Pharmaceutical composition for treating cat infectious peritonitis and preparation method thereof

Abstract

The invention discloses a pharmaceutical composition for cat infectious peritonitis. The pharmaceutical composition comprises or is prepared from the following components: GS-441524 or pharmaceutically acceptable salt thereof, a solubilizer, an acid-base regulator and water. The high solubility of the GS-441524 can be effectively maintained in a relatively high pH value range (2 to 4), so that the stability and the injection safety of the GS-441524 are improved.

Description

Technical Field

[0001] The present invention relates to the field of veterinary medicine, and in particular to a pharmaceutical composition for treating feline infectious peritonitis and a preparation method thereof. Background Art

[0002] Feline infectious peritonitis (FIP) is a chronic infectious disease caused by a pathogenic coronavirus, characterized by peritonitis and ascites. It is widespread worldwide, particularly in the United States and Europe. It occurs in cats of all ages and sexes, with a high seroprevalence in healthy cats. FIP is also associated with a high mortality rate.

[0003] GS-441524 (CAS: 1191237-69-0) is a 1'-cyano-substituted adenine C-nucleoside ribose analogue and a precursor of pharmacologically active nucleoside triphosphate molecules. These analogues act as alternative substrates and RNA chain terminators for viral RNA-dependent RNA polymerases. It has antiviral activity against many RNA viruses, including the zoonotic severe acute respiratory syndrome (SARS) coronavirus. It has not yet been licensed or approved for marketing in any country in the world, and its safety and efficacy have not been proven. However, in vitro and animal models, GS-441524 has demonstrated safety and efficacy in treating naturally occurring FIP cats. Studies have shown that GS-441524 is a safe and effective treatment for FIP, with a recommended therapeutic dose of 4.0 mg / kg subcutaneously every 24 hours for at least 12 weeks. The disclosed structural formula of the GS-441524 compound is as follows:

[0004]

[0005] Studies on the chemical properties of GS-441524 have revealed that its solubility in water is extremely low, with dissolution being pH-dependent within the pH range of 1-10. It is highly soluble in acidic media, but its solubility decreases with increasing pH. Developing an injectable formulation for GS-441524 based on these properties must address the solubility issue. Existing technology has developed an injectable formulation by dissolving GS-441524 in 5% ethanol, 30% propylene glycol, 45% PEG 400, and 20% water (pH 1.5, containing HCl) under acidic conditions. However, this formulation not only has a very low pH but also contains a large amount of alcohol. The solution has a certain viscosity, making it difficult to filter and complex to prepare. Furthermore, when injected into cats, it can cause immediate pain at the injection site, and the cats may even growl and become agitated.

[0006] Therefore, it is very meaningful to provide a more stable pharmaceutical composition containing GS-441524 for felines, especially cats infected with infectious peritonitis. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a pharmaceutical composition containing GS-441524, which has better stability and administration compliance.

[0008] In a first aspect, the present invention provides a pharmaceutical composition for treating feline infectious peritonitis, wherein the pharmaceutical composition comprises or is prepared from the following components: GS-441524 or a pharmaceutically acceptable salt thereof, a solubilizer, an acidifier, an alkalizer, and water.

[0009] In some embodiments, the water is water for injection.

[0010] In some embodiments, the solubilizing agent is a cyclodextrin.

[0011] Preferably, the cyclodextrin is β-cyclodextrin.

[0012] More preferably, the β-cyclodextrin is methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin or sodium sulfobutyl-β-cyclodextrin (SBECD).

[0013] Most preferably, the β-cyclodextrin is sodium sulfobutyl-β-cyclodextrin (SBECD).

[0014] In some embodiments, the acidifying agent comprises hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, maleic acid, tartaric acid, or lactic acid.

[0015] Preferably, the acidifying agent is hydrochloric acid.

[0016] In some embodiments, the alkalizing agent comprises sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, aqueous ammonia, or triethanolamine.

[0017] Preferably, the alkalizing agent is sodium hydroxide.

[0018] In some embodiments, the pH of the pharmaceutical composition is 2-4.

[0019] In some embodiments, the content of GS-441524 or a pharmaceutically acceptable salt thereof is 10-15 mg / mL, preferably 12-13 mg / mL, and more preferably 12.5 mg / mL.

[0020] In some embodiments, the mass ratio of GS-441524 or a pharmaceutically acceptable salt thereof to the solubilizer is 1:10 to 1:20, preferably 1:10-1:16.

[0021] In some embodiments, the pharmaceutical composition comprises or is made of the following components: GS-441524 or a pharmaceutically acceptable salt thereof, sodium sulfobutyl-β-cyclodextrin, hydrochloric acid, sodium hydroxide and water, the content of GS-441524 or a pharmaceutically acceptable salt thereof is 10-15 mg / mL, the mass ratio of GS-441524 or a pharmaceutically acceptable salt thereof to sodium sulfobutyl-β-cyclodextrin is 1:10 to 1:20, and the pH of the pharmaceutical composition is 2-4.

[0022] Preferably, the content of GS-441524 or a pharmaceutically acceptable salt thereof is 12-13 mg / mL, more preferably 12.5 mg / mL.

[0023] Preferably, the mass ratio of GS-441524 or a pharmaceutically acceptable salt thereof to sodium sulfobutyl-β-cyclodextrin is 1:10 to 1:16.

[0024] Preferably, the water is water for injection.

[0025] In a second aspect, the present invention provides a method for preparing the pharmaceutical composition of the first aspect, comprising the following steps:

[0026] Step 1: Add the initial volume of water for injection to the container, add the solubilizer to the water for injection, and stir until dissolved;

[0027] Step 2: Add acidifier to adjust the pH to an appropriate level;

[0028] Step 3: Add GS-441524 and stir until dissolved;

[0029] Step 4: Add an alkalizer to adjust the pH to an appropriate level;

[0030] Step 5: Add appropriate amount of water for injection to reach the final batch volume and mix to obtain a homogeneous solution;

[0031] Step 6: Sterilize and filter the solution through a microporous filter membrane, and dispense the filtered solution into glass bottles, stopper and cap the bottles to obtain the final product.

[0032] In some embodiments, in step 2), the acidifier is used to adjust the pH value to 1.2-1.6, preferably, to 1.3-1.5, and most preferably, to 1.4.

[0033] In some embodiments, in step 4), an alkalizing agent is used to increase the pH value to 2.0-4.0.

[0034] In some embodiments, in step 6), the pore size of the microporous filter membrane is 0.2-0.25 μm, more preferably 0.22 μm.

[0035] In a third aspect, the present invention provides use of the pharmaceutical composition described in the first aspect or the pharmaceutical composition prepared by the preparation method described in the second aspect in preparing a drug for treating and / or preventing feline infectious peritonitis.

[0036] Sodium sulfobutyl-β-cyclodextrin is the sulfonated modified sodium salt of β-cyclodextrin, also known as sodium sulfobutyl ether-β-cyclodextrin. It is an anionic, highly water-soluble cyclodextrin derivative that can well incorporate drug molecules to form non-covalent complexes, thereby improving drug stability, water solubility, and safety, reducing nephrotoxicity, alleviating drug hemolysis, controlling drug release rate, and masking unpleasant odors.

[0037]

[0038] (Sodium sulfobutyl-β-cyclodextrin, CAS: 182410-00-0)

[0039] Table 1 shows some relevant information of SBECD

[0040] name Sodium sulfobutyl-β-cyclodextrin (SBECD) Molecular weight About 2163g / mole (degree of substitution 6.5) Solubility More than 800 mg / mL in water CAS number 182410-00-0

[0041] To assess the solubility range of GS-441524 at feasible pH values, a series of solutions containing a fixed 20% (w / v) SBECD concentration were prepared. The solubility of GS-441524 in SBECD solutions at various pH values (1 to 6) was investigated. Table 2 shows the solubility of GS-441524 in 20% SBECD solutions at various pH values.

[0042] Table 2 Solubility of GS-441524 in 20% SBECD solution at different pH values

[0043] pH GS-441524 solubility (mg / ml) 1 38.9 2 14.1 3 2.1 4 1.1 5 0.5 6 0.3

[0044] The degree of supersaturation of a 20% (w / v) acidic solution of SBECD was also evaluated. When the pH of a 20% (w / v) SBECD aqueous solution (pH = 2.0) containing 12.5 mg / ml of GS-441524 was raised, it was unexpectedly found that GS-441524 did not precipitate with the increase in pH, but rather maintained a relatively good supersaturation state until the pH exceeded 4, at which point precipitation occurred.

[0045] The above study provides an important discovery that SBECD has a significant solubilization effect. For example, a dose of GS-441524 greater than 10 mg can be dissolved in 1 ml of solution. More importantly, this solubilization effect can be achieved under weakly acidic conditions with a pH greater than 2.

[0046] Beneficial effects:

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] The present invention unexpectedly discovered that sodium sulfobutyl-β-cyclodextrin (SBECD) can effectively maintain the high solubility of GS-441524 at a relatively high pH range (2-4), thereby improving the stability and injection safety of GS-441524. The outstanding advantages are mainly manifested in the following aspects:

[0049] 1. The pH value is increased, and cats are more compliant with injections;

[0050] 2. The dissolution effect is better, and no organic solvent is needed during the dissolution process;

[0051] 3. Safer for clinical use and fewer side effects;

[0052] 4. The product is more stable and maintains supersaturation for a long time without precipitation. DETAILED DESCRIPTION

[0053] The following examples are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the scope of protection of the present invention is not limited thereto. This section further describes the present invention in detail in conjunction with specific implementation cases, and its technical features and advantages will be clearly reflected in the description. It should be pointed out that the embodiments are only exemplary descriptions and are not intended to limit the scope of the claims of the present invention. Any detailed adjustments, equivalent replacements, or adaptive improvements based on the core principles of the present invention fall within the substantive protection scope of the present invention.

[0054] Comparative Example 1 (Reference 1: DOI: 10.1177 / 1098612X19825701, Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019)

[0055] Table 3 Composition of Comparative Example 1

[0056] solvent Proportion% Batch size GS-441524 1.25 25g ethanol 5 100mL Propylene glycol 30 600mL PEG400 45 900mL 2mol / LHCl appropriate amount appropriate amount Add water 20 400mL

[0057] Preparation process:

[0058] Prepare the solvent according to the above proportions. Evenly mix ethanol, propylene glycol, and PEG400 in the prescribed ratio. Adjust the pH to 1.5 with 2 mol / L HCl and dilute to volume with water for injection. Dilute the API to a concentration of 12.5 mg / mL with the above solvent. Stir until suspended. Place the solution in an ultrasonic bath for 5-20 minutes until clarified. Filter through a 0.2 μm microporous membrane and aliquot to obtain the finished product.

[0059] During the preparation process of this embodiment, it was found that the solvent had high viscosity and high filtration resistance, which made the operation difficult. In addition, ethanol had a certain volatility, which was not conducive to quantitative determination.

[0060] Example 1

[0061] Table 4 Composition (GS-441524: SBECD = 1:10)

[0062]

[0063]

[0064] Preparation process:

[0065] 1) Add 80% water for injection into the container, add the solubilizer sodium sulfobutyl-β-cyclodextrin into the water for injection, and stir until dissolved;

[0066] 2) Add 2 mol / L hydrochloric acid solution to adjust the pH value to 1.3;

[0067] 3) Add GS-441524 and stir until dissolved;

[0068] 4) Add 2% sodium hydroxide solution to adjust the pH value to 2.0;

[0069] 5) Add water for injection to make up the volume and mix to obtain a uniform solution;

[0070] 6) Sterilize and filter the solution through a 0.22 μm microporous filter membrane, and dispense the filtered solution into glass bottles, stopper and cap the bottles to obtain the final product.

[0071] Example 2

[0072] Table 5 Composition (GS-441524: SBECD = 1:16)

[0073] solvent Proportion% Batch size GS-441524 1.25 25g SBECD 20 400g 2mol / LHCl appropriate amount appropriate amount 2% NaOH appropriate amount appropriate amount Add water to 100 2L

[0074] Preparation process:

[0075] 1) Add 80% water for injection into the container, add the solubilizer sodium sulfobutyl-β-cyclodextrin into the water for injection, and stir until dissolved;

[0076] 2) Add 2 mol / L hydrochloric acid solution to adjust the pH value to 1.4;

[0077] 3) Add GS-441524 and stir until dissolved;

[0078] 4) Add 2% sodium hydroxide solution to adjust the pH value to 3.0;

[0079] 5) Add water for injection to make up the volume and mix to obtain a uniform solution;

[0080] 6) Sterilize and filter the solution through a 0.22 μm microporous filter membrane, and dispense the filtered solution into glass bottles, stopper and cap the bottles to obtain the final product.

[0081] Example 3

[0082] Table 6 Composition (GS-441524: SBECD = 1:20)

[0083] solvent Proportion% Batch size GS-441524 1.25 25g SBECD 25 500g 2mol / LHCl appropriate amount appropriate amount 2% NaOH appropriate amount appropriate amount Add water to 100 2L

[0084] Preparation process:

[0085] 1) Add 70% water for injection into the container, add the solubilizer sodium sulfobutyl-β-cyclodextrin into the water for injection, and stir until dissolved;

[0086] 2) Add 2 mol / L hydrochloric acid solution to adjust the pH value to 1.5;

[0087] 3) Add GS-441524 and stir until dissolved;

[0088] 4) Add 2% sodium hydroxide solution to adjust the pH value to 3.5;

[0089] 5) Add water for injection to make up the volume and mix to obtain a uniform solution;

[0090] 6) Sterilize and filter the solution through a 0.22 μm microporous filter membrane, and dispense the filtered solution into glass bottles, stopper and cap the bottles to obtain the final product.

[0091] Example 4 Solubilization Effect Comparison

[0092] To evaluate the solubilization effect of the GS-441524 pharmaceutical composition for injection on GS-441524, 2% sodium hydroxide solution was added to the preparations of Comparative Example 1 and Examples 1-3 to observe the pH value when the drug was precipitated.

[0093] Table 7 Initial / precipitation pH values of different compositions

[0094] Composition Initial pH value of the preparation pH value of the preparation when it precipitates Comparative Example 1 1.9 2.0 Example 1 2.0 3.0 Example 2 3.0 3.5 Example 3 3.5 4.0

[0095] According to the results in Table 7, Comparative Example 1 can only maintain the drug supersaturation state under a lower pH value, while the present invention can maintain the drug supersaturation state under a higher pH value.

[0096] Example 5 Comparison of stability in low temperature / freeze-thaw test

[0097] To evaluate the stability of the GS-441524 pharmaceutical composition for injection, the preparations of Comparative Example 1 and Examples 1-3 were subjected to a low-temperature / freeze-thaw test. In the low-temperature test, the preparations were first placed in a refrigerator at 2-8°C for 2 days, then placed at 40°C for 2 days, and this cycle was repeated three times before sampling and detecting the drug content. In the freeze-thaw test, the preparations were first placed in a refrigerator at -20°C for 2 days, then placed at 40°C for 2 days, and this cycle was repeated three times before sampling and detecting the drug content.

[0098] Table 8 Comparison of active ingredient content before and after low temperature / freeze-thaw test

[0099] Composition Initial content of preparation Content of preparation after low temperature Content of preparation after freeze-thaw Comparative Example 1 96.9 85.7 80.5 Example 1 98.6 99.1 98.4 Example 2 100.2 99.9 99.6 Example 3 99.6 99.5 99.3

[0100] According to the results in Table 8, the embodiments of the present invention have good stability and can maintain a high effective dose even under adverse conditions.

[0101] Example 6 Clinical Study Comparison

[0102] In order to evaluate the clinical effect of the GS-441524 pharmaceutical composition for injection in treating feline infectious peritonitis, a clinical comparative study was conducted on the preparations of Comparative Example 1 and Example 2.

[0103] Methods: Twenty cats (all 6-9 months old) were prospectively recruited and started treatment after symptoms of peritoneal infection (fever, loss of appetite, jaundice, and peritoneal effusion) became apparent. They were randomly divided into two groups of 10 cats each. Group 1 received 4 mg / kg of the formulation of Comparative Example 1 subcutaneously every 24 hours, while Group 2 received the formulation of Example 2 at the same dose subcutaneously every 24 hours for at least 10 weeks. Laboratory test results, veterinary evaluations, and owner assessments were recorded.

[0104] Results: All 20 cats survived to 6 months. Signs of successful treatment included resolution of fever, effusion, and clinical signs of peritonitis during the first half of treatment, and normalization of globulin concentrations and sustained weight gain during the second half of treatment. Group 1 recovered after 10 weeks of treatment, but three cats required secondary treatment due to recurrence of peritonitis symptoms. The injection process occasionally caused discomfort and skin irritation at the injection site, and owners reported crying out when their skin was pulled during follow-up. Group 2 recovered after 8 weeks of treatment. The cats were calm and cooperative during the injection process, and there were no relapses within 6 months, demonstrating a positive recovery in health.

[0105] Conclusion: The preparations of Example 1 and Example 2 are both effective in treating feline infectious peritonitis, but the preparation of Example 2 is better tolerated, has a low recurrence rate, is better recovered, and has almost no sequelae.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating feline infectious peritonitis, characterized in that: The pharmaceutical composition comprises or is made of the following components: GS-441524 or a pharmaceutically acceptable salt thereof, a solubilizer, an acidifier, an alkalizer and water. Preferably, the water is water for injection.

2. The pharmaceutical composition according to claim 1, characterized in that The solubilizing agent is cyclodextrin, preferably β-cyclodextrin.

3. The pharmaceutical composition according to claim 2, characterized in that The β-cyclodextrin is methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin or sodium sulfobutyl-β-cyclodextrin (SBECD), preferably sodium sulfobutyl-β-cyclodextrin (SBECD).

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The acidulant includes hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, maleic acid, tartaric acid, lactic acid, preferably hydrochloric acid.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that The alkalizing agent includes sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, ammonia water, triethanolamine, preferably sodium hydroxide.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that The content of GS-441524 or a pharmaceutically acceptable salt thereof is 10-15 mg / mL, preferably 12-13 mg / mL, more preferably 12.5 mg / mL; and / or, The mass ratio of GS-441524 or a pharmaceutically acceptable salt thereof to the solubilizer is 1:10 to 1:20, preferably 1:10-1:

16.

7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that The pharmaceutical composition comprises or is made of the following components: GS-441524 or a pharmaceutically acceptable salt thereof, sodium sulfobutyl-β-cyclodextrin, hydrochloric acid, sodium hydroxide and water, the content of the GS-441524 or a pharmaceutically acceptable salt thereof is 10-15 mg / mL, the mass ratio of the GS-441524 or a pharmaceutically acceptable salt thereof to sodium sulfobutyl-β-cyclodextrin is 1:10 to 1:20, and the pH of the pharmaceutical composition is 2-4.

8. The pharmaceutical composition according to claim 7, characterized in that The content of GS-441524 or a pharmaceutically acceptable salt thereof is 12-13 mg / mL, more preferably 12.5 mg / mL; and / or, The mass ratio of GS-441524 or a pharmaceutically acceptable salt thereof to sodium sulfobutyl-β-cyclodextrin is 1:10 to 1:16; and / or The water is water for injection.

9. A method for preparing the pharmaceutical composition according to any one of claims 1 to 8, comprising the following steps: Step 1: Add the initial volume of water for injection to the container, add the solubilizer to the water for injection, and stir until dissolved; Step 2: Add acidifier to adjust the pH to an appropriate level; Step 3: Add GS-441524 and stir until dissolved; Step 4: Add an alkalizer to adjust the pH to an appropriate level; Step 5: Add appropriate amount of water for injection to reach the final batch volume and mix to obtain a homogeneous solution; Step 6: sterilize and filter the solution through a microporous filter membrane, and dispense the filtered solution into glass bottles, stoppering and capping them to obtain the final product; Preferably, in step 2), the acidifier is used to adjust the pH value to 1.2 to 1.6, more preferably, to 1.3 to 1.5, and most preferably, to 1.4; Preferably, in step 4), the alkalizing agent is used to raise the pH value to 2.0-4.0; Preferably, in step 6), the pore size of the microporous filter membrane is 0.2-0.25 μm, more preferably 0.22 μm.

10. Use of the pharmaceutical composition according to any one of claims 1 to 8 or the pharmaceutical composition obtained by the preparation method according to claim 9 in preparing a medicament for treating and / or preventing feline infectious peritonitis.

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