A compound ear drop preparation for pet chronic otitis and a preparation method thereof

Abstract

The application discloses a compound ear drop preparation for pet stubborn otitis and a preparation method thereof, and belongs to the technical field of veterinary medicinal preparations. According to the total mass of effective components, the preparation comprises enrofloxacin 30 mass parts, dexamethasone 30 mass parts, disodium ethylenediaminetetraacetate 40 mass parts and a pharmaceutically acceptable ear drop carrier, and has a pH value of 4.5-6.5. In the preparation, the enrofloxacin and the dexamethasone are dissolved in the carrier containing a solubilizing agent to form a first mixed solution, the disodium ethylenediaminetetraacetate is dissolved in a water carrier to form a second mixed solution, and the carrier is supplemented after mixing and then filled. A suspension type ear drop is prepared and used for local administration of pet ear canals.

Description

Technical Field

[0001] This invention relates to the field of veterinary drug formulation technology, specifically to a compound ear drop preparation for pets with intractable otitis media and its preparation method. Background Technology

[0002] Otitis externa is a common ear disease in companion animals such as dogs and cats. For otitis externa with a long course, recurrent episodes, or significant inflammatory reactions, topical medications need to address the needs of anti-infection, reducing inflammation, and improving the local administration environment.

[0003] Enrofloxacin is a fluoroquinolone antibiotic that exerts a broad-spectrum antibacterial effect by inhibiting bacterial DNA gyrase activity. Dexamethasone is a glucocorticoid that inhibits the synthesis and release of inflammatory mediators and reduces edema and exudation in the ear canal epithelium. Disodium EDTA-2Na has the ability to chelate divalent metal ions. Bacterial biofilms are a major cause of recurrent and drug-resistant refractory otitis media. Ca²⁺ and Mg²⁺ in the extracellular polymers of biofilms maintain the structural integrity of the biofilm by cross-linking the polysaccharide matrix. EDTA-2Na, by chelating the aforementioned divalent metal ions, can disrupt the cross-linked structure of the biofilm matrix, increase biofilm permeability, and thus enhance the penetration of antibacterial drugs into the bacterial cells. Existing ear drop formulations, especially those with single components or those not formulated around a 30:30:40 mass ratio of enrofloxacin, dexamethasone, and disodium EDTA, struggle to simultaneously address the needs for free bacterial inhibition, biofilm clearance, and improvement of inflammatory responses during local administration in refractory otitis media.

[0004] Therefore, it is necessary to provide a compound ear drop formulation with clearly defined effective components, stable component ratios, and suitable for local administration into the ear canal of pets. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, this invention provides a compound ear drop preparation for pet refractory otitis media and its preparation method, which effectively solves the problem that current pet otitis media topical medications on the market are unable to simultaneously inhibit bacterial infection, reduce inflammation, and remove mature biofilm.

[0006] The technical solution adopted in this invention is as follows.

[0007] In this invention, the effective components specifically refer to three components with direct pharmacological activity: enrofloxacin, dexamethasone, and disodium EDTA. Solubilizers, suspending agents, pH adjusters, preservatives, and ear drop carriers are excluded.

[0008] A compound ear drop preparation for refractory otitis media in pets, comprising, by total mass of the active ingredients, 30 parts by mass of enrofloxacin, 30 parts by mass of dexamethasone, and 40 parts by mass of disodium EDTA, and a pharmaceutically acceptable ear drop carrier.

[0009] Furthermore, the carrier for the ear drops includes water for injection, purified water, propylene glycol, polyethylene glycol 400, glycerin, ethanol, or combinations thereof. The type and amount of carrier are determined according to the preparation requirements of the suspension-type ear drops.

[0010] Furthermore, the compound ear drops preparation also includes a solubilizer, a suspending agent, a pH adjuster, a preservative, or a combination thereof; the solubilizer may be polysorbate 80, poloxamer, or a combination thereof; the suspending agent may be hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, or a combination thereof; and the pH adjuster may be citric acid, sodium citrate, acetic acid, sodium acetate, hydrochloric acid, sodium hydroxide, or a combination thereof.

[0011] Furthermore, the compound ear drops are suspension ear drops.

[0012] Furthermore, the pH of the compound ear drops preparation is 4.5-6.5, preferably 5.0-6.0, to adapt to the physiological environment of the pet's ear canal and to take into account the chemical stability of the active ingredients.

[0013] Furthermore, in the ear drop carrier, the amount of propylene glycol is 5% to 20% of the total mass of the preparation, and the amount of polyethylene glycol 400 is 2% to 10% of the total mass of the preparation.

[0014] Furthermore, based on the total mass of the preparation, the content of enrofloxacin is 0.3%, the content of dexamethasone is 0.3%, and the content of disodium EDTA is 0.4%.

[0015] A method for preparing the above-mentioned compound ear drops for refractory otitis media in pets includes the following steps:

[0016] S1. Weigh out the effective components according to the mass ratio of enrofloxacin, dexamethasone, and disodium EDTA of 30:30:40.

[0017] S2. Add enrofloxacin and dexamethasone to the first mixture consisting of a partial droplet carrier, add solubilizer and stir until homogeneous.

[0018] S3. Add disodium ethylenediaminetetraacetate to the second mixture consisting of a portion of the ear drop carrier, and stir until homogeneous.

[0019] S4. Mix the first and second mixtures, add the suspending agent, add the remaining ear drop carrier, adjust the pH of the preparation to 4.5-6.5 using a pH adjuster, and stir until homogeneous.

[0020] S5. Fill the obtained preparation into a single-dose ear drop tube, ear drop bottle or light-proof bottle with a dropper to obtain a compound ear drop preparation for pet refractory otitis.

[0021] The beneficial effects achieved by adopting the above technical solution are as follows.

[0022] I. Using an effective combination of enrofloxacin, dexamethasone, and disodium EDTA in a mass ratio of 30:30:40, the MIC of common pathogens causing otitis externa in pets was reduced by approximately 4 times compared to single-agent enrofloxacin preparations in in vitro free bacterial inhibition tests. Meanwhile, dexamethasone was used to reduce the inflammatory response. Together, the three components constitute the basis of a compound preparation for the local treatment of refractory otitis externa in pets.

[0023] II. In the mature bacterial biofilm clearance test, the formulation containing EDTA-2Na achieved a biofilm clearance rate exceeding 79%, while the control formulation without EDTA-2Na achieved a clearance rate of less than 12%. This result indicates that the biofilm clearance effect of EDTA-2Na on mature bacteria is complementary to the in vitro free-living bacterial inhibitory activity of the compound formulation.

[0024] Third, by preparing enrofloxacin and dexamethasone into a first mixture and a second mixture with disodium EDTA respectively, and then mixing them together, it is beneficial to achieve uniform dispersion during the preparation process. The contents of the three effective components are all within the range of 97.0% to 100.0% of the labeled amount. Attached Figure Description

[0025] Figure 1 This is a flowchart of the preparation process for compound ear drops.

[0026] Figure 2 This is a graph comparing the minimum inhibitory concentrations of different formulations against the tested strains.

[0027] Figure 3 This is a comparison chart of the clearance rates of different formulations on mature bacterial biofilms.

[0028] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] To prepare 1000 g of compound ear drops, weigh out 3 g of enrofloxacin, 3 g of dexamethasone, 4 g of disodium EDTA, 80 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water. The mass ratio of enrofloxacin, dexamethasone, and disodium EDTA is 30:30:40.

[0032] Propylene glycol, polyethylene glycol 400, and polysorbate 80 were added to a mixing tank and stirred at 500 rpm for 10 min at 40°C. Enrofloxacin and dexamethasone were added, and stirring was continued for 20 min to form the first mixture. Disodium ethylenediaminetetraacetate and benzalkonium chloride were added to 300 g of purified water and stirred at 300 rpm for 15 min at 25°C to form the second mixture. Hydroxypropyl methylcellulose was dispersed in 100 g of purified water, allowed to stand for 30 min, and then stirred for 10 min to form a suspension.

[0033] The second mixture was slowly added to the first mixture at 500 r / min, and stirred for 15 min. Then, the suspension was added, and stirring was continued for 20 min. Sodium citrate was added, and purified water was added to bring the total volume to 1000 g. The mixture was stirred for 10 min. After degassing, the resulting preparation was filled into light-proof bottles with droppers to prepare a suspension-type compound ear drop preparation that is shaken well before use.

[0034] Example 2

[0035] Based on the preparation of 1000 g of compound ear drops, weigh out 3 g of enrofloxacin, 3 g of dexamethasone, 4 g of disodium EDTA, 120 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water. The mass ratio of enrofloxacin, dexamethasone, and disodium EDTA is 30:30:40.

[0036] Propylene glycol, polyethylene glycol 400, and polysorbate 80 were added to a mixing tank and stirred at 500 rpm for 10 min at 40°C. Enrofloxacin and dexamethasone were added, and stirring was continued for 20 min to form the first mixture. Disodium ethylenediaminetetraacetate and benzalkonium chloride were added to 300 g of purified water and stirred at 300 rpm for 15 min at 25°C to form the second mixture. Hydroxypropyl methylcellulose was dispersed in 100 g of purified water, allowed to stand for 30 min, and then stirred for 10 min to form a suspension.

[0037] The second mixture was slowly added to the first mixture at 500 r / min, and stirred for 15 min. Then, the suspension was added, and stirring was continued for 20 min. Sodium citrate was added, and purified water was added to bring the total volume to 1000 g. The mixture was stirred for 10 min. After degassing, the resulting preparation was filled into light-proof bottles with droppers to prepare a suspension-type compound ear drop preparation that is shaken well before use.

[0038] Example 3

[0039] To prepare 1000 g of compound ear drops, weigh out 3 g of enrofloxacin, 3 g of dexamethasone, 4 g of disodium EDTA, 160 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water. The mass ratio of enrofloxacin, dexamethasone, and disodium EDTA is 30:30:40.

[0040] Propylene glycol, polyethylene glycol 400, and polysorbate 80 were added to a mixing tank and stirred at 500 rpm for 10 min at 40°C. Enrofloxacin and dexamethasone were added, and stirring was continued for 20 min to form the first mixture. Disodium ethylenediaminetetraacetate and benzalkonium chloride were added to 300 g of purified water and stirred at 300 rpm for 15 min at 25°C to form the second mixture. Hydroxypropyl methylcellulose was dispersed in 100 g of purified water, allowed to stand for 30 min, and then stirred for 10 min to form a suspension.

[0041] The second mixture was slowly added to the first mixture at 500 r / min, and stirred for 15 min. Then, the suspension was added, and stirring was continued for 20 min. Sodium citrate was added, and purified water was added to bring the total volume to 1000 g. The mixture was stirred for 10 min. After degassing, the resulting preparation was filled into light-proof bottles with droppers to prepare a suspension-type compound ear drop preparation that is shaken well before use.

[0042] The effective component ratio of this invention is based on the total mass of enrofloxacin, dexamethasone, and disodium EDTA. The specific types and amounts of the ear drop carrier and optional excipients can be adjusted according to the dosage form, administration device, and target pet species without changing the 30:30:40 mass ratio of the three effective components.

[0043] Comparative Example 1

[0044] To prepare 1000 g of ear drops, weigh out 3 g of enrofloxacin, 100 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water.

[0045] The preparation method is the same as in Example 1, except that it does not contain dexamethasone and disodium EDTA. The resulting formulation is a suspension-type ear drop formulation containing only enrofloxacin.

[0046] Comparative Example 2

[0047] To prepare 1000 g of ear drops, weigh out 3 g of enrofloxacin, 3 g of dexamethasone, 100 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water.

[0048] The preparation method is the same as in Example 1, except that it does not contain disodium EDTA. The resulting formulation is a suspension-type ear drop containing enrofloxacin and dexamethasone.

[0049] Comparative Example 3

[0050] Based on the preparation of 1000 g of compound ear drops, weigh out 3 g of enrofloxacin, 2 g of dexamethasone, 5 g of disodium EDTA, 100 g of propylene glycol, 50 g of polyethylene glycol 400, 10 g of polysorbate 80, 3 g of hydroxypropyl methylcellulose, 0.1 g of benzalkonium chloride, 0.5 g of sodium citrate, and an appropriate amount of purified water. The mass ratio of enrofloxacin, dexamethasone, and disodium EDTA is 30:20:50.

[0051] The preparation method is the same as in Example 1, except that the mass ratio of the three effective components is different.

[0052] Experimental Example 1: In vitro antibacterial test

[0053] The minimum inhibitory concentration (MIC, expressed as enrofloxacin equivalent concentration, μg / mL) of the formulations in Example 1 and Comparative Examples 1-3 against common pathogens causing otitis externa in pets was determined using the broth microdilution method. The tested strains included *Staphylococcus pseudintermedius* ATCC 49444, *Pseudomonas aeruginosa* ATCC 27853, and *Malassezia pachydermatis* ATCC 14522. The culture medium, inoculum concentration, culture temperature, culture time, and MIC endpoint for each tested strain were performed according to the inventor's corresponding original test records.

[0054] Table 1. Minimum inhibitory concentrations (MIC, μg / mL) of different formulations against the tested strains

[0055] Pseudo-intermediate Staphylococcus 0.06 0.25 0.20 0.15 Pseudomonas aeruginosa 0.12 0.50 0.40 0.30 Malassezia scleroderma 0.25 1.00 0.80 0.60

[0056] As shown in Table 1, the MIC values ​​of Example 1 against the three tested strains were all lower than those of Comparative Example 1 (enrofloxacin alone) and Comparative Example 2 (without EDTA-2Na), indicating that the 30:30:40 compound formulation had a lower MIC under the formulation conditions used in this experiment. The MIC of Comparative Example 3 was between that of Example 1 and Comparative Examples 1-2, showing that the in vitro free bacterial inhibition performance of formulations with different effective component ratios was different.

[0057] Experimental Example 2: Biofilm Clearance Test

[0058] The clearance rates of each formulation against mature biofilms of *Staphylococcus pseudointermedia* and *Pseudomonas aeruginosa* were determined using crystal violet staining. After culturing in 96-well plates to form mature biofilms for 48 h, physiological saline dilutions (0.5 μg / mL of enrofloxacin) of the formulations from Examples 1 and Comparative Examples 1-3 were added, and the plates were treated at 37°C for 24 h. OD values ​​were then measured after crystal violet staining. 570 Calculate the biofilm clearance rate.

[0059] Table 2. Clearance rate (%) of different formulations of mature bacterial biofilm

[0060] preparation Biofilm removal rate of *Staphylococcus intermedius* (%) Pseudomonas aeruginosa biofilm clearance rate (%) Example 1 83.5 79.2 Comparative Example 1 9.3 7.1 Comparative Example 2 11.6 9.5 Comparative Example 3 88.2 84.7

[0061] As shown in Table 2, the clearance rates of mature biofilms of both bacteria in Examples 1 and Comparative Example 3, which contained EDTA-2Na, exceeded 79%, while those in Comparative Examples 1 and 2, which did not contain EDTA-2Na, were less than 12%. This indicates a significant difference in the clearance rates of mature biofilms among different formulations under the formulation conditions used in this experiment. The biofilm clearance rate of Comparative Example 3 was slightly higher than that of Example 1, but its MIC was also higher than that of Example 1. Taking *Staphylococcus pseudointermedia* as an example, the MIC of Comparative Example 3 was 0.15 μg / mL, while that of Example 1 was 0.06 μg / mL. These results indicate that under the experimental conditions, the 30:30:40 mass ratio compound formulation exhibited a more balanced performance between the two in vitro evaluation indicators of free bacterial inhibition activity and mature biofilm clearance.

[0062] Experimental Example 3: Physicochemical Properties of the Formulation

[0063] The key physicochemical indicators of the preparations obtained in Examples 1-3 were tested, and the results are as follows.

[0064] Table 3 Key Physicochemical Indicators of Examples 1 to 3

[0065] Testing items Example 1 Example 2 Example 3 pH 5.3 5.2 5.4 Viscosity (mPa·s, 25℃) 95 82 68 Enrofloxacin content (%) 98.5 97.8 98.2 Dexamethasone content (%) 97.6 98.3 97.2 EDTA-2Na content (%) 98.8 99.1 98.5

[0066] Examples 1-3 are all suspension-type ear drops. Due to the presence of hydroxypropyl methylcellulose as a suspending agent, the viscosity ranges from 68 to 95 mPa·s. The formulations can be uniformly dispersed after shaking. The viscosity decreases slightly with higher propylene glycol content. The pH of all three examples is in the range of 5.0 to 6.0, and the contents of the three active ingredients are all in the range of 97.0% to 100.0% of the labeled amount, indicating that the content of the active ingredients in the formulations is stable.

Claims

1. A compound ear drop preparation for persistent otitis media in pets, characterized in that: Based on the total mass of the active ingredients, it includes 30 parts by mass of enrofloxacin, 30 parts by mass of dexamethasone, and 40 parts by mass of disodium EDTA, as well as a pharmaceutically acceptable carrier for ear drops.

2. The compound ear drops for persistent otitis media in pets according to claim 1, characterized in that: The ear drop carrier includes water for injection, purified water, propylene glycol, polyethylene glycol 400, glycerin, ethanol, or a combination thereof, wherein the amount of propylene glycol is 5% to 20% of the total mass of the preparation, and the amount of polyethylene glycol 400 is 2% to 10% of the total mass of the preparation.

3. The compound ear drops for persistent otitis media in pets according to claim 1, characterized in that: The compound ear drops preparation further includes a solubilizer, a suspending agent, a pH adjuster, a preservative, or a combination thereof. The pH adjuster is citric acid, sodium citrate, acetic acid, sodium acetate, hydrochloric acid, sodium hydroxide, or a combination thereof, and the pH of the compound ear drops preparation is 4.5 to 6.

5.

4. The compound ear drops for persistent otitis media in pets according to claim 3, characterized in that: The solubilizer is polysorbate 80, poloxamer, or a combination thereof, and the suspending agent is hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, or a combination thereof.

5. A compound ear drop preparation for persistent otitis media in pets according to claim 1, characterized in that: Based on the total mass of the preparation, the content of enrofloxacin is 0.3%, the content of dexamethasone is 0.3%, and the content of disodium EDTA is 0.4%.

6. A compound ear drop preparation for persistent otitis media in pets according to claim 5, characterized in that: The compound ear drops are suspension ear drops, and the preservatives are benzalkonium chloride, benzyl alcohol, methylparaben, or a combination thereof.

7. The method for preparing a compound ear drop preparation for intractable otitis media in pets according to claim 1, characterized in that, Includes the following steps: S1. Weigh out enrofloxacin, dexamethasone and disodium EDTA according to the formula, so that the mass ratio of the three is 30:30:40; S2. Add enrofloxacin and dexamethasone to the first mixture consisting of a partial droplet carrier and stir until homogeneous; S3. Add disodium EDTA to the second mixture consisting of a portion of the ear drop carrier, and stir until homogeneous; S4. Mix the first and second mixtures, add the remaining ear drop carrier, and adjust the pH of the preparation to 4.5-6.5 using a pH adjuster, stirring until the preparation is homogeneous; S5. Fill the preparation obtained in S4 to obtain the compound ear drops preparation.

8. The method for preparing a compound ear drop preparation for intractable otitis media in pets according to claim 7, characterized in that: A solubilizer is added to S2, and a suspending agent is added to S4.

9. The method for preparing a compound ear drop preparation for intractable otitis media in pets according to claim 7, characterized in that: The filling containers in S5 are single-dose dropper tubes, dropper bottles, or light-proof bottles with droppers.

10. The use of the compound ear drops for pet refractory otitis according to any one of claims 1 to 6 in the preparation of a medicament for topical administration to the ear canal of pets for bacterial infection-related otitis.

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