PHARMACEUTICAL COMPOSITION IN ORAL FORM FOR TREATMENT OF FELINE INFECTIOUS PERITONITIS, CONSISTING OF INHIBITOR OF RNA-DEPENDENT RNA POLYMERASE AND PROTEASE INHIBITOR 3CLpro

Abstract

FIELD: pharmaceuticals; veterinary medicine.SUBSTANCE: veterinary pharmaceutical composition for the treatment of feline infectious peritonitis (FIP). The composition is made in a form for oral administration and contains as active ingredients: an inhibitor of RNA-dependent RNA polymerase – (2R, 3R, 4S, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile (vefinavir) in an amount of 20-50 mg, a protease inhibitor 3CLpro-monosodium salt (βS)-α-hydroxy-β-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidinepropanesulfonic acid (prinvenavir) in the amount of 60-80 mg and 1,3-thiazol-5-ylmethyl-N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate (ritonavir) in the amount of 20-40 mg.EFFECT: reliable treatment of FIP in cats with minimal side effects and a low risk of relapse.3 cl, 5 dwg, 3 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of veterinary medicine, in particular to the treatment of infectious diseases in cats. In a non-limiting embodiment, the present invention provides a pharmaceutical composition in oral form for the antiviral therapy of feline infectious peritonitis, which comprises a combination of:

[0003] - Vefinavir (VS-121354) nucleoside analogue, inhibitor of RNA-dependent RNA polymerase of the virus ((2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5 (hydroxymethyl) oxolane-2-carbonitrile);

[0004] - Prinvenavir (VS-821303) protease inhibitor 3CLpro ((βS)-α-hydroxy-β-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidine propanesulfonic acid, monosodium salt).

[0005] To enhance the antiviral activity of the proposed combination, an option with the addition of Ritonavir (1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate) is proposed.

[0006] Technical level

[0007] Currently, antiviral drugs that inhibit feline coronavirus (FCoV) are being developed for the treatment of feline infectious peritonitis (FIP). However, their efficacy and safety are difficult to assess due to the lack of centralized studies and adequate clinical data. A drawback of these known drugs is the painful, prolonged subcutaneous injections required, typically 12 weeks or longer, which causes discomfort for both owners and animals.

[0008] Subcutaneous injections over such a long period of time can lead to side effects such as malignancies, clinical abscesses, and necrosis. The long duration of therapy is due to the relatively rapid occurrence of disease relapses (4-6 weeks), which requires the use of high doses of the drug over a long period. Therefore, a combination drug in an oral form was developed, providing convenient and painless administration of the drug. A combination of several FCoV inhibitors allows for increased efficacy, reduced dosage, shortened therapy duration, and minimized the likelihood of relapse. The use of a combination drug containing a combination of FCoV inhibitors (GC376 and GS-441524) is known in the source CN 110215456 A dated June 25.2019, a drawback of this invention is the injection form of administration, which is very painful and can lead to side effects. The solution presented in this application is intended for oral administration and, in one embodiment, contains three active ingredients.

[0009] The use of an analogue of Vefinavir (VS-121354) for the treatment of FIP is known (RU 2791737 C1 dated 03 / 13 / 23, RU 2780097 C1 dated 09 / 19 / 2022, CN 111135184 A dated 03 / 05 / 2020, CN 113185519 A dated 07 / 30 / 2020), including compositions containing GS-441524 in combination with GC376 (which are analogues of Vefinavir (VS-121354) and Prinvenavir (VS-821303) (CN 110215456 A dated 06 / 25 / 2019, CN 111135166 A from 05.03.2020).

[0010] Essence of the invention

[0011] The present invention discloses a pharmaceutical composition in oral form consisting of a combination of two active substances:

[0012] - Vefinavir (VS-121354) is a nucleoside analogue, an inhibitor of the RNA-dependent RNA polymerase of the virus ((2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5(hydroxymethyl)oxolane-2-carbonitrile),

[0013] - Prinvenavir (VS-821303) is a 3CLpro protease inhibitor ((βS)-α-hydroxy-β-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidine propanesulfonic acid, monosodium salt).

[0014] To enhance the antiviral activity of the proposed combination, an option with the addition of Ritonavir (1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate) is proposed.

[0015] Feline Corona Virus (FCoV) is a single-stranded, positive-sense RNA virus belonging to the family Coronaviridae, genus Alphacoronavirus, species Alphacoronavirus 1. FCoV is divided into types I and II based on in vitro growth, genomic properties, and antigenicity. FCoV has two biotypes: feline enteric coronavirus (FECV), which replicates primarily in intestinal epithelial cells, and feline infectious peritonitis virus (FIPV), which causes the disease feline infectious peritonitis (FIP). Both types I and II can exist as each biotype. FIPV is considered to be a virulent form of FECV [1]. FCoV is widespread among cats: according to various estimates, from 40 to 95% of cats worldwide are carriers of this virus [2, 3].Cats infected with FECV typically show no symptoms during the initial viral infection but may occasionally experience brief bouts of diarrhea and / or mild upper respiratory tract clinical signs, with spontaneous recovery. Cats infected with FECV typically mount an immune response that produces antibodies against the virus within 7-10 days of infection. Approximately 5-10% of cats infected with FECV have one or more mutations in the virus that alter its biological behavior [4]. As a result, white blood cells (macrophages / neutrophils) are infected with the virus, form an immune complex, and spread throughout the cat's body. This mechanism of action technically causes FIPV. However, FIPV is not the only virus that can replicate systemically: FECV has also been shown to replicate systemically in healthy cats and cats without FIP ​​[5, 6].An intense inflammatory response to FIPV develops rapidly around blood vessels in tissues and organs, spreading through infected complexes, often to the peritoneal cavity, kidneys, or brain, causing peritonitis and central nervous system damage. Mortality from FIP can reach 95%, and the average survival time for untreated cats is only eight to nine days. Most cats infected with FCoV shed the virus for two to three months [7], either continuously or intermittently [8], and then cease shedding. Immunity is short-lived, as cats can be reinfected with the same or a different FCoV strain within a few weeks [9]. Once infection is detected, supportive therapy is usually used: force-feeding, intravenous fluids to correct dehydration, anti-inflammatory drugs, etc.Antiviral therapy with drugs that inhibit feline FCoV proliferation in cells is also becoming widespread. However, a drawback of these drugs is the need for painful subcutaneous injections over a long period of time, typically 12 weeks or longer. Subcutaneous injections over such a long period can lead to side effects such as malignant neoplasms, clinical abscesses, and necrosis.

[0016] The technical objective is to create a drug with improved therapeutic activity in cats in the fight against FIP / FIB, including the creation of a drug for oral administration.

[0017] To solve this technical problem, a pharmaceutical composition in oral form was developed containing Vefinavir (VS-121354) and Prinvenavir (VS-821303) as active ingredients, in one version with the addition of Ritonavir to enhance antiviral activity.

[0018] The combination of vefinavir (VS-121354) and prinvenavir (VS-821303) inhibits FCoV proliferation in cells, while ritonavir enhances this effect by inhibiting CYP3A4. This combination provides more potent antiviral activity than either drug alone, allowing low doses to achieve effective inhibitory effects and significant synergistic effects. The oral administration method ensures painless, simple, and convenient administration.

[0019] Vefinavir (VS-121354) is a 1'-cyano-substituted adenine-C-ribose nucleoside analogue that inhibits viral RNA synthesis. It serves as an alternative substrate for viral RNA-dependent RNA polymerases and RNA chain terminator and has antiviral activity against many RNA viruses, including FIPV. The mechanism of action is that Vefinavir (VS-121354) is phosphorylated to nucleoside monophosphate via a cellular kinase and then converted to an active triphosphate metabolite, an analogue of the nucleoside triphosphate (NTP) structure. In the synthesis of viral RNA, the active structural analogue of NTP acts as a competition for natural NTP and competition for natural nucleoside (ATP, TTP, CTP, GTP) for participation in RNA transcription

[10] . When Vefinavir (VS-121354) is inserted into the transcription product, the transcription will be interrupted in advance, thus inhibiting the transcription process of viral RNA.Vefinavir (VS-121354) has a good therapeutic effect on cats infected with FIPV, but is not ideal and requires long-term administration [10, 11, 12, 13, 14, 15, 16]. The chemical name of Vefinavir (VS-121354) is (2R,3R,4S,5R)-2-(4-Aminopyrollo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile. The molecular formula is C. 12 H 13 N5O4.

[0020] Prinvenavir (VS-821303) is a broad-spectrum 3C-like protease (3CLpro) inhibitor that binds to the active site of viral 3C protease, blocking its catalytic activity, and thus inhibits coronavirus replication in vivo

[17] . After viral entry into host cells, FCoV produces viral polyproteins consisting of nonstructural viral proteins, which are subsequently cleaved into mature functional viral proteins by virally encoded 3C-like protease (3CLpro) and papain-like protease. The released viral proteins form a replicase-transcriptase complex, which is required for RNA replication and transcription of subgenomic RNAs, so inhibition of viral proteases blocks viral replication. Prinvenavir (VS-821303) has a good therapeutic effect in cats infected with FIPV [18, 19, 20], but it poorly penetrates the blood-brain barrier, allowing the virus to survive in the nervous system.It has been shown that even a 5-fold increase in the drug dose (from 10 mg / kg to 50 mg / kg) can slow down, but not eliminate, the neurological symptoms of the disease

[21] . At the same time, it has been demonstrated that Vefinavir (VS-121354) overcomes the blood-ophthalmic and blood-brain barriers - a single subcutaneous administration of Vefinavir (VS-121354) at a dose of 10 mg / kg resulted in levels of the original Vefinavir (VS-121354) inside the eye and in the CNS being close to or exceeding the effective level of 1 μM

[10] . The chemical name of Prinvenavir (VS-821303) is (βS)-α-hydroxy-β-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidine propanesulfonic acid, monosodium salt. Molecular formula: C. 21 H 30 N3NaO8S.

[0021] Ritonavir is a potent inhibitor of cytochrome P450 3A4 (CYP3A4). Ritonavir is currently one of the most potent CYP3A4 inhibitors in routine clinical use and is often used to deliberately increase the oral availability of drugs that are otherwise extensively metabolized by CYP3A4. Similarly, ritonavir is used as a pharmacokinetic enhancer to increase the exposure of the COVID-19 protease inhibitor nirmarelvir, a CYP3A4 substrate [22, 23]. The chemical name of Ritonavir is 1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate). Molecular formula: C 37 H 48 N6O5S2.

[0022] Based on the results discussed above, both vefinavir (VS-121354) and prinvenavir (VS-821303) are effective drugs in the treatment of FIPV, despite their distinctly different mechanisms of inhibition of viral replication. However, the use of vefinavir (VS-121354) or prinvenavir (VS-821303) alone requires a long treatment period (12 weeks or more), which is uncomfortable for both animals and their owners. In this regard, a pharmaceutical composition in oral form has been developed containing a combination of feline coronavirus inhibitors Vefinavir (VS-121354) and Prinvenavir (VS-821303), with the addition of Ritonavir to increase bioavailability and without the addition of Ritonavir for use in animals with contraindications and / or intolerance to this drug.The combination of Vefinavir (VS-121354) and Prinvenavir (VS-821303) inhibits FCoV proliferation in cells, with the combined therapeutic effect being significantly higher than that achieved with either drug alone. Ritonavir acts as a pharmacokinetic enhancer, increasing the peak plasma concentration of Prinvenavir (VS-821303) and the concentration of Prinvenavir (VS-821303) measured immediately before each dose, thereby increasing overall drug exposure. These effects can be used as a coronavirus inhibitor in cats for the treatment of FIP. Its advantages include good efficacy, high safety, reduced viral resistance to drugs, and fewer adverse drug effects. The oral administration method ensures painless, simple, and convenient administration.

[0023] The technical result, when using the claimed invention, is the effective treatment of FIP / IP in cats with minimal side effects and a low probability of relapse.

[0024] The technical result is achieved by producing an oral antiviral composition containing the RNA-dependent RNA polymerase inhibitor Vefinavir (VS-121354) and the 3CLpro protease inhibitor Prinvenavir (VS-821303) as active ingredients. This improves the consumer properties of the finished dosage form and increases therapeutic efficacy due to the synergistic action of the two active ingredients. One embodiment proposes enriching the resulting pharmaceutical composition with Ritonavir to enhance its antiviral activity.

[0025] Essence of the invention

[0026] The pharmaceutical composition contains two active ingredients: two direct-acting antiviral agents—the 3CLpro protease inhibitor Prinvenavir (VS-821303) and the RNA-dependent RNA polymerase inhibitor Vefinavir (VS-121354). It also contains excipients: microcrystalline cellulose, polyvinylpyrrolidone, magnesium stearate, and flavoring. In one embodiment, the resulting composition contains a pharmacokinetic enhancer (CYP3A4 inhibitor)—Ritonavir.

[0027] In some embodiments, the pharmaceutical composition is presented in tablet form. In some embodiments, the pharmaceutical composition is presented as a tightly packed powder in gelatin capsules. In some embodiments, the pharmaceutical composition is presented as a jelly tablet / lozenge.

[0028] In some embodiments, the pharmaceutical composition comprises Vefinavir (VS-121354) in an amount of 20 to 50 mg.

[0029] In some embodiments, the pharmaceutical composition comprises Prinvenavir (VS-821303) in an amount of 60 to 80 mg.

[0030] In some embodiments, the pharmaceutical composition comprises Ritonavir in an amount of 20 to 40 mg.

[0031] In some embodiments, the invention relates to a method for the antiviral therapy of wet or effusive feline infectious peritonitis.

[0032] In some embodiments, the invention relates to a method for the antiviral treatment of feline infectious peritonitis of the dry or non-effusive form.

[0033] Literature:

[0034] 1. Pedersen NC A review of feline infectious peritonitis virus infection: 1963-2008. J Feline Med Surg., 2009, vol. 11(4), p. 225-258. doi:10.1016 / j.jfms.2008.09.008.

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[0047] 14. Pedersen N.C., Perron M., Bannasch M., Montgomery E., Murakami E., Liepnieks M., Liu H. Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis. J. Feline Med. Surg. 2019, vol. 21, p. 271-281. doi: 10.1177 / 1098612X19825701.

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[0050] 17. Chang H.W., de Groot R.J., Egberink H.F., Rottier P.J. Feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene. J Gen Virol. 2010, vol. 91, p. 415-420. doi: 10.1099 / vir.0.016485-0.

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[0052] 19. Perera K.D., Rathnayake A.D., Liu H., Pedersen N.C., Groutas W.C., Chang K.O., Kim Y. Characterization of amino acid substitutions in feline coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease inhibitor. Vet Microbiol. 2019, vol. 237, 108398. doi: 10.1016 / j.vetmic.2019.108398.

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[0057] Перечень фигур

[0058] Fig. 1. The Karnofsky scale, modified for cats by Hartmann and Kuffer (1998), using a classification from 0% (dead) to 50% (normal health). It was used to assess the general condition and well-being of cats.

[0059] Fig. 2. Results of the study of the proposed antiviral composition based on Vefinavir (VS-121354) and Prinvenavir (VS-821303) on animals with a confirmed diagnosis of feline infectious peritonitis (FIP), therapy for 84 days.

[0060] Fig. 3. Results of the study of the proposed antiviral composition based on Vefinavir (VS-121354) and Prinvenavir (VS-821303) on animals with a confirmed diagnosis of feline infectious peritonitis (FIP), therapy for 45 days.

[0061] Fig. 4. Results of the study of the proposed antiviral composition based on Vefinavir (VS-121354) and Prinvenavir (VS-821303) with the addition of Ritonavir on animals with a confirmed diagnosis of feline infectious peritonitis (FIP), therapy for 45 days.

[0062] Fig. 5. The result of determining the antibodies of feline coronavirus infection FCoV.

[0063] Information confirming the possibility of implementing the invention:

[0064] Example 1

[0065] Study of antiviral activity with a therapy duration of 84 days.

[0066] Cats were treated orally for 84 days (from day 0 to day 84). The dosage of the compound was selected based on the described studies with vefinavir (VS-121354) and prinvenavir (VS-821303) analogues and was adjusted depending on the emerging side effects. Cats were treated orally by administering tablets containing 45 mg of vefinavir (VS-121354) and 60 mg of prinvenavir (VS-821303) every 24 hours. The dosage was adjusted daily to a specific weight, and the tablets were always administered at the same time, on an empty stomach. Half an hour after tablet administration, the cats were given food.

[0067] Nine cats with FIP were included in the experiment, including 7 with the wet form of FIP and 2 with the non-exudative form of FIP. Cats were aged from 6 to 24 months. The primary diagnostic method for FIP in the exudative form is PCR of effusion fluid and the Rivalta test (Rivalta test); for the non-exudative (neurological, ophthalmological) form, PCR of material obtained during fine-needle aspiration biopsy of a regional lymph node. All 9 cats tested positive for feline coronavirus (FCoV). A single dose of the composition contained: Vefinavir (VS-121354) - 15 mg / kg / 24 h and Prinvenavir (VS-821303) - 20 mg / kg / 24 h, the control group was not included in the study due to the fact that in the absence of treatment, mortality from FIP reaches 90%.

[0068] Animals were recruited for the study sequentially, as a suitable patient was presented to a veterinarian. Only first-time patients were included in the study, those receiving treatment for the given diagnosis for the first time and not yet receiving any antiviral or other therapy other than supportive care. All cats were hospitalized within the first 8 days (days 0 through 7). Before administration of the pharmaceutical composition, on day 0, each cat underwent a medical history and a complete examination. The study parameters were assessed during the first 8 days and on days 14, 30, 45, 60, and 84. The examination included body weight measurement, abdominal ultrasound, complete blood count, urinalysis, blood biochemistry (including symmetric dimethylarginine (SDMA) and serum amyloid A (SAA)), and measurement of blood viral load, effusion viral load (in case of effusion accessible for drainage), and serum FCoV antibodies.

[0069] The Karnofsky scale (Fig. 1), modified for cats by Hartmann and Kuffer (1998), was used to assess the general condition and well-being of cats, using a classification from 0% (dead) to 50% (normal health).

[0070] A general clinical and biochemical blood test was performed using automated analyzers (DF50 Vet, Dymond, China; Miura 300, ISE srl, Italy). A differential blood test was performed on blood smears stained with Haema Quick Staining / Diff-Quick if hematological parameters were abnormal. Cats were discharged on day 7, and owners continued daily administration of the study drug. During the treatment period, cats were not allowed outdoors. Feline coronavirus RNA load was detected by PCR in blood and effusion samples (while present) for several days.

[0071] Already 45 days after the start of therapy, 100% of animals that underwent diagnostic testing using the PCR method to detect the causative agent of FIP / FIP (RNA of feline coronavirus infection (FCoV)) showed a negative result.

[0072] All nine cats were considered clinically healthy after treatment completion on day 84. All animals remained alive 14-30 days after treatment, with no relapses. The study demonstrated the safety and positive effects of a composition containing vefinavir (VS-121354) and prinvenavir (VS-821303) as the main active ingredients.

[0073] Figure 2 presents summary data from a study of the proposed antiviral composition in animals with a confirmed diagnosis of FIP. It was demonstrated that clinical improvement occurs by day 30 of therapy and subsequently leads to recovery. It was established that with the proposed regimen of oral administration of the pharmaceutical composition, complete recovery of the animals occurs, as reflected by the absence of the FIP / FIP pathogen (feline coronavirus (FCoV) RNA) in the animals' bodies and a clinical picture characterizing the animals as healthy. During a follow-up examination 14-30 days after completion of treatment with the proposed composition, no adverse effects were observed in any animal after discontinuation of the drug or signs of disease relapse.

[0074] Example 2

[0075] Study of antiviral activity with a therapy duration of 45 days.

[0076] Cats were treated orally for 84 days (days 0–84). The dosage of the compound was selected based on described studies with vefinavir (VS-121354) and prinvenavir (VS-821303) analogues and was adjusted depending on the emerging side effects. Cats were treated orally by administering tablets containing 45 mg of vefinavir (VS-121354) and 60 mg of prinvenavir (VS-821303) every 24 hours. The dosage was adjusted daily to a specific weight, and the tablets were always administered at the same time, on an empty stomach. Half an hour after tablet administration, the cats were given food.

[0077] Six cats with FIP were included in the experiment, including four with the wet form of FIP and two with the non-exudative form of FIP. Cats were aged from 6 to 24 months. The primary diagnostic method for FIP in the exudative form is PCR of effusion fluid and the Rivalta test (Rivalta test); for the non-exudative (neurological, ophthalmological) form, PCR of material obtained during fine-needle aspiration biopsy of a regional lymph node. All six cats tested positive for feline coronavirus (FCoV). A single dose of the composition contained: Vefinavir (VS-121354) - 15 mg / kg / 24 h and Prinvenavir (VS-821303) - 20 mg / kg / 24 h, the control group was not included in the study due to the fact that in the absence of treatment, mortality from FIP reaches 90%.

[0078] Animals were recruited for the study sequentially, as a suitable patient was presented to a veterinarian. Only first-time patients were included in the study, those receiving treatment for the given diagnosis for the first time and not yet receiving any antiviral or other therapy other than supportive care. All cats were hospitalized within the first 8 days (days 0 to 7). Before administration of the pharmaceutical composition, on day 0, each cat underwent a medical history and a complete examination. The study parameters were assessed during the first 8 days and on days 14, 21, 28, and 45. The examination included body weight measurement, abdominal ultrasound, complete blood count, urinalysis, blood biochemistry (including symmetric dimethylarginine (SDMA) and serum amyloid A (SAA)), and measurement of blood viral load, effusion viral load (in case of effusion accessible for drainage), and serum FCoV antibodies.

[0079] The Karnofsky scale (Fig. 1), modified for cats by Hartmann and Kuffer (1998), was used to assess the general condition and well-being of cats.

[0080] A general clinical and biochemical blood test was performed using automated analyzers (DF50 Vet, Dymond, China; Miura 300, ISE srl, Italy). A differential blood test was performed on blood smears stained with Haema Quick Staining / Diff-Quick if hematological parameters were abnormal. Cats were discharged on day 7, and owners continued daily administration of the study drug. During the treatment period, cats were not allowed outdoors. Feline coronavirus RNA load was determined by PCR in blood and effusion samples (as long as they were present) for several days.

[0081] Forty-five days after the start of therapy, 100% of animals tested negative for the FIP / FIP pathogen (feline coronavirus (FCoV) RNA) using PCR. All animals were considered clinically healthy at the end of treatment, on day 45. All animals remained alive 30-45 days after the end of treatment, with no relapses. The study demonstrated the safety and beneficial effects of a composition containing vefinavir (VS-121354) and prinvenavir (VS-821303) as the main active ingredients.

[0082] Figure 3 presents summary data from a study of the proposed antiviral composition in animals with a confirmed diagnosis of FIP. Improvement in the clinical picture was demonstrated by day 21 of therapy and subsequently led to recovery. Complete recovery was observed in animals treated with the proposed oral pharmaceutical composition, as evidenced by the absence of the FIP / FIP pathogen (feline coronavirus (FCoV) RNA) in the animals' bodies and a clinical picture characterizing the animals as healthy. A follow-up examination 30-45 days after completion of treatment with the proposed composition revealed no adverse effects or signs of disease relapse in any animal.

[0083] Example 3

[0084] Study of the antiviral activity of the obtained composition with the addition of Ritonavir with a duration of therapy of 45 days.

[0085] Cats were treated orally for 45 days (from day 0 to day 45). The dosage of the compound was selected based on the described studies with ritonavir and analogs of vefinavir (VS-121354) and prinvenavir (VS-821303) and was adjusted depending on the emerging side effects. Cats were treated orally by administering tablets containing 45 mg of vefinavir (VS-121354), 60 mg of prinvenavir (VS-821303), and 35 mg of ritonavir, every 24 hours. The dosage was adjusted daily to a specific weight; the drug was always administered at the same time, on an empty stomach. Half an hour after tablet administration, the cats were given food.

[0086] 6 cats with FIP were included in this experiment, 4 of which had the wet form of FIP and 2 had the non-exudative form of FIP. Cats were aged from 6 to 24 months. The main method for diagnosing FIP in the exudative form is PCR of effusion fluid, Rivalta test (Rivalta test); in the non-exudative (neurological, ophthalmological) form: PCR of material obtained by fine-needle aspiration biopsy of the regional lymph node. All 6 cats tested positive for feline coronavirus (FCoV). A single dose of the composition contained: Vefinavir (VS-121354) - 15 mg / kg / 24 h; Prinvenavir (VS-821303) - 20 mg / kg / 24 h; Ritonavir - ≈11.7 mg / kg / 24 h, the control group was not included in the study.

[0087] Animals were recruited for the study sequentially, as a suitable patient was presented to a veterinarian. Only first-time patients were included in the study, those receiving treatment for the given diagnosis for the first time and not yet receiving any antiviral or other therapy other than supportive care. All cats were hospitalized within the first 8 days (days 0 through 7). Before administration of the pharmaceutical composition, on day 0, each cat underwent a medical history and a complete examination. The study parameters were assessed during the first 8 days and on days 14, 21, 28, and 45. The examination included body weight measurement, abdominal ultrasound, complete blood count, urinalysis, blood biochemistry (including symmetric dimethylarginine (SDMA) and serum amyloid A (SAA)), and measurement of blood viral load, effusion viral load (in case of effusion accessible for drainage), and serum FCoV antibodies.

[0088] The Karnofsky performance status scale modified for cats by Hartmann and Kuffer (1998) was used to assess the general condition and well-being of cats, using a classification from 0% (dead) to 50% (normal health). General clinical and biochemical blood tests were performed using automated analyzers (DF50 Vet, Dymond, China; Miura 300, ISE srl, Italy). Differential blood tests were performed on blood smears stained with Haema Quick Staining / Diff-Quick if hematological parameters were abnormal. Cats were discharged on day 7, and the owners continued daily administration of the study drug. Cats were not allowed outside during the treatment period. Feline coronavirus RNA load was detected by PCR for several days in blood and effusion samples (as long as they were present).

[0089] At the end of treatment, 45 days after the start of therapy, all animals (6 cats) tested negative for FIP / IP. The animals were considered clinically healthy.

[0090] Figure 4 presents summary data from a study of the proposed antiviral composition in animals with a confirmed diagnosis of FIP. Improvement in the clinical picture was demonstrated by day 14 of therapy and subsequently led to recovery. Complete recovery was observed in animals treated with the proposed oral pharmaceutical composition, as evidenced by the absence of the FIP / FIP pathogen (feline coronavirus (FCoV) RNA) in the animals' bodies and a clinical picture characterizing the animals as healthy. A follow-up examination 30-45 days after completion of treatment with the proposed composition revealed no adverse effects or signs of disease recurrence in any animal.

[0091] Ninety days after the end of therapy, a diagnostic test was performed by PCR of blood to detect the causative agent of FIP / IP in the blood of animals, and an enzyme-linked immunosorbent assay (ELISA) of the blood serum of animals was performed to determine the titer of antibodies to feline coronavirus infection FCoV. Venous blood was taken as the material for diagnostic PCR and ELISA tests. 100% of the animals that underwent diagnostic testing by PCR had a negative result. ELISA analysis of blood serum to determine the titer of IgG antibodies to feline coronavirus infection FCoV showed the results shown in Fig. 5. Moreover, 83.33% of cats have an antibody titer of 1:3200; 16.67% of cats have an antibody titer of 1:1600. Thus, 83.33% of animals diagnosed using the ELISA method have a titer of antibodies to feline coronavirus infection FCoV within the diagnostic limit.Moreover, 16.67% of animals have a titer of antibodies to FCoV, which indicates, with a high degree of probability, the release of the virus into the external environment.

Claims

1. A veterinary pharmaceutical composition for the treatment of infectious peritonitis in cats, formulated for oral administration and containing as active ingredients: an inhibitor of RNA-dependent RNA polymerase - (2R, 3R, 4S, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile (vefinavir) in an amount of 20 to 50 mg, a protease inhibitor 3CLpro - monosodium salt of (βS)-α-hydroxy-β-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidinepropanesulfonic acid (prinvenavir) in an amount of 60 to 80 mg and 1,3-thiazol-5-ylmethyl-N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate (ritonavir) in quantities from 20 to 40 mg.

2. A veterinary pharmaceutical composition according to claim 1, characterized in that it contains vefinavir in an amount of 45 mg, prinvenavir in an amount of 60 mg, and ritonavir in an amount of 35 mg.

3. A veterinary pharmaceutical composition according to any one of paragraphs 1, 2, characterized in that it is made in the form of a tablet, a tightly packed powder in gelatin capsules or in the form of a jelly tablet or lozenge.

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