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

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.

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 the treatment of 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 the main 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.

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