 17.6 h) in humans [30]. When administered orally to cats, the time to peak plasma concentrations (Tmax) is comparable to humans, averaging 15 hours. The estimated total apparent volume of distribution in healthy humans is approximately 19.2-22.1 l / kg and the systemic clearance is 0.026-0.042 l / h / kg [31], while the apparent mean ± SD volume of distribution in cats is 17.4 ± 4 .08 l / kg and the apparent clearance is 0.060 ± 0.020 l / h / kg when calculated at 0 to 96 h. Plasma protein binding was 98 % in healthy human volunteers and patients with uncomplicated falciparum malaria [30] and also 99 % in cat plasma from clinically normal cats and FIP cat plasma [19]. In humans, the elimination half-life of mefloquine is approximately 20 days in healthy subjects, 10 to 14 days in patients with uncomplicated falciparum malaria [32,33], and 20 days in cases involving severe malaria [33,34]. In humans, a loading dose and then treatment once a week is recommended [35,36]. Mefloquine is slowly excreted from the body via faeces and urine [31,37]. In our study, we estimated that the elimination half-life of mefloquine in clinically healthy cats is approximately 224 hours or approximately 9.3 days, similar to healthy humans. The elimination half-life calculation was based on 24 to 96 h time periods only; further studies, with sampling at a single dose of mefloquine longer than 96 hu in cats, may provide a clearer result.

Oral absorption was increased when mefloquine was administered with food. Pharmacokinetic analysis showed that the mean plasma concentration was higher (4.06 ug / ml) after 240 h when mefloquine was administered with food, compared to the plasma concentration of 2.71 ug / ml after 15 h when mefloquine was administered without food. . Other factors affecting the higher plasma concentrations after 240 hours include the cumulative effect of the drug when administered multiple times and the possible enterohepatic circulation of the drug. In humans, the presence of food in the gastrointestinal tract affects the pharmacokinetic properties of mefloquine by significantly increasing the rate and extent of absorption [38]. 

Mefloquine at 10 μM was shown to show significant inhibition against two FCoV biotypes, FIPV WSU 79-1146 (FIPV1146) and FECV WSU 79-1683 (FECV1683), obtained from the American Type Culture Collection (Virginia, USA), [17] . As the molecular weight of mefloquine is 378 g / mol, a plasma concentration of 10 μM = 3.78 μg / ml is achieved using 10 μM mefloquine [17]. This study showed that a single oral dose of mefloquine ~ 12.5 mg / kg achieved a maximum plasma concentration (Cmax) of 2.71 μg / ml. A higher dose of mefloquine may be required to inhibit FIP. However, it is possible that Cmax will be much higher when mefloquine is administered with food, as reported after 12 to 24 hours. The evaluation of the efficacy of mefloquine against FCoV deserves a clinical study. In addition, mefloquine has been shown to inhibit cytopathic effects in cells infected with SARS-CoV-2a coronavirus (GX_P2X), making it a potential drug for use in cats with SARS-CoV-2 infection [27].

Chloroquine, a 4-aminoquinoline with a similar mode of action to mefloquine [39], has shown an inhibitory effect against FIPV replication and an anti-inflammatory effect in vitro and improved the clinical evaluation of experimentally induced FIP in cats [40]. Although mefloquine has been shown to inhibit FIPV in vitro [17], its clinical efficacy in cats with FIP remains unknown. However, chloroquine caused an increase in ALT levels when used in FIP-infected cats. In this study, mefloquine did not increase ALT levels. Although some cats had elevated ALT levels prior to the mefloquine dose, no further increase was observed after four doses of mefloquine twice a week. Hydroxychloroquine has been studied in a clinical study for the treatment of COVID-19 in humans [41] and its antiviral properties against FIPV in vitro have also been recently investigated [42]. At when used with recombinant feline IFN-ω, hydroxychloroquine has shown increased antiviral activity against FIPV infection [42]. Further clinical studies are needed to verify its clinical efficacy and safety in cats with coronavirus or calicivirus infection. Side effects of mefloquine in humans are common, with 47-90 % people having some mild to moderate side effects [30,43,44]. The incidence of adverse events decreases with long-term use, from 44 % during the first 4 months to 19 % after one year [30,45]. The most common side effects include neuropsychiatric effects [46-48], gastrointestinal dysfunction [49], dermatological symptoms [50], haematological changes [51] or cardiovascular dysfunction [30,33]. In humans, nausea and vomiting are common side effects and may be dose and age dependent, with younger children being most at risk [49,52]. In this study, two cats vomited after the first administration of mefloquine without food. One cat (cat F) was successfully re-administered mefloquine after feeding and was therefore re-enrolled in the study. Cat C was also re-administered a second dose of mefloquine (day 4); however, this cat refused food before the second dose. Mefloquine was therefore dosed without food. This cat vomited again and was excluded from the study. No further vomiting was observed when mefloquine was administered with food. No other clinical side effects were observed in cats in our study. However, our cats were observed for only 14 days. Any delayed or long-term side effects of mefloquine in cats remain unknown. It is also possible that the incidence of side effects could be reduced with long-term administration, as observed in humans [30,45]. 

The cause of the lower plasma concentration curve of Cat E during the first dosing interval (0-96 h) remained unknown (Figures 1 and 2). The age difference may explain the lower plasma concentration curve of cats D and E, as these two cats are younger than the others (3 years versus 6-7 years). Decreased hepatic clearance, increased volume of lipid-soluble drugs with extended half-lives and increased oral bioavailability have been proposed to explain why older people have different pharmacokinetics compared to younger adults, and these causes could potentially contribute to differences in cat plasma concentrations. D and E [53,54]. However, no increase in volume of distribution and prolonged half-life were observed in older cats (cats A, B, C and G). Although human mefloquine blood levels during pregnancy are lower than in non-pregnant adults, there were no age-related differences in plasma mefloquine concentrations in the pharmacokinetic profiles [55,56]. Interestingly, maximum blood concentrations are 2-3 times higher in Asian adults compared to non-Asian volunteers, and the reason for this ethnic difference is unclear [30,57]. It is thought that a lower volume of distribution may have contributed to higher plasma concentrations due to lower body fat or differences in enterohepatic drug circulation in Asian volunteers [58]. In our study, it was not possible to identify differences in plasma concentrations between gender, castration, and body weight. Cat E also had normal biochemical analytes before treatment, at 168 and 336 h. Liver dysfunction causing altered drug metabolism is unlikely; however, it cannot be completely ruled out without determining bile acid levels before and after treatment to assess liver function. In humans, mefloquine is metabolised by cytochrome P450 3A (CYP 3A) in the liver [59]. In cats, CYP 3A activity was found to be lower in cats compared to cats [60]. However, the special plasma concentration curve of cat E cannot be explained. Another explanation could be that cat E could vomit without being observed after the first administration of mefloquine. 

The difference in pharmacokinetic profile is observed in healthy people compared to malaria-infected people. In humans, plasma mefloquine concentrations are 2-3 times higher in uncomplicated falciparum malaria compared to healthy volunteers. Uncomplicated malaria falciparum also has a shortened half-life [30,31,33,61]. The cause is not fully understood. One possible cause of the shortened half-life in patients with malaria is a decrease in enterohepatic circulation and greater fecal clearance. Another explanation for the differences in pharmacokinetic profile between the two groups is the difference in mefloquine binding to plasma proteins. Mefloquine is highly bound to plasma proteins, especially acute phase proteins such as alpha-1-acid glycoprotein (AGP) [62]. An increase in AGP in malaria is thought to lead to an increase in plasma protein binding of mefloquine, which affects the apparent volume of distribution [61]. High levels of AGP have been demonstrated in experimentally induced FIP [63] and naturally infected cats with FIP [64,65] and are commonly used in practice as a diagnostic tool for FIP [66]. Thus, it is possible that high levels of AGP and potentially other acute phase proteins in FIP-infected cats increase mefloquine binding to plasma proteins, altering the pharmacokinetic profile in these cats. Plasma protein binding of mefloquine in clinically normal and FIP-infected cats was investigated in vitro; however, the difference was ambiguous due to the unknown biological variability of the assay [19]. Further studies on the pharmacodynamics and pharmacokinetics of mefloquine are required in cats with FIP. 

During this study, a trend of increasing SDMA without a change in creatinine was observed in all cats (Figure 3). SDMA has been shown to be an early renal biomarker compared to creatinine [67-69] and increases in acute renal injury and chronic kidney disease [70]. Elevated serum SDMA concentrations in cats have been associated with decreased renal function as measured by glomerular filtration rate (GFR) [71]. Based on the results of our study, it is possible that mefloquine may cause decreased renal function in cats. Renal toxicity from antimalarial administration is rare in humans [72]. Another explanation for the elevated SDMA concentrations is the effect of general anesthesia and the cumulative effect of isoflurane during the study. Serum SDMA levels measured after induction of anesthesia (17.11 g / dl) have been shown to be significantly higher than levels measured before induction of anesthesia (12.39 g / dl) [73]. As cats were anesthetized with isoflurane during blood collection, this could potentially contribute to increased SDMA concentrations. Blood collection, mefloquine dosing, and cat recruitment were commissioned from an external designated animal research facility (Invetus Pty, Ltd., Casino, NSW, Australia) due to lack of subject availability, and this study was performed according to Invetus standard operating procedures. 

One limitation of this study was that not all cats had normal liver enzymes prior to treatment. Three cats had elevated ALT, AST and ALP levels before treatment. Because blood collection, pre-treatment blood tests, and treatment were performed at an external facility, investigators were unaware of elevated liver parameters prior to treatment. Investigators were also not involved in the recruitment process for these cats and the drug history and previous records of these cats were not known. Nevertheless, there was no significant increase in ALT and ALP after mefloquine treatment. ALT and ALP remained unchanged after 336 hours in one cat (cat G). The plasma mefloquine concentration curve in cat G did not differ significantly from the other cats (cats A, B and D). 

Another limitation is the small number of cats in our study. Only five cats had complete mefloquine plasma concentrations, and only four cats were included in the pharmacokinetic analysis (the low cat E concentration curve was omitted). Despite the small number of cats used in the analysis, an important description of the mefloquine drug profile in a clinically normal cat was provided. This preliminary information is crucial for all other research projects that involve the use of mefloquine in cats. 

Conclusions

The study provides preliminary data on the pharmacokinetic profile of mefloquine in cats and provides useful information for planning clinical trials of mefloquine for the treatment of cats with feline coronavirus (including FIP) and feline calicivirus infections and, if necessary, COVID-19 to potentially reduce virus shedding. . Further studies on its therapeutic effects are needed to determine the therapeutic benefit of mefloquine in cats with these diseases.

Author's contributions: Conceptualization, JMN and MG; methodology, MG and BK; software, BK and MG; validation, MG and BK; formal analysis, MG and BK; investigation, MG, BK and JY; sources, MG and BK; data curators, MG, JY and BK; preparation of the original proposal, JY; writing - review and editing, JY, MG and JMN; visualization, MG and JY; supervision, JMN and MG; project administration, MG; fundraising, JMN, MG and BK All authors have read and agreed to the published version of the manuscript.

Financing: This research was funded by the Winn Feline Foundation, grant number 2019_027, a donation from the estate of Christine Gai Atkins and a reference from Lesley Muir of the Sydney School of Veterinary Science.

Acknowledgments: The authors are grateful to Invetus for supporting the recruitment of cats, providing shelter, medication and taking the blood of cats.

Conflict of interests: The authors do not indicate any conflict of interest. The funders did not participate in the study design, data collection, analysis or interpretation, manuscript writing or decision to publish the results.

Some veterinarians around the world are already using mefloquine as an off-label treatment for FIP, while formal research and clinical trials continue.

Dosage according to one of the following researchers: “The dose used is 10 to 12 mg / kg orally twice a week and must be given with a small amount of food to prevent vomiting. This is usually equivalent to Lariam 1/4 250 mg tablets in many cats. The drug shows good penetration into the central nervous system in humans, so we hope that it may be more beneficial for severe neurological cases of FIP. "

Researchers are interested in all cats with FIP in treatment. Please contact me privately via email at defeatfip@verizon.net. Well thank you.

Susan E Gingrich, Founder of the Bria FIP Research Fund, December 12, 2020

Note: The entire pack of Lariam with 8 tablets (each containing 250 mg of mefloquine) costs about CZK 700 (approx. € 27). It would therefore be a very inexpensive form of FIP treatment. A two-week treatment for one cat is therefore cheaper than CZK 100 / € 4.

References

  1. Felten, S .; Hartmann, K. Diagnosis of Feline Infectious Peritonitis: A Review of the Current Literature. Viruses 2019, 11, 1068.
  2. Pedersen, NC; Allen, CE; Lyons, LA Pathogenesis of feline enteric coronavirus infection. J. Feline Med. Surg. 2008, 10, 529–541.
  3. Addie, D .; Belak, S .; Boucraut-Baralon, C .; Egberink, H .; Frymus, T .; Gruffydd-Jones, T .; Hartmann, K .; Hosie, MJ; Lloret, A .; Lutz, H .; et al. Feline infectious peritonitis. ABCD guidelines on prevention and management. J. Feline Med. Surg. 2009, 11, 594–604.
  4. Pedersen, NC A review of feline infectious peritonitis virus infection: 1963–2008. J. Feline Med. Surg. 2009, 11, 225–258.
  5. Pedersen, NC, An update on feline infectious peritonitis: Diagnostics and therapeutics. Vet. J. 2014, 201, 133–141.
  6. Tsai, HY; Chueh, LL .; Lin, CN; Su, BL Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan. J. Feline Med. Surg. 2011, 13, 74–80.
  7. Hugo, TB; Heading, KL Prolonged survival of a cat diagnosed with feline infectious peritonitis by immunohistochemistry. Can. Vet. J. 2015, 56, 53–58.
  8. Ishida, T .; Shibanai, A .; Tanaka, S .; Uchida, K .; Mochizuki, M. Use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis. J. Feline Med. Surg. 2004, 6, 107–109.
  9. Kim, Y .; Liu, H .; Galasiti Kankanamalage, AC; Weerasekara, S .; Hua, DH; Groutas, WC; Chang, KO; Pedersen, NC Reversal of the progression of fatal coronavirus infection in cats by a broad ‐ spectrum coronavirus protease inhibitor. PLoS Pathog. 2016, 12, e1005531.
  10. Kim, Y .; Mandadapu, SR; Groutas, WC; Chang, KO Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C ‐ like protease. Antiviral Res. 2013, 97, 161–168.
  11. Murphy, BG; Perron, M .; Murakami, E .; Bauer, K .; Park, Y .; Eckstrand, C .; Liepnieks, M .; Pedersen, NC The nucleoside analog GS ‐ 441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet. Microbiol. 2018, 219, 226–233.
  12. Pedersen, NC; Kim, Y .; Liu, H .; Galasiti Kankanamalage, AC; Eckstrand, C .; Groutas, WC; Bannasch, M .; Meadows, JM; Chang, KO Efficacy of a 3C ‐ like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J. Feline Med. Surg. 2018, 20, 378–392.
  13. 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, 271–281.
  14. Hsieh, LE; Lin, CN; Su, BL; Jan, TR; Chen, CM; Wang, CH; Lin, DS; Lin, CT; Chueh, LL, Synergistic antiviral effect of Galanthus nivalis agglutinin and nelfinavir against feline coronavirus. Antiviral Res. 2010, 88, 25–30.
  15. Strasfeld, L .; Chou, S., Antiviral drug resistance: Mechanisms and clinical implications. Infect. Dis. Clin. North. Am. 2010, 24, 413–437.
  16. Radford, AD; Addie, D .; Belak, S .; Boucraut-Baralon, C .; Egberink, H .; Frymus, T .; Gruffydd-Jones, T .; Hartmann, K .; Hosie, MJ; Lloret, A .; Lutz, H .; Marsilio, F .; Pennisi, MG; Thiry, E .; Truyen, U .; Horzinek, MC, Feline calicivirus infection. ABCD guidelines on prevention and management. J. Feline Med. Surg. 2009, 11 (7), 556–564.
  17. McDonagh, P .; Sheehy, PA; Norris, JM Identification and characterization of small molecule inhibitors of feline coronavirus replication. Vet. Microbiol. 2014, 174, 438–447.
  18. Izes, AM; Kimble, B .; Norris, JM; Govendir, M. In vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush ‐ tailed possum (Trichosurus vulpecula). PLoS One 2020, 15, e0230975.
  19. Izes, AM Comparative studies of in vitro hepatic metabolism of mefloquine by feline microsomes and those of other selected species. PhD Thesis, The University of Sydney, Camperdown, NSW, Australia, November 2019.
  20. Schwartz, DE; Weber, W .; Richard-Lenoble, D .; Gentilini, M., Kinetic studies of mefloquine and of one of its metabolites, Ro 21‐5104, in the dog and in man. Acta. Too much. 1980, 37, 238–242.
  21. Remple, JD Intracellular Hematozoa of Raptors: A Review and Update. J. Avian Med. Surg.2004, 18, 75– 88.
  22. Grill, ML; Vanstreels, RE; Wallace, R .; Garcia-Parraga, D .; Braga, EM; Chitty, J .; Catao-Dias, JL; Madeira de Carvalho, LM Malaria in penguins - current perceptions. Avian Pathol. 2016, 45, 393–407.
  23. Hamaguchi, M .; Suzuki, K .; Fujita, H .; Uzuka, T .; Matsuda, H .; Shishido-Hara, Y .; Arai, S .; Nakamura, T .; Kikuchi, S .; Nakamichi, K .; et al. Successful treatment of non ‐ HIV progressive multifocal leukoencephalopathy: Case report and literature review. J. Neurol. 2020, 267, 731–738.
  24. Nambirajan, A .; Suri, V .; Kataria, V .; Sharma, MC; Goyal, V. Progressive multifocal leukoencephalopathy in a 44 ‐ year old male with idiopathic CD4 + T ‐ lymphocytopenia treated with mirtazapine and mefloquine. Neurol. India 2017, 65, 1061–1064.
  25. McDonagh, P .; Sheehy, PA; Fawcett, A .; Norris, JM, Antiviral effect of mefloquine on feline calicivirus in vitro. Vet. Microbiol. 2015, 176, 370–377.
  26. Balasubramanian, A .; Teramoto, T .; Kulkarni, AA; Bhattacharjee, AK; Padmanabhan, R. Antiviral activities of selected antimalarials against dengue virus type 2 and Zika virus. Antiviral Res. 2017, 137, 141–150.
  27. Fan, HH; Wang, LQ; Liu, WL; An, XP; Liu, ZD; He, XQ; Song, LH; Tong, YG Repurposing of clinically approved drugs for the treatment of coronavirus disease 2019 in a 2019 ‐ novel coronavirus (2019nCoV) related coronavirus model. Chin. Med. J. (Engl) 2020, 133 (9), 1051–1056.
  28. Brickelmaier, M .; Lugovskoy, A .; Kartikeyan, R .; Reviriego-Mendoza, MM; Allaire, N .; Simon, K .; Frisque, RJ; Gorelik, L. Identification and characterization of mefloquine efficacy against JC virus in vitro. Antimicrob. Agents Chemother. 2009, 53, 1840–1849.
  29. Owen, A .; Janneh, O .; Hartkoorn, RC; Chandler, B .; Bray, PG; Martin, P .; Ward, SA; Hart, CA; Khoo, SH; Back, DJ In vitro synergy and enhanced murine brain penetration of saquinavir coadministered with mefloquine. J. Pharmacol. Exp. Ther. 2005, 314, 1202–1209.
  30. Palmer, KJ; Holliday, SM; Brogden, RN Mefloquine. A review of its antimalarial activity, pharmacokinetic properties and therapeutic efficacy. Drugs 1993, 45, 430–475.
  31. Karbwang, J .; White, NJ, Clinical pharmacokinetics of mefloquine. Clin. Pharmacokinet. 1990, 19, 264–279.
  32. Desjardins, RE; Pamplin, CL, 3rd; von Bredow, J .; Barry, KG; Canfield, CJ Kinetics of a new antimalarial, mefloquine. Clin. Pharmacol. Ther. 1979, 26, 372–379.
  33. Karbwang, J .; Na ‐ Bangchang, K., Clinical application of mefloquine pharmacokinetics in the treatment of P falciparum malaria. Fundam. Clin. Pharmacol. 1994, 8, 491–502.
  34. Juma, FD; Ogeto, JO Mefloquine disposition in normals and in patients with severe Plasmodium falciparum malaria. Eur. J. Drug Metab. Pharmacokinet. 1989, 14, 15–17.
  35. Tan, KR; Arguin, PM; Arguin, T. Travel related infectious disease. In Centers for Disease Control and PRevention. CDC Yellow Book 2020: Health information for international travel Available online: https://www.cbc.gov/malaria/travelers/drugs.html (Accessed on 21st April2020)
  36. Centers for Disease Control and Prevention, Malaria, how to choose a drug to prevent Malaria. Available online: https://www.cdc.gov/malaria/travelers/drugs.html (accessed on 21st April 2020).
  37. White, NJ Clinical pharmacokinetics of antimalarial drugs. Clin. Pharmacokinet. 1985, 10, 187–215.
  38. Crevoisier, C .; Handschin, J .; Barre, J .; Roumenov, D .; Kleinbloesem, C. Food increases the bioavailability of mefloquine. Eur. J. Clin. Pharmacol. 1997, 53, 135–139.
  39. Anthony, HA; Parija, SC Antimalarial drug resistance: An overview. Too much. Parasitol. 2016, 6, 30–41.
  40. Takano, T .; Katoh, Y .; Doki, T .; Hohdatsu, T. Effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo. Antiviral Res. 2013, 99, 100–107.
  41. Lundstrom, K. Coronavirus Pandemic ‐ Therapy and Vaccines. Biomedicine 2020, 8, 109.
  42. Takano, T .; Satoh, K .; Doki, T .; Tanabe, T .; Hohdatsu, T. Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection. Viruses 2020, 12, 576.
  43. Lobel, HO; Bernard, KW; Williams, SL; Hightower, AW; Patchen, LC; Campbell, CC Effectiveness and tolerance of long ‐ term malaria prophylaxis with mefloquine. Need for a better dosing regimen. JAMA 1991, 265, 361–364.
  44. Tin, F .; Hlaing, N .; Lasserre, R. Single ‐ dose treatment of falciparum malaria with mefloquine: Field studies with different doses in semi ‐ immune adults and children in Burma. Bull. World Health Organ. 1982, 60, 913– 917.
  45. Lobel, HO; Miani, M .; Eng, T .; Bernard, KW; Hightower, AW; Campbell, CC Long ‐ term malaria prophylaxis with weekly mefloquine. Lancet 1993, 341, 848–851.
  46. Gribble, FM; Davis, TM; Higham, CE; Clark, A .; Ashcroft, FM The antimalarial agent mefloquine inhibits ATP ‐ sensitive K ‐ channels. Br. J. Pharmacol. 2000, 131, 756–760.
  47. Ringqvist, A .; Bech, P .; Glenthoj, B .; Petersen, E. Acute and long ‐ term psychiatric side effects of mefloquine: A follow ‐ up on Danish adverse event reports. Travel Med. Infect. Dis. 2015, 13, 80–88.
  48. Ritchie, EC; Block, J .; Nevin, RL Psychiatric side effects of mefloquine: Applications to forensic psychiatry. J. Am. Acad. Psychiatry Law 2013, 41, 224–235.
  49. Lee, SJ; Ter Kuile, FO; Price, RN; Luxemburger, C .; Nosten, F. Adverse effects of mefloquine for the treatment of uncomplicated malaria in Thailand: A pooled analysis of 19, 850 individual patients. PLoS One 2017, 12, e0168780.
  50. Smith, HR; Croft, AM; Black, MM Dermatological adverse effects with the antimalarial drug mefloquine: A review of 74 published case reports. Clin. Exp. Dermatol. 1999, 24, 249–254.
  51. Stracher, AR; Stoeckle, MY; Giordano, MF Aplastic anemia during malarial prophylaxis with mefloquine. Clin. Infect. Dis. 1994, 18, 263–264.
  52. Magnussen, P .; Bygbjerg, IC Treatment of Plasmodium falciparum malaria with mefloquine alone or in combination with iv quinine at the Department of Communicable and Tropical Diseases, Rigshospitalet, Copenhagen 1982–1988. Day. Med. Bull. 1990, 37, 563–564.
  53. Klotz, U. Pharmacokinetics and drug metabolism in the elderly. Drug Metab. Rev. 2009, 41, 67–76.
  54. Mangoni, AA; Jackson, SH Age ‐ related changes in pharmacokinetics and pharmacodynamics: Basic principles and practical applications. Br. J. Clin. Pharmacol. 2004, 57, 6–14.
  55. Singhasivanon, V .; Chongsuphajaisiddhi, T .; Sabcharoen, A .; Attanath, P .; Webster, HK; Wernsdorfer, WH; Sheth, UK; Djaja Lika, I. Pharmacokinetics of mefloquine in children aged 6 to 24 months. Eur. J. Drug Metab. Pharmacokinet. 1992, 17, 275–279.
  56. Vieira, JL; Borges, LM; Ferreira, MV; Rivera, JG; Gomes Mdo, S. Patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria. Braz. J. Infect. Dis. 2016, 20, 482–486.
  57. Karbwang, J .; Bunnag, D .; Breckenridge, AM; Back, DJ The pharmacokinetics of mefloquine when given alone or in combination with sulphadoxine and pyrimethamine in Thai male and female subjects. Eur. J. Clin. Pharmacol. 1987, 32, 173–177.
  58. Looareesuwan, S .; White, NJ; Warrell, DA; Forgo, I .; Dubach, UG; Ranalder, UB; Schwartz, DE Studies of mefloquine bioavailability and kinetics using a stable isotope technique: A comparison of Thai patients with falciparum malaria and healthy Caucasian volunteers. Br. J. Clin. Pharmacol. 1987, 24, 37–42.
  59. Fontaine, F .; de Sousa, G .; Burcham, PC; Duchene, P .; Rahmani, R. Role of cytochrome P450 3A in the metabolism of mefloquine in human and animal hepatocytes. Life Sci. 2000, 66, 2193–2212.
  60. Shah, SS; Sanda, S .; Regmi, NL; Sasaki, K .; Shimoda, M. Characterization of cytochrome P450 ‐ mediated drug metabolism in cats. J. Vet. Pharmacol. Ther. 2007, 30, 422–428.
  61. Reuter, SE; Upton, RN; Evans, AM; Navaratnam, V .; Olliaro, PL Population pharmacokinetics of orally administered mefloquine in healthy volunteers and patients with uncomplicated Plasmodium falciparum malaria. J. Antimicrob. Chemother. 2015, 70, 868–876.
  62. Zsila, F.; Visy, J .; Mady, G .; Fitos, I. Selective plasma protein binding of antimalarial drugs to alpha1 ‐ acid glycoprotein. Bioorg. Med. Chem. 2008, 16, 3759–3772.
  63. Stoddart, ME; Whicher, JT; Harbor, DA Cats inoculated with feline infectious peritonitis virus exhibit a biphasic acute phase plasma protein response. Vet. Rec. 1988, 123, 622–624.
  64. Duthie, S .; Eckersall, PD; Addie, DD; Lawrence, CE; Jarrett, O. Value of alpha 1 ‐ acid glycoprotein in the diagnosis of feline infectious peritonitis. Vet. Rec. 1997, 141, 299–303.
  65. Giordano, A .; Spagnolo, V .; Colombo, A .; Paltrinieri, S. Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection. Vet. J. 2004, 167, 38–44.
  66. Paltrinieri, S .; Giordano, A .; Tranquillo, V .; Guazzetti, S. Critical assessment of the diagnostic value of feline alpha1 ‐ acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach. J. Vet. Diagn. Invest. 2007, 19, 266–272.
  67. Hall, JA; Yerramilli, M .; Obare, E .; Yerramilli, M .; Jewell, DE Comparison of serum concentrations of symmetric dimethylarginine and creatinine as kidney function biomarkers in cats with chronic kidney disease. J. Vet. Intern. Med. 2014, 28, 1676–1683.
  68. Charged, MB; Lees, GE; Boggess, MM; Yerramilli, M .; Obare, E .; Yerramilli, M .; Rakitin, A .; Aguiar, J .; Relford, R. Symmetric dimethylarginine assay validation, stability, and evaluation as a marker for the early detection of chronic kidney disease in dogs. J. Vet. Intern. Med. 2015, 29, 1036–1044.
  69. Yerramilli, M .; Farace, G .; Quinn, J .; Yerramilli, M. Kidney disease and the nexus of chronic kidney disease and acute kidney injury: the role of novel biomarkers as early and accurate diagnostics. Vet. Clin. North. Am. Small Anim. Pract. 2016, 46, 961–993.
  70. Dahlem, DP; Neiger, R .; Schweighauser, A .; Francey, T .; Yerramilli, M .; Obare, E .; Steinbach, SML Plasma symmetric dimethylarginine concentration in dogs with acute kidney injury and chronic kidney disease. J. Vet. Intern. Med. 2017, 31, 799–804.
  71. Braff, J .; Obare, E .; Yerramilli, M .; Elliott, J .; Yerramilli, M. Relationship between serum symmetric dimethylarginine concentration and glomerular filtration rate in cats. J. Vet. Intern. Med. 2014, 28, 1699– 1701.
  72. Wiwanitkit, V. Antimalarial drug and renal toxicity. J. Nephropharmacol. 2016, 5, 11–12.
  73. Namba, S .; Kitamura, R .; Amaha, T .; Befu, M .; Zama, T .; Moriwaki, T .; Kumono, S .; Shichijo, S. Impact of general anesthesia on serum symmetric dimethylarginine concentration in cats, American Association of Feline Practitioners Conference 2018, Charlotte, NC, USA, 27th – 30th September 2018
Read "Pharmacokinetic profile of oral mefloquine in clinically normal cats: Preliminary in vivo study of the potential treatment of feline infectious peritonitis (FIP)."

Rapid remission of non-fusive FIP uveitis during treatment with oral adenosine analogue and feline omega interferon

Diane D. Addie, Johanna Covell-Ritchie, Oswald Jarrett, and Mark Fosbery
Original article: Rapid Resolution of Non-Effusive Feline Infectious Peritonitis Uveitis with an Oral Adenosine Nucleoside Analogue and Feline Interferon Omega
27.10.2020; Translation 23.3.2021

Abstract

This is the first report of successful treatment of a case of non-fusive FIP uveitis using the oral form of the adenosine analog and feline interferon omega, and alpha-1 acid glycoprotein (AGP) as an indicator of recovery. A 2-year-old neutered Norwegian Forest Cat has been affected by uveitis, keratic clots, mesenteric lymphadenopathy and weight loss. He was hypergammaglobulinemic and had non-regenerative anemia. Feline coronavirus (FCoV) RNA was detected by thin-needle aspiration of mesenteric lymph nodes using polymerase chain reaction reverse transcription (RT-PCR) - non-fusive FIP was diagnosed. Prednisolone acetate eye drops were administered three times daily for 2 weeks. Treatment with an oral form of an adenosine analogue (Mutian) was started. After 50 days of Mutian treatment, the cat gained more than one kilogram, the globulin level decreased from 77 to 51 g / l, and the hematocrit increased from 22 to 35%; uveitis subsided and vision improved. Serum AGP levels decreased from 3100 to 400 μg / ml (within normal limits). Symmetric dimethylarginine (SDMA) was above normal at 28 μg / dl, declining to 14 μg / dl at the end of treatment; It is not known whether the increase in SDMA was due to FIP lesions in the kidney or Mutian. After cessation of Mutian treatment, low doses of the oral form of recombinant feline interferon omega were started - recovery of the cat continued.

Keywords: feline infectious peritonitis, coronavirus, uveitis, Mutian, adenosine analog, feline interferon omega, mesenteric lymph nodes, alpha-1 acid glycoprotein, symmetric dimethylarginine

1. Introduction

Feline coronavirus (FCoV) is a highly infectious enteric virus that causes subclinical infection or diarrhea in most infected cats. [1], but in about 10% it causes potentially fatal immune-mediated monocyte-associated granulomatous vasculitis [2], known as feline infectious peritonitis (FIP). FCoV is an alpha coronavirus with positive polarity, a member of the Coronaviridae family of the Nidovirales family. [3].

Although treatment of FIP with an injectable nucleoside analog has been described in the past [4] and cure of enteric FCoV infection by oral form of adenosine analog (Mutian, Nantong Biotechnology, China) [5], this is the first reported case of recovery of a cat with a systemic FCoV infection - i.e. FIP - using Mutian.

2. Case study

Skywise was a 2-year-old neutered male Norwegian Forest Cat from a household with five cats; he developed uveitis in the remaining eye (in the right, he lost his left eye as a kitten) and occasional diarrhea. Its iris was unnaturally colored and grainy fat precipitates were visible (Figure 1a).

Figure 1
Cat's eye before (a) and (b) after treatment with Mutian and topical steroids. The cat had uveitis and keratic clots (a). Right (b): the same eye after 7 days of systemic prednisolone, 2 weeks of topical prednisolone and 7 weeks of Mutian treatment, showing almost complete resolution of uveitis.

His medical history included bringing two 11-month-old Norwegian Forest Cats (Link and Zelda) into the home three months ago and the onset of uveitis five days after revaccination (Leucofeligen, Virbac, France). Skywise was tested for FCoV antibodies and was found to have a very high titer above 10,240 (Idexx Laboratories, Wetherby, UK) three weeks before presentation due to the contact cat (Paddy) being persistently pyrexic and malaise. . Paddy was negative for FeLV p27 antigens and FIV antibodies (Idexx Laboratories, Wetherby, UK), but his anti-FCoV antibody titer was found to be very high (Table 1), so all other domestic cats were tested for FCoV antibodies to decided whether they should be isolated from Paddy, but since they were also found to have high FCoV antibody titers (Table 1), segregation was not necessary.

Day −29Day −19Day 7Day 41
Skywise2 years - Norwegian Forest Cat (NFC) FIP > 10,240CT 30Neg
Paddy2 years - NFC, persistent pyrexia, lethargy> 10,240 CT 18Neg
Oliver8 years old - Domestic shorthair cat > 10,240CT 20Neg
Link1 year - NFC > 10,240CT 20Neg
Zelda1 year - NFC 640NegNeg
Table 1
Feline coronavirus (FCoV) antibody titer and qRT-PCR C resultsT.
CT - Cycle threshold for FCoV qRT-PCR performed on a stool sample. The days are relative to the first presumed diagnosis of feline infectious peritonitis (FIP) at Skywise on day 0 (April 18, 2020); ie. its FCoV antibody titer was tested 19 days before the diagnosis of FIP. Mutian treatment was started for Skywise on day 6 and for other FCoV infected cats on day 18 for 5 days.

At presentation, the patient's body weight was 2.89 kg (Figure 2) and the fitness score was 2/9. Ultrasound confirmed the absence of abdominal or thoracic effusion, but mesenteric lymphadenopathy was present. Mesenteric lymph node (MLN) aspirate was collected by ultrasound-guided thin-needle aspiration biopsy (FNA) and tested for FCoV reverse transcription polymerase chain reaction (qRT-PCR) (Idexx Laboratories, Wetherby, UK). The assay was positive for the M1058L mutation and negative for the S1060A mutation [6].

Figure 2
Weight before, during and after FIP recovery and timeline. The weight of the cat is shown in the graph above: Day 0 represents the day of presentation with uveitis at the clinic. Revaccination was performed 5 days before the onset of uveitis, but the cat's weight was not recorded before that day, so it is not known whether she lost weight before revaccination or whether there was weight loss after revaccination; ie. whether the FIP was pre-vaccinated or whether the FIP was due to revaccination itself. The cat lost 30 g of weight during systemic treatment with prednisolone (days 0 to 6), then only topical steroids were administered. Mutian treatment was between days 6 and 56; after starting Mutian treatment, the cat's weight increased rapidly and weight gain continued even after Mutian was replaced by feline interferon omega, until the cat's weight reached about 4.35 kg.

The blood results are shown in Table 2. Hematology revealed mild to moderate non-regenerative anemia with hematocrit (HCT) 22%, lymphocyte count 2.07 × 109/ la by eosinopenia below the detection level (reported as 0.0 × 109/ l). Biochemical analysis of the blood showed hyperglobulinemia (77.2 g / l) and an albumin to globulin (A: G) ratio of 0.31. Bilirubin was 11 micromoles per liter (μmol / l) (0.64 mg / dl). Alpha-1 acid glygoprotein (AGP) was increased to 3100 μg / ml (Idexx Laboratories, Wetherby, UK) (normal level is ≤ 500 μg / ml [7]).

AGPTPAlbGlobA: GBilirubinHctLymphocytesALTAPGGTSDMACreatUrea
Ref. range<500 μg / mL57-89 g / L22-40 g / L28-51 g / L> 0.8 *0–15 μmol / L30–52%0.92–6.88 × 10912-130 U / L14-111 U / L0–4 U / L0-14 μg / dL71–212 μmol / L5.7–12.9 mmol / L
Day 0 ND11120910.221125.70.9564NDNDNDNDND
Day 2 310010324770.31ND22.02.07NDNDNDNDNDND
Day 18 7008326570.46634.43.6127320ND889.6
Day 41 4008029510.57535.13.856740028938.3
Day 62 4007830480.60535.22.6461400191066.5
Day 90 3007831470.70640.04.15814001411310.0
Day 153 4006734331.03135.24.4981462ND **987.8
Table 2
Laboratory data related to FIP treatment monitoring.
* A: G above 0.8 has a strong negative predictive value for FIP [8]. ** SDMA was not repeated because urine analysis was normal. AGP - alpha-1 acid glycoprotein; TP - total protein; Alb - albumin; Glob - globulin; A: G - ratio of albumin to globulin; Hct - hematocrit; ALT - alanine aminotransferase; AP - alkaline phosphatase; GGT - gamma glutamyltransferase; SDMA - symmetric dimethylarginine; Creat - creatinine; ND = not determined.

Only parameters that have been monitored repeatedly are shown in this table; others for which few (or no) results were available (eg aspartate aminotransferase) or which were not relevant to the procedure in this case are omitted. FCoV antibody titers and faecal FCoV qRT-PCR results are shown in Table 1. Day 0 is the day of FIP diagnosis in the first cat; Mutian was administered between days 6 and 56, after which the cat was treated with 100,000 units of the oral form of feline interferon omega.

Systemic treatment was initiated with prednisolone 5 mg for uveitis (Table 3). The dose was reduced to 2.5 mg sid, then stopped after 7 days and replaced with topical prednisolone-acetate 1% eye suspension (Pred Forte®, Allergan, Dublin, Ireland) applied every 8 hours for two weeks. On the sixth day after the presentation, treatment with an oral adenosine analogue began (Mutian 200, Nantong Biotechnology, Nantong, China). [5] at a dose of 8 mg / kg every 24 hours in divided doses: this was twice the normal dose, in an effort to ensure penetration into the eyeball. S-adenosyl-L-methionine supplementation was recommended to support the liver during treatment with Mutian. The dose of Mutian was reduced to 6 mg / kg on day 25, but by then the weight of the cat had increased, so the same number of capsules per day was required.

TreatmentDayOphthalmoscopic findings
Systemic prednisolone 0Day of presentation to the primary veterinarian.
1Eye examined using a standard ophthalmoscope. Cornea unchanged, anterior chamber slightly cloudy, but sedation would be required for a detailed examination. Cat blind and stressed.
2Blood samples and aspirates of mesenteric lymph nodes with a thin needle taken under sedation.
Topically prednisolone acetate 1% tid 4 Keratin (protein) precipitated on the lens, which partially covers the fundus. Pupillary light reflex present but reduced.
Mutian 8mg / kg for 19 days, then 6mg / kg 6The cat's caregiver said he thought the cat could see.
17The cat's caregiver said the cat was chasing a bird.
18Uveitis recedes, anterior chamber clear. Ceramic precipitates as before.
41Uveitis has largely subsided, keratic precipitates still present. The fundus was visible, the chorioretinitis had subsided, and the cat's high temperament made detailed examination impossible. Excellent visual sensitivity.

Table 3
Timeline of treatment and resolution of uveitis.

To evaluate the susceptibility of the FCoV strain to the antiviral drug used, faecal samples were subjected to an FCoV qRT-PCR test (Veterinary Diagnostic Services, University of Glasgow, Scotland) [5]; the result was weakly positive in the threshold cycle (CT) 30, then negative, indicating that the virus was sensitive to the antiviral (Table 1). The faeces of three of the other four domestic cats were also positive for FCoV RNA (Table 1).

After starting Mutian treatment, the cat's weight increased rapidly, while it decreased by 30 g (2.89-2.86 kg) during prednisolone treatment (Figure 2). After 40 days of Mutian, the cat gained one kilogram (Figure 2). The dose of Mutian was adjusted during treatment in accordance with weight gain.

Blood tests were repeated on day 13 of Mutian treatment (18 days after diagnosis) (Table 2). Most importantly, the anemia reversed, HCT increased from 22.0 to 34.4%, and bilirubin decreased from 11 to 6 μmol / L. Globulin decreased to 57 g / l and albumin increased by 2 g / l, so the albumin: globulin ratio increased from 0.31 to 0.46, which was still a small but still improvement (Figure 3). Clinical examination revealed that iris color had returned to normal (Figure 1, Table 3) and vision improved; the caregiver reported an increase in activity - playing with other cats - and that the cat was trying to catch a bird. His droppings regained their normal consistency and frequency.

Figure 3
Timeline of treatment in relation to globulin albumin levels, showing a decrease in globulin (and thus total protein) levels, an increase in albumin and an albumin to globulin ratio. Improvement in these parameters continued after discontinuation of Mutian and feline interferon omega therapy.

The dose of Mutian was reduced to 6 mg / kg on day 20 and treatment with Mutian was stopped after 7 weeks for two reasons: AGP levels returned to normal (400 μg / ml - normal is <500 μg / ml), and because SDMA rose to 28 μg / dl (reference range is below 14 μg / dl). Recombinant feline interferon omega (rFeIFN-ω; Virbagen Omega, Virbac, France) started at 100,000 units every 24 hours orally. SDMA decreased to 19 μg / dl on day 62 (one week after the end of Mutian treatment) and to 14 μg / dl on day 90, which was 35 days after the end of Mutian treatment (Table 2). Urine analysis performed at days 62, 90, and 153 revealed no abnormalities, and the specific gravity was 1,050, 1,055, and 1,050, respectively. Weight gain continued after stopping Mutian treatment and using feline interferon omega (Figure 1), albeit at a slower rate (average 9g / day instead of 23g / day).

Cohabiting cats were successfully treated with Mutian at a dose of 4 mg / kg for five days to prevent coronavirus secretion and thus recurrent recovery of the recovered cat; the faeces of all five cats were confirmed as negative by RT-PCR (Table 1).

3. Discussion

The successful treatment of FIP with antiviral drugs was first published by Dr. Pedersen [4,9]. Cat owners can obtain various anti-FCoV drugs, including Mutian, an oral form of adenosine analog, over the Internet. However, such medicines are not licensed for veterinary use, which puts treating veterinarians in a dilemma: they cannot prescribe or supply such treatment, but they can help clients who choose to take these medicines by providing diagnostic and routine supportive care. It is particularly useful to make an accurate diagnosis that will prevent the treatment of about 40% cats that would otherwise be misdiagnosed (data not shown). Our view is that it would be more appropriate for cat owners to use such drugs under proper veterinary supervision, even if these products are unlicensed. The purpose of this case study is to report what has worked for us in monitoring a case of non-fusible FIP treated with Mutian followed by feline interferon.

Diagnosis of non-fusive FIP is challenging because the symptoms are diverse and the list of differential diagnoses is often lengthy. Dunbar et al. 2019 [10], reported that detection of FCoV RNA from Mesenteric Lymph Node (MLN) FNA (Fine Needle Aspiration) is 96% specific for FIP diagnostics. Unfortunately, the lab initially erroneously performed the FCoV RT-PCR test on a blood sample from day 2, instead of the MLN FNA as required - and the test turned negative. FCoV RT-PCR on blood samples is not useful in the diagnosis of FIP because most cats with FIP are not viremic at the time of clinical manifestations. [11] and also because about 5% cats do not test positive for FIP [12,13] because FCoV-infected cats undergo transient viremia [12,13,14,15]. FCoV RNA was found in feline MLN FNA and revealed to be positive for the M1058L mutation, which was further evidence to support the diagnosis of FIP [6,11]; although substitution with methionine for leucine at position 1058 in the FCoV spike protein was found in 89% tissue samples from 14 cats without FIP, suggesting that the mutation indicates systemic spread of FCoV from the gut rather than a virus with the potential to cause FIP [16].

Despite laboratory delays in confirming the diagnosis of FIP, we initiated Mutian treatment based on history, clinical signs, blood scores, and elevated AGP, which had previously been shown to be very specific for distinguishing FIP from other diseases with similar manifestations. [17]. There was an urgency to start treatment because the FIP male staging score was 6, indicating death within two weeks. [18]. The HCT of the cat decreased rapidly (from 25.7 to 22% in two days), which proved to be an indicator of imminent near death: Tsai et al., (2011) [18] found that HCT levels drop dramatically 2 weeks before death and bilirubin increases one week before death.

The history of the cat was typical of many cases of FIP: two new 11-month-old purebred cats were introduced into the household, which were probably the source of a recent coronavirus infection; the cat was vaccinated five days before the onset of uveitis. Introducing new cats and vaccinations are stressful for cats - it is known that stress causes FIP in cats infected with FCoV [19] and Riemer et al., (2016) [20] found that vaccination was a suspected stress factor in 6.9 % cases of FIP. Unfortunately, the weight of the cat between the introduction of new cats and vaccination was not recorded, so it is not known whether he lost weight during revaccination (ie was subclinically ill with FIP) or whether weight loss started later. This case nevertheless illustrates that vaccination of FCoV-infected cats may pose a risk to the development of FIP, and that optimal vaccination should be performed after the cat has stopped shedding the virus, which occurs within a few months for most FCoV type 1 infections. [21 ].

Clinical signs of weight loss, appetite, diarrhea and uveitis were also consistent with the diagnosis of non-fusive FIP. FIP is a major cause of uveitis in young cats - ophthalmologists from the North Carolina Veterinary School diagnosed FIP in 19 of 120 cats (15.8%) with uveitis [22]. Uveitis was reported in 17 of 59 (29%) cats with non-fusive FIP [23].

Although treatment with an injectable nucleoside analogue has been documented previously in the treatment of FIP [4], to our knowledge, oral treatment of FIP with an adenosine analogue has not been previously reported, although it has been used to treat FCoV infection in asymptomatic or diarrhea cats. [5]. The patient was given twice the normal therapeutic dose of FIP (ie, 8 mg / kg vs. 4 mg / kg) to potentially increase the concentration of the drug in the eye and to rule out any virus that might be present in the brain because only 20% of the absorbed drug passes through the blood-brain barrier (T Xue, personal communication). The response to treatment was rapid, dramatic and extremely positive. Although we tried to reduce the dose of Mutian as soon as possible in the event of possible side effects, in practice the amount of medication did not change significantly because the cat gained weight at the same time. Pedersen et al., (2019) [4] stated that the simplest indicator of response to treatment was weight gain, which was staggering in this case, as the cat gained 1.1 kg during the first 50 days of treatment. In contrast, he lost 30g during the week on prednisolone, before starting treatment with Mutian.

In two studies, cats with FIP treated with corticosteroids had a median survival of 7.5 days [24] and 8 days [25], which is significantly shorter than the 21 - day average reported by Tsai et al., 2011 [18] (although the potential adverse effects of biopsies on survival need to be considered in the two studies and many deaths were due to secondary bacterial infection due to extensive corticosteroid-induced immunosuppression [25]). In addition, prednisolone treatment was previously found to shorten survival in FIP cats that were co-treated with polyprenyl immunostimulant. [23]. Therefore, in our case, systemic corticosteroids were replaced by topical corticosteroids for the treatment of uveitis.

We do not know if it was Mutian, systemic and topical steroids, or a combination of all treatments that affected the overall cure of uveitis. The cat owner reported a partial return of vision on day 6, which was the day Mutian treatment was started, suggesting that steroids were responsible for return vision (Table 3). Legendre et al., (2017) [23] reported progress in 3 of 17 cats with ocular FIP treated with polyprenyl immunostimulant (PI). Their initial symptoms included anterior uveitis (all three cats), clots of ceramic origin (one cat), iris discoloration (one cat) and anisocoria (one cat). In two cats, anterior uveitis significantly improved or resolved after 2 months of PI treatment without corticosteroids. In the third cat, uveitis did not improve; the cat received topical ocular corticosteroids at the same time as PI and the eyes were enucleated [23]. Whether or not Mutian alone can affect the treatment of FIP-associated uveitis without current topical corticosteroids remains to be seen. Systemic corticosteroids appear to have little or no significance in these cases.

No clinical side effects were observed during treatment with Mutian and the biochemical parameters of the liver and kidney were within the reference range, with the exception of plasma symmetric dimethylarginine (SDMA), which increased (Table 2). SDMA is an early indicator of kidney disease [26,27]. There are three possible explanations for increased SDMA: first, it may be due to FIP lesions in the kidneys. Azotemia is more likely in non-effusive than effusive FIP [20], but as far as we know, no study has been conducted to determine whether SDMA is rising in cats with FIP - veterinary pathologists speculate that transient FCoV infection of the kidneys may be the cause of interstitial nephritis observed in many older cats (W Jarrett and S Toth, personal communication ). Second, predisposition to the breed may be a factor - Birm cats have been found to have elevated SDMA levels. [28], but no study has been performed on SDMA levels in Norwegian Forest Cats and in cats, SDMA levels have fallen to normal levels, suggesting that the increased SDMA levels did not appear to be related to his breed. Third, increased SDMA may have been a side effect of Mutian treatment - SDMA decreased within seven days after the end of Mutian treatment and returned to normal within 35 days after the end of Mutian treatment. However, because we did not test it earlier during the disease, we do not know if it was increased before the deployment of Mutian. During treatment, cat creatinine and urea were always within normal limits, although a progressive increase in creatinine was observed (Table 2), which will be closely monitored in the future.

Because the recommended time of administration of GS-441524 was 84 days [4], cat owners using other antivirals have been treating their cats with FIP for the same length of time. The optimal duration of FIP oral Mutian treatment has not been established. However, this case illustrates that treatment can be stopped after a much shorter cycle if laboratory indicators for FIP return to normal. We believe that in our case, complete recovery occurred after 50 days of treatment for the following reasons - uveitis subsided, the cat recovered from anemia, lymphopenia reversed, bilirubin decreased and AGP decreased permanently. Nevertheless, we did not want to leave the cat completely without antiviral coverage, so a low oral dose of rFeIFN-omega was used as described above. [29]. This drug has antiviral and immunomodulatory properties and was an optional treatment for FIP with meloxicam prior to the onset of specific antiviral drugs against FCoV. [30]. The cat continued to gain weight with rFeIFN-omega, although more slowly than before, stabilized at 4.35 kg after 100 days (Figure 2) and hematological parameters continued to improve (Table 2, Figure 3).

FIP relapse did not occur in this - or in other - cases whose treatment we monitored. We attribute our success primarily to assessing the effectiveness of an antiviral agent against the FCoV strain in an infected cat by monitoring the amount of virus excreted in the stool (by qRT-PCR) to ensure that the viral load decreased in response to the antiviral agent; second, preventing FIP-cured re-infection by identifying other sources of viruses in the same household and stopping FCoV shedding by Mutian [5]; third, short-term treatment with a dose that eliminated the virus from the brain; fourth, after treatment with a nucleoside analogue (or protease inhibitor) by initiating a low dose of oral feline interferon until the FCoV antibody titer has decreased - an indicator that no coronavirus remains in the body.

Skywise and his roommates are alive and well 6 months after diagnosis.

4. Conclusions

To our knowledge, this is the first published case of curing a non-fusive FIP cat using an oral adenosine analogue, followed by a recombinant feline interferon, and AGP used as a recovery marker from FIP. To protect the liver and monitor liver enzymes, we recommend taking SAMe with Mutian. SDMA levels in future cases following this FIP treatment protocol require further investigation. We suggest that systemic corticosteroids be contraindicated in the treatment of FIP for non-palliative purposes.

Acknowledgement

We would like to thank the reviewers for their time in thoroughly reviewing our work and for their helpful comments: Table 1 and Table 3 were added thanks to their comments.

Author's contributions

Conceptualization, DDA and JC-R .; methodology, MF, OJ and DDA .; validation, OJ, DDA, JC-R. and MF .; formal analysis, MF and DDA .; investigation, MF and JC-R .; sources, JC-R. and MF .; data management, DDA, JC-R. and MF .; writing - preparation of the original proposal, DDA; writing - reviews and edits, OJ, DDA, JC-R. and MF .; visualization, DDA and JC-R .; supervision, DDA and JC-R .; project administration, JC-R., MF. financing acquisition, JC-R. and DDA All authors have read and agreed to the published version of the manuscript.

Financing

Cat treatment and lab tests were funded by JC-RDDA Thank you subscribers www.catvirus.com for support during this study and we are very grateful to the Angelica Fund donor for the payment of publication fees.

References

  1. Addie DD, Toth S., Murray GD, Jarrett O. The risk of feline infectious peritonitis in cats naturally infected with feline coronavirus. Am. J. Vet. Res. 1995;56: 429–434. [PubMed] [Google Scholar]
  2. Kipar A., May H., Menger S., Weber M., Leukert W., Reinacher M. Morphologic features and development of granulomatous vasculitis in feline infectious peritonitis. Vet. Pathol. 2005;42: 321–330. doi: 10.1354 / vp.42-3-321. [PubMed] [CrossRef] [Google Scholar]
  3. De Groot RJ, Baker SC, Baric R., Enjuanes L., Gorbalenya AE, Holmes KV, Perlman S., Poon L., Rottier PJM, Talbot PJ, et al. Family Coronaviridae. In: Adams MJ, Carstens EB, Lefkowitz EJ, editors. Ninth Report International Committee on Taxonomy of Viruses; King AMQ. Elsevier; Amsterdam, The Netherlands: 2012. pp. 806–828. [Google Scholar]
  4. 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: 271–281. doi: 10.1177 / 1098612X19825701. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  5. Addie DD, Curran S., Bellini F., Crowe B., Sheehan E., Ukrainchuk L., Decaro N. Oral Mutian® X stopped faecal feline coronavirus shedding by naturally infected cats. Res. Vet. Sci. 2020;130: 222–229. doi: 10.1016 / j.rvsc.2020.02.012. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  6. Chang HW, Egberink HF, Halpin R., Spiro DJ, Rottier PJ Spike protein fusion peptide and feline coronavirus virulence. Emerg. Infect. Dis. 2012;18: 1089–1095. doi: 10.3201 / eid1807.120143. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  7. Duthie S., Eckersall PD, Addie DD, Lawrence CE, Jarrett O. Value of alpha1-acid glycoprotein in the diagnosis of feline infectious peritonitis. Vet. Rec. 1997;141: 299–303. doi: 10.1136 / vr.141.12.299. [PubMed] [CrossRef] [Google Scholar]
  8. Shelly SM, Scarlett-Kranz J., Blue JT Protein electrophoresis on effusions from cats as a diagnostic test for feline infectious peritonitis. J. Am. Anim. Hosp. Assoc. 1988;24: 495–500. [Google Scholar]
  9. Pedersen NC, Kim Y., Liu H., Galasiti KAC, Eckstrand C., Groutas WC, Bannasch M., Meadows JM, Chang KO Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J. Feline Med. Surg. 2018;20: 378–392. doi: 10.1177 / 1098612X17729626. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  10. Dunbar D., Kwok W., Graham E., Armitage A., Irvine R., Johnston P., McDonald M., Montgomery D., Nicolson L., Robertson E., et al. Diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative polymerase chain reaction from mesenteric lymph node fine needle aspirates. J. Feline Med. Surg. 2019;21: 910–921. doi: 10.1177 / 1098612X18809165. [PubMed] [CrossRef] [Google Scholar]
  11. Felten S., Leutenegger CM, Balzer HJ, Pantchev N., Matiasek K., Wess G., Egberink H., Hartmann K. Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum / plasma of cats to diagnose feline infectious peritonitis. BMC Vet. Res. 2017;13: 228. doi: 10.1186 / s12917-017-1147-8. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  12. Fish EJ, Diniz PPV, Juan YC, Bossong F., Collisson EW, Drechsler Y., Kaltenboeck B. Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California. J. Feline Med. Surg. 2018;20: 295–301. doi: 10.1177 / 1098612X17705227. [PubMed] [CrossRef] [Google Scholar]
  13. Simons FA, Vennema H., Rofina JE, Pol JM, Horzinek MC, Rottier PJ, Egberink HF A mRNA PCR for the diagnosis of feline infectious peritonitis. J. Virol. Methods. 2005;124: 111–116. doi: 10.1016 / j.jviromet.2004.11.012. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  14. Herrewegh AAPM, de Groot RJ, Cepica A., Egberink HF, Horzinek MC, Rottier PJM Detection of feline coronavirus RNA in feces, tissue, and body fluids of naturally infected cats by reverse transcriptase PCR. J. Clin. Microbiol. 1995;33: 684–689. doi: 10.1128 / JCM.33.3.684-689.1995. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  15. Tasker S. Diagnosis of feline infectious peritonitis: Update on evidence supporting available tests. J. Feline Med. Surg. 2018;20: 228–243. doi: 10.1177 / 1098612X18758592. [PubMed] [CrossRef] [Google Scholar]
  16. Porter E., Tasker S., Day MJ, Harley R., Kipar A., Siddell SG, Helps CR Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis. Vet. Res. 2014;45: 49. doi: 10.1186 / 1297-9716-45-49. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  17. Giori L., Giordano A., Giudice C., Grieco V., Paltrinieri S. Performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases. J. Small Anim. Pract. 2011;52: 152–157. doi: 10.1111 / j.1748-5827.2011.01042.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  18. Tsai HY, Chueh LL, Lin CN, Su BL Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan. J. Feline Med. Surg. 2011;13: 74–80. doi: 10.1016 / j.jfms.2010.09.014. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  19. Rohrer C., Suter PF, Lutz H. The diagnosis of feline infectious peritonitis (FIP): A retrospective and prospective study. Kleinterpraxis. 1993;38: 379–389. [Google Scholar]
  20. Riemer F., Kuehner KA, Ritz S., Sauter-Louis C., Hartmann K. Clinical and laboratory features of cats with feline infectious peritonitis – A retrospective study of 231 confirmed cases (2000–2010) J. Feline Med. Surg. 2016;18: 348–356. doi: 10.1177 / 1098612X15586209. [PubMed] [CrossRef] [Google Scholar]
  21. Addie DD, Jarrett JO Use of a reverse-transcriptase polymerase chain reaction for monitoring feline coronavirus shedding by healthy cats. Vet. Rec. 2001;148: 649–653. doi: 10.1136 / vr.148.21.649. [PubMed] [CrossRef] [Google Scholar]
  22. Jinks MR, English RV, Gilger BC Causes of endogenous uveitis in cats presented to referral clinics in North Carolina. Vet. Ophthalmol. 2016;19(Suppl. S1): 30–37. doi: 10.1111 / vop.12324. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  23. Legendre AM, Kuritz T., Galyon G., Baylor VM, Heidel RE Polyprenyl Immunostimulant Treatment of Cats with Presumptive Non-Effusive Feline Infectious Peritonitis in a Field Study. Front. Vet. Sci. 2017;4: 7. doi: 10.3389 / fvets.2017.00007. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  24. Fischer Y., Ritz S., Weber K., Sauter-Louis C., Hartmann K. Randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis. J. Vet. Intern. Med. 2011;25: 1270–1276. doi: 10.1111 / j.1939-1676.2011.00806.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  25. Ritz S., Egberink H., Hartmann K. Effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis. J. Vet. Intern. Med. 2007;21: 1193–1197. doi: 10.1111 / j.1939-1676.2007.tb01937.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  26. Jepson RE, Syme HM, Vallance C., Elliott J. Plasma asymmetric dimethylarginine, symmetric dimethylarginine, l-arginine, and nitrite / nitrate concentrations in cats with chronic kidney disease and hypertension. J. Vet. Intern. Med. 2008;22: 317–324. doi: 10.1111 / j.1939-1676.2008.0075.x. [PubMed] [CrossRef] [Google Scholar]
  27. Relford R., Robertson J., Clements C. Symmetric Dimethylarginine: Improving the Diagnosis and Staging of Chronic Kidney Disease in Small Animals. Vet. Clin. U.S. Small Anim. Pract. 2016;46: 941–960. doi: 10.1016 / j.cvsm.2016.06.010. [PubMed] [CrossRef] [Google Scholar]
  28. Paltrinieri S., Giraldi M., Prolo A., Scarpa P., Piseddu E., Beccati M., Graziani B., Bo S. Serum symmetric dimethylarginine and creatinine in Birman cats compared with cats of other breeds. J. Feline Med. Surg. 2018;20: 905–912. doi: 10.1177 / 1098612X17734066. [PubMed] [CrossRef] [Google Scholar]
  29. Gil S., Leal RO, McGahie D., Sepúlveda N., Duarte A., Niza MM, Tavares L. Oral Recombinant Feline Interferon-Omega as an alternative immune modulation therapy in FIV positive cats, clinical and laboratory evaluation. Res. Vet. Sci. 2014;96: 79–85. doi: 10.1016 / j.rvsc.2013.11.007. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  30. Addie D. Feline coronavirus infections. In: Green C., editor. Infectious Diseases of the Dog and Cat. Elsevier; Maryland Heights, MO, USA: 2012. pp. 92–108. [Google Scholar]
Read "Rapid remission of non-fusive FIP uveitis during treatment with oral adenosine analogue and feline omega interferon"

Antiviral therapy using the adenosine nucleoside analog GS-441524 in cats with clinically diagnosed neurological feline infectious peritonitis.

Original article: Antiviral treatment using the adenosine nucleoside analogue GS ‐ 441524 in cats with clinically diagnosed neurological feline infectious peritonitis; 5/22/2020; Translation 19.2.2021

Peter J. Dickinson, Michael Bannasch, Sara M. Thomasy, Vishal D. Murthy, Karen M. Vernau, Molly Liepnieks, Elizabeth Montgomery, Kelly E. Knickelbein, Brian Murphy, Niels C. Pedersen

Abstract

Feline infectious peritonitis (FIP) is caused by a mutant feline enteric coronavirus biotype. The resulting FIP virus (FIPV) in the case of the non-fusion form commonly causes pathologies of the central nervous system (CNS) and the eyes. More than 95% cats with FIP succumb to the disease several days to months after diagnosis, despite various historically used treatments. Recently developed antiviral drugs have shown promise in the treatment of non-neurological FIPs, but data on the treatment of neurological cases of FIP are limited. Four cases of naturally occurring FIP with CNS involvement were treated with the antiviral nucleoside analog GS-441524 (5-10 mg / kg) for at least 12 weeks. Cats were monitored continuously by physical, neurological and ophthalmological examinations. One cat underwent magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analysis, including FCoV titer, RT-PCR, and ophthalmic examination using Fourier optical coherence tomography and in vivo confocal microscopy (IVCM). All cats responded positively to treatment. The three cats are still alive (528, 516 and 354 days after the start of treatment) with normal results of physical and neurological examinations. One cat was euthanized 216 days from the start of treatment after relapses after primary and secondary treatment. In 1 case, the cure of the disease was determined on the basis of normalization of MRI and CSF findings and examination of cranial and caudal segment disease using ocular imaging methods. Treatment with GS-441524 shows its clinical efficacy and may lead to clearance and long-term regression of neurological FIP. The doses required for CNS disease may be higher than the doses used for non-neurological FIPs.

Keywords: antiviral, cat, coronavirus, ophthalmology

Abbreviations
AGalbuin / globulin
CNScentral nervous system
CSFcerebrospinal fluid
ELISAenzyme immunoabsorption test
FCoVfeline coronavirus
FD ‐ OCTFourier optical coherence tomography
FeLVfeline leukemia virus
FIPfeline infectious peritonitis
FIPVfeline infectious peritonitis virus
FIVfeline immunodeficiency virus
HIVhuman immunodeficiency virus
IFAindirect immunofluorescence test
IgGimmunoglobulin G
IgMimmunoglobulin M
IVCMin vivo confocal microscopy
LMlarge mononuclear
MRImagnetic resonance imaging
ODoculus dexter, right eye
OSoculus sinister, left eye
OUoculus uterque, both eyes
PLRpupillary light reflex
RT-PCRreverse transcriptase polymerase chain reaction
SMsmall mononuclear
TNCCtotal number of nucleated cells
TPtotal protein

1. Introduction

The experimental treatments were approved by the Institutional Committee on the Care and Use of Animals and the Clinical Trials Evaluation Committee of the Veterinary Medical University School of the University of California, Davis. GS-441524 was provided by Gilead Sciences Inc. (Foster City, CA), as previously described. 1, 2 The clinical diagnosis of FIP was based on a combination of characteristic information, medical history, disease symptoms, laboratory test results including hyperglobulinemia, decreased albumin: globulin (AG) ratio and FCoV antibody titers (indirect immunofluorescence test [IFA]), Fuller Laboratories, Fullerton, California) 3 and response to virus-specific treatment. The status of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) was determined for FeLV antigen and FIV antibody by ELISA (IDEXX, Westbrook, Maine). In one case, repeated advanced diagnostic testing was performed, including MRI, CSF analysis, CSF FCoV RT ‐ PCR (Real-time PCR Research and Diagnostics Core Facility, UC Davis, Davis, CA) and serology, FD ‐ OCT and IVCM.

2. Case 1

A neutered male of an 8-month-old blue Siamese cat obtained as a kitten from a rescue group. History of several months of lethargy and decreased appetite and monthly progressive pelvic limb ataxia confirmed by neurological examination. The cat weighed 3.0 kg, which was 1 kg less than the female sibling. Serum biochemistry abnormalities included increased total protein concentration (8.9 g / dl; reference interval 6.3-8.8 g / dl) with an AG ratio of 0.53 (albumin, 3.1 g / dl; reference interval 2, 6-3.9 g / dl; globulin), 5.8 g / dl; reference interval, 3.0-5.9 g / dl). Tests for FeLV and FIV antibody titers and Toxoplasma gondii IgM and IgG (Protatek, Mesa, Arizona) were negative and the FCoV antibody titer was positive at 1:12 800. OU). Ultrasound of the abdomen showed circumferential hyperechoic lines at the renal corticomedullary junctions, linear hyperechoic lines parallel to the luminal surface of the jejunum and ileum, and enlarged colic and mesenteric lymph nodes. The cat was treated with 5 mg / kg GS-441524 SC once daily for 14 weeks. Appetite and activity, including jumping on elevated surfaces, improved within 4 days. The total serum protein concentration after the end of treatment was 7.8 g / dl with an AG ratio of 0.77. Neurological examination was normal and body weight was 5.1 kg. The cat remains clinically normal at the time of writing 528 days after the start of treatment.

3. Case 2

The annual neutered domestic shorthair cat born to the rescued feral cat had a 3-month history of anterior uveitis and a weekly history of lethargy, altered behavior, tail twitching, generalized seizures, decreased appetite, and pelvic dysphagia and ataxia. The cat weighed 3.7 kg. At the neurological examination, the cat was dulled by generalized ataxia, decreased postural responses in the left thoracic and right pelvic limbs, decreased physiological OU nystagmus, and decreased nasal perception on the right side. Approach response was reduced in both eyes, anisocoria (mydriasis in the left eye [oculus sinister, OS]) was present with reduced direct and consensus pupillary light reflex (PLR) with OS illumination. Ophthalmic examination revealed anterior uveitis of the OU with retinal detachment of the OS and retinal vasculitis of the OU. The cat weighed 3.7 kg and serum biochemistry abnormalities included an increased total protein concentration (8.6 g / dl; reference interval, 6.6-8.4 g / dl) with an AG ratio of 0.48 (albumin, 2.8 g reference interval, 2.2-4.6 g / dl; globulin, 5.8 g / dl; reference interval, 2.8-5.4 g / dl), increased total bilirubin concentration (1.8 mg / dl; reference interval, 0.0-0.2 mg / dl) and increased AST activity (128 IU / dl) L; reference interval, 17-58 IU / L). The feline leukemia virus, FIV and heartworm antigen test (SNAP Feline Triple Test, IDEXX, Westbrook, Minnesota) was negative. Abdominal palpation showed enlarged mesenteric lymph nodes. The cat was treated with 5 mg / kg GS-441524 SC, once daily for 14 weeks. The body weight at the end of treatment was 5.9 kg. The level of mentation and activity improved significantly 48 hours after the start of treatment. After 3 weeks of treatment, neurological and ophthalmic symptoms were unobservable except for mild intermittent anisocoria and chorioethinal scars OU. Previously reported abnormalities in serum biochemistry resolved with a total serum protein concentration of 8.1 g / dl and an AG ratio of 0.77. Three weeks after the end of the treatment, the cat weighed 6.4 kg, physical and neurological examinations were found and the total serum protein concentration was 7.0 mg / dl with an AG ratio of 0.84. The cat remains clinically normal at the time of writing 516 days after the start of treatment.

4. Case 3

The 18-month-old neutered domestic shorthair cat obtained from the shelter had a 3-month history of eye disease, a 3-week progressive history of lethargy and anorexia, and a several-day history of progressive limb paresis. The cat was treated with prednisolone-acetate 1% OU eye drops, every 6 hours for 5 days before presentation. At the neurological examination, the cat showed inappropriate behavior and hypersensitivity to cranial palpation. The cat was non-ambulatory paraparetetic with reduced pelvic limb reflexes. The blink reflex lacked OU with anisocoria (pupil of medium extent in the right eye [oculus dexter, OD], mydriasis OS). The pupillary light reflex lacked OD due to posterior synechiae and absent OS with direct or indirect illumination. Reflections for OU glare and vision were present and the cat showed reduced vision under photopic conditions. Ophthalmic examination revealed uveitis and OU hyperviscosity syndrome. The cat weighed 2.6 kg. Serum biochemistry abnormalities included increased total protein concentration (11.7 g / dl; reference interval 6.3-8.8 g / dl) with an AG ratio of 0.2 (albumin, 2.0 g / dl; reference interval 2, 6-3.9 g / dl; globulin), 9.7 g / dl; reference interval, 3.0-5.9 g / dl). Tests for FeLV and FIV were negative and the FCoV antibody titer was positive at 1: 6400. Ultrasound of the abdomen showed hepatosplenomegaly, small kidneys with indistinct corticomedullary junctions, and enlarged mesenteric lymph nodes. The cat was treated with 5 mg / kg GS-441524 SC, once daily for 15 weeks. After 1 month of treatment, uveitis improved, but is still present and the cat was outpatient paraparetetic with normal segmental reflexes. The cat weighed 3.3 kg and the AG ratio was 0.55. After 2 months of treatment, mild signs of active anterior ODitis were present, but there was only a slight improvement in outpatient paraparesis. Body weight increased to 3.7 kg and the AG ratio was 0.67. After 15 weeks of treatment, there were only minimal signs of uveitis OD and ambulatory paraparesis, which was static during the previous 4 weeks, improved. The cat weighed 4.0 kg and the AG ratio was 0.76. After cessation of treatment, lethargy, anorexia and anisocoration returned within 36 hours. Treatment was resumed at 5 mg / kg GS-441524 SC once daily and symptoms resolved within 24 hours. The signs remained static during the 12 weeks of the second round of treatment, but the reduced activity reappeared after the end of treatment. The cat was euthanized in part due to increased resistance to drug administration. Histopathological evaluation after autopsy showed multifocal chronic dysfunctional meningitis, encephalomyelitis and ventriculitis, lymphocyte, histiocytic uveitis and choroiditis OU and interstitial nephritis. Positive coronavirus immunohistochemical immunoreactivity (FIP antibody - V3-70, Custom Monoclonals International, Sacramento, CA) was identified within histiocytes associated with lesions in the brain, kidney and eye.

5. Case 4

A seven-month-old sterilized female domestic shorthair cat adopted from a shelter had a three-week history of lethargy and anorexia and a two-week history of ataxia and crouching. At the neurological examination, the cat had an ataxic gait that was worse in the pelvic limbs. Postural responses were reduced in the pelvic limbs. Anisocoria (midrange OD, miotic OS) was present with incomplete PLR OUs. Blink reflexes, dazzling reflexes and vision were present by the OU. The cat weighed 2.7 kg. Brain magnetic resonance imaging has shown multifocal T2-weighted hyperintensity in the entire parenchyma, most severely in the midbrain and thalamus. Post-contrast T1-weighted images showed diffuse thickening and thickening of the meninges and brainstem with marked ventriculomegaly (Figure 1). Cerebrospinal fluid collected from the cerebellomedular cistern was xantochromic with a total nucleated cell count (TNCC) of 888 / μL (reference interval, <3 cells / μL) and a total CSF protein concentration of 1790 mg / dl (reference interval, <25 mg / dl). Serum and CSF titers of FCoV antibodies were positive at> 1:20 480 and real-time TaqMan RT-PCR for FCoV in CSF was positive with a threshold cycle (Ct) value of 18.87. Tests for FeLV and FIV were negative. Serum biochemistry and CBC abnormalities included a total protein concentration of 8.5 g / dl (reference interval, 6.6-8.4 g / dl), an AG ratio of 0.37 (albumin 2.3 g / dl; reference interval 2.2 -4.6 g / dl, globulin 6.2 g / dl, reference interval 2.8-5.4 g / dl), total bilirubin concentration 0.5 mg / dl (reference interval 0.0-0.2 mg / dl), anemia (hematocrit, 25.8%; reference interval, 30 -50%) and lymphopenia (835 / μL; reference interval, 1000-7000 / μL). Ultrasound of the abdomen showed hyperechoic kidneys and retroperitoneal fat, several enlarged lymph nodes, and mild peritoneal effusion. An ophthalmological examination of the FD ‐ OCT and IVCM revealed anterior uveitis with keratinous clots present in the OU at the back; ocular hypertension (25 mmHg OD, 11 mmHg OS) and chorioretinitis were also identified by OD (Figure 2). The cat was treated with 5 mg / kg GS-441524 SC, once daily for 4 weeks and prednisolone acetate 1% eye drops OU q8h and dorzolamide 2% eye drops OD q8h during the first 3 weeks of GS-441524 treatment. Activity and mentation improved within 24 hours after onset. treatment. After 4 weeks, the ophthalmic disease improved significantly (Figure 2), but ataxia was still present and the cat lost 0.2 kg of body weight (Figure 3). Serum total protein concentration was steadily increased (8.6 g / dl) with an improved AG ratio of 0.72; lymphopenia and anemia resolved. Due to lack of weight gain and persistent neurological deficits, the dose of GS-441524 was increased to 8 mg / kg SC, once daily for another 10 weeks (a total of 14 weeks). The cat also received a two-week course of prednisolone 1 mg / kg PO every 24 hours. Increased activity and willingness to jump on elevated surfaces were observed within 24 hours, and 1 week after the end of GS-441524 treatment, neurological examination was negative and no active ophthalmic disease was detected. Body weight was increased to 3 kg and total serum protein concentration was normal (7.6 g / dl) with an AG ratio of 0.8. Repeated magnetic resonance imaging (Figure 1) showed minimal increase in meningeal contrast, but ventriculomegaly increased. Repeated CSF RT ‐ PCR for FCoV RNA was negative and TNF in CSF decreased compared to the previous count, but still high at 224 / μL. Due to the evidence from the cerebrospinal fluid analysis that the infection was still active, the dose of GS-441524 was further increased to 10 mg / kg SC, once daily for another 5 weeks (a total of 19 weeks). The cat remained clinically normal with increased activity during this period and body weight increased to 4.7 kg (Figure 3). Immediately after the end of treatment, neurological and ophthalmological examinations remained unchanged and repeated MRI was found in addition to persistent ventriculomegaly. Repeated CSF analysis showed a continued decrease in TNCC (8 cells / μL) and total protein concentration (85 mg / dl), negative RT ‐ PCR for FCoV RNA, and a reduced 1: 128 FCoV antibody titer. Approximately 8 months after the start of treatment and 3 months after the end of treatment, the MRI did not change from the previous image, except for less severe ventriculomegaly. Cerebrospinal fluid analysis showed TNCC 6 cells / μL, total protein concentration 52 mg / dl, negative RT ‐ PCR for FCoV RNA and static antibody titer FCoV 1: 128. Serum total protein concentration was 7.1 g / dl with AG 0 ratio , 97. No signs of active inflammation were observed on ophthalmology, although focal areas of retinal dilution were identified. The cat remains clinically normal at the time of writing 354 days after the start of treatment.

Figure 1
Sequential magnetic resonance imaging from case 4. The lines represent selected postcontrast (gadolinium) T1 weighted transverse images of the brain obtained in one imaging sequence. Routine cerebrospinal fluid analysis at the time of imaging is shown in white for each imaging time point: TNCC = total number of nucleated cells in CSF (cells / μl); TP = total protein CSF (mg / dl); N = neutrophils, SM = small mononuclear cells, LM = large mononuclear cells. The characteristic neutrophil pleocytosis resolved during treatment. Additional CSF analyzes related to FCoV detection are presented in yellow for each time point: PCR = FCoV RT-PCR result [positive (+) or negative (-)]; Dilution ratio = FCoV antibody titer in cerebrospinal fluid. For each imaging sequence, the time points and doses of GS-441524 provided prior to imaging are described. The initial marked increase in meningeal pain contrast resolves after treatment with GS-441524 and does not recur after treatment. Ventriculomegaly, which is present after the initial response to treatment, slowly resolved upon subsequent imaging. The reduction in abnormalities in the CSF analysis findings in parallel corresponded to reduced abnormalities in MR imaging.
Figure 2
Sequential multimodal imaging of cranial and caudal segments from case 4. On the presentation of (A, B) predilation and (G, H) post-dilatation photographs of cranial segments showing mild diffuse corneal edema, pigmented ceramic clots, rubeosis iridis, detail of cloudy iris with flashes in aqueous humor and incomplete dilatation of the OU; dyskorrhea with incomplete pupillary dilatation due to caudal synechia OS (H) was also observed. Ceramic clots were also visualized by OS biomicroscopy with slit lamp (V), corneal FD-OCT (M) and endothelial IVCM (X, arrows); increased corneal thickness was also observed with FD-OCT (X). Imaging of the retina and choroid by FD ‐ OCT revealed a cellular infiltrate in the choroid (P, arrow), which was visible as a hyporeflective lesion with infrared photography (S). 0.8 months after the start of GS-441524 treatment, photographs of pre- (C, D) and post-dilatation (I, J) cranial segments of the clear cornea and cranial ventricle OU, isocoria, reduced rubeosis iridis and complete dilatation of the pupil. A significant decrease in pigmented ceramic precipitates was observed in slit lamp (W), corneal FD ‐ OCT (N) and IVCM endothelial (Y, arrow) biomicroscopy. Normal retinal and choroid morphology is observed in FD ‐ OCT (Q), although hyporeflective lesions remain on infrared imaging (T). At 7.6 months, pre- (E, F) and post-dilatation (K, L) photographs of the cranial segment showed clear corneas and cranial chambers of the OU, isocorrium, normal iris morphology, and post-inflammatory pigment on OS lens capsules. Corneal FD-OCT (O) lacks ceramic precipitates. In FD ‐ OCT and infrared imaging, dilution of the dorsal peripheral retina (R, arrow) was present with loss of normal stratification but no cellular infiltrate or retinal detachment (U).
Figure 3
Case 4 body weight development plotted with time-initiated GS-441524 treatment. Drug dose changes are indicated by red arrows. After increasing the dose of the drug above the initial dose of 5 mg / kg, an increase in body weight was observed and was accompanied by a remission of clinical signs.

6. Discussion

Infectious feline peritonitis is a major cause of mortality in young cats and a common cause of neurological disease. Several experimental treatments have not shown consistent efficacy against FIP, and cats are euthanized or die several days to months after the development of clinical disease, especially when FIP affects the CNS. Fortunately, drugs aimed at replicating RNA viruses in important human diseases, such as human immunodeficiency virus (HIV), hepatitis C and Ebola, have provided a model for treating viral diseases in other species, such as FIP. GS-441524 is a 1'-cyano-substituted adenine C-nucleoside ribose analog that inhibits viral RNA synthesis. GS-441524 and the 3C-Like viral protease inhibitor have demonstrated efficacy against FIPV in experimentally induced and naturally occurring FIP. However, preliminary studies suggest that treatment of ocular and CNS forms of FIP may be more complex due to limited drug passage across the blood-blood and blood-brain barriers. A high rate of relapse of FIP with CNS involvement has been observed in protease inhibitor-based therapy, while GS-441524 has become more promising in the treatment of ocular and neurological FIP. An initial clinical study with GS-441524 on naturally occurring, non-neurological FIP used doses of 2 mg / kg, which appeared to be insufficient for cats that developed neurological symptoms during treatment. However, 2 cats that developed neurological disease at this dose appeared to respond to 4 mg / kg. 4 cases of neurological FIP from our study were treated with a dose of 5 mg / kg, with duration of treatment and subsequent dose increase based on clinical reactions. A dose of 5 mg / kg, SC, once daily for 12 to 14 weeks was sufficient to treat 2 less severe neurological cases of FIP (cases 1, 2), but repeated cycles of 5 mg / kg in the most severely clinically affected cat (case 3 ) only resulted in improvement in clinical symptoms with rapid clinical regression after cessation of treatment. This therapeutic failure resulted in a gradual increase in dose from 5 to 10 mg / kg in case 4. The in vitro 50% effective concentration (EC50) for GS-441524 to prevent viral cytopathic effects was reported at 0.8 μM with complete inhibition of viral replication at 10 μM and partial inhibition at 1 μM. 1 Limited pharmacokinetic studies in cats from the same study showed that CSF concentrations of GS-441524 were approximately 20% in plasma and a dose of 10 mg / kg resulted in CSF concentrations of 0.8 to 2.7 μM. These data correspond to the limited efficacy associated with doses of 5 mg / kg in cases 3 and 4 and the apparent efficacy associated with increasing the dose to 8 to 10 mg / kg in cases 4. To further define the optimal dose of GS-441524 in cats with neurological FIP, extended pharmacokinetic studies in healthy and affected cats with intact and impaired blood-brain barrier function. As in previous reports, limited adverse reactions associated with long-term use of GS-441524 have been observed. Local skin reactions and discomfort after SC injection were the only clinically relevant adverse reactions, but this was a major factor influencing the euthanasia decision. 3. Although responses to treatment were measurable by MRI, CSF analysis, and eye imaging, clinical responses to treatment at the appropriate dose were equally useful, with rapid improvements in mentation, appetite, and activity, which were generally observed within 24 to 36 years. hours. Increased body weight and the ability to jump on elevated objects and surfaces were also considered consistent indicators of effective treatment. GS-441524 is not available for routine clinical use, but reported cases suggest that FIP affecting the CNS may be treatable with appropriate antiviral drugs. The development of similar antiviral drugs for clinical use should be considered a priority for this historically deadly disease.

References

  1. Murphy BG, Perron M, Murakami E, et al. The nucleoside analog GS ‐ 441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet Microbiol. 2018; 219: 226-233. [PMC free article] [PubMed] [Google Scholar]
  2. Pedersen NC, Perron M, Bannasch M, et al. 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: 271-281. [PMC free article] [PubMed] [Google Scholar]
  3. Pedersen NC. An update on feline infectious peritonitis: diagnostics and therapeutics. Vet J. 2014; 201: 133–141. [PMC free article] [PubMed] [Google Scholar]
  4. Pedersen NC. A review of feline infectious peritonitis virus infection: 1963‐2008. J Feline Med Surg. 2009; 11: 225-258. [PMC free article] [PubMed] [Google Scholar]
  5. Pedersen NC, Kim Y, Liu H, et al. Efficacy of a 3C ‐ like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2018; 20: 378-392. [PMC free article] [PubMed] [Google Scholar]
  6. Kim Y, Liu H, Galasiti Kankanamalage AC, et al. Reversal of the progression of fatal coronavirus infection in cats by a broad ‐ Spectrum coronavirus protease inhibitor. PLoS Pathog. 2016; 12: e1005531. [PMC free article] [PubMed] [Google Scholar]
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