A lincomycin hydrochloride and spectinomycin hydrochloride microemulsion and a preparation method thereof

Abstract

The present application relates to a kind of lincomycin hydrochloride and spectinomycin hydrochloride microemulsion and its preparation method, the microemulsion includes the following components: lincomycin hydrochloride, spectinomycin hydrochloride, emulsifier, coemulsifier, antioxidant, complexing agent, isotonic agent, injection oil, injection water and osmotic pressure regulator, the microemulsion prepared by the present application has the advantages such as small average particle size, uniform distribution, high encapsulation efficiency and high stability.

Description

Technical Field

[0001] This invention relates to the field of veterinary antibiotics, and in particular to a microemulsion of lincomycin hydrochloride and spectinomycin hydrochloride and its preparation method. Background Technology

[0002] Clindamycin hydrochloride, also known as clindamycin, is a lincomycin antibiotic and a derivative of lincomycin. Clindamycin hydrochloride has strong antibacterial activity and a broad antibacterial spectrum. It is active against Gram-positive aerobic bacteria, streptococci, and staphylococci, and also against various anaerobic bacteria, chlamydia, mycoplasma, and hookworms.

[0003] Spectinomycin is an aminoglycoside antibiotic produced by Streptomyces and is used to treat infections caused by Gram-negative bacteria and mycoplasma. Studies have shown that spectinomycin is effective in treating and preventing livestock and poultry diseases caused by Streptococcus, Erysipelothrix rhusiopathiae, Staphylococcus aureus, Pasteurella, Escherichia coli, Salmonella, and mycoplasma, and is particularly effective against mixed infections of mycoplasma and Escherichia coli.

[0004] Lincomycin and spectinomycin, also known as lincomycin combination, have a synergistic effect when combined, exhibiting a broad antibacterial spectrum, rapid action, low toxicity, and no adverse effects on chickens and pigs at therapeutic doses. It is highly effective against piglet diarrhea, mycoplasmal pneumonia in pigs, and chronic respiratory disease in chickens caused by Mycoplasma gallisepticum.

[0005] Existing formulations of lincomycin and spectinomycin combination drugs include premixes, tablets, suspensions, soluble powders, and aqueous injections. Both lincomycin and spectinomycin have low oral bioavailability, but are well absorbed via injection, resulting in high bioavailability, wide tissue distribution, and rapid onset of action. However, spectinomycin has a short stability period in aqueous solution; according to J. Szunyog et al., spectinomycin aqueous solution is unstable. Under acidic conditions, spectinomycin hydrolyzes to actinamine, losing its biological activity. Under alkaline conditions, the α-keto-hemiacetal junction of actinamine and actinospectose is easily broken and rearranged, forming the degradation product actinospectinoic acid, which rapidly decreases potency and pH value. The stability of lincomycin hydrochloride aqueous solution is slightly worse; the glucosinolate bonds in its structure are easily hydrolyzed under acidic or metallic conditions, affecting the stability of the solution. Therefore, the stability of lincomycin and spectinomycin combination injections is a challenge for many manufacturers.

[0006] CN101829082B discloses a method for preparing veterinary spectinomycin hydrochloride and lincomycin hydrochloride injections. This method requires first encapsulating spectinomycin hydrochloride before preparing it into an injection. However, the preparation process requires heating to 50°C to dissolve cyclodextrin and then rapidly cooling to 4°C, which places high demands on the equipment. CN102048748B discloses a lincomycin and spectinomycin compound suspension injection and its preparation method, but it uses organic solvents to prepare the injection, and as an oil suspension, it is highly irritating to animals.

[0007] Therefore, the development of a stable, low-irritant, highly encapsulated, and conveniently administered lincomycin hydrochloride and spectinomycin hydrochloride compound injection is of great significance for the application and promotion of lincomycin and spectinomycin compound, especially in the prevention and treatment of piglet diarrhea and mycoplasmal pneumonia in pigs. Summary of the Invention

[0008] The purpose of this invention is to provide a lincomycin hydrochloride and spectinomycin hydrochloride microemulsion to solve the technical problems of poor stability, high irritation and low encapsulation efficiency in the prior art.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned microemulsion;

[0010] Another objective of this invention is to provide the application of the above-mentioned microemulsion in the treatment of diseases such as diarrhea in piglets and mycoplasmal pneumonia in pigs.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0012] A microemulsion of lincomycin hydrochloride and spectinomycin hydrochloride is provided, comprising lincomycin hydrochloride, spectinomycin hydrochloride, emulsifier, co-emulsifier, antioxidant, complexing agent, isotonic agent, oil for injection, and water for injection, characterized in that the components are in the following weight ratios: 0.5-7.5% lincomycin hydrochloride, 1-15% spectinomycin hydrochloride, 1-3% emulsifier, 0.01-2% co-emulsifier, 0.01%-1% complexing agent, 0.01-2% antioxidant, 2-5% isotonic agent, 10-30% oil for injection, and the remainder being water for injection;

[0013] In one embodiment, the amount of lincomycin hydrochloride is preferably 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or 7.5 wt%, and the amount of spectinomycin hydrochloride is preferably 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, or 15 wt%.

[0014] In one embodiment, the emulsifier is selected from one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, or synthetic phospholipids, and the amount of the emulsifier is preferably 1 wt%, 1.5 wt%, 2 wt%, or 3 wt%.

[0015] In one embodiment, the co-emulsifier is selected from one or more combinations of PEG400, PEG600, oleic acid, linoleic acid, and palmitic acid, and the amount of the co-emulsifier is preferably 0.01wt%, 0.02wt%, 0.03wt%, 0.05wt%, 0.5wt%, 0.6wt%, 1wt%, 1.5wt%, or 2wt%.

[0016] In one embodiment, the complexing agent is selected from disodium EDTA, and the amount of disodium EDTA used is preferably 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.05 wt%, 0.5 wt%, or 1 wt%.

[0017] In one embodiment, the antioxidant is selected from one or more of vitamin E, propyl gallate, tert-butyl-p-hydroxyanisole, ascorbic acid, sodium bisulfite, and sodium metabisulfite, and the amount of said antioxidant is preferably 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.05 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, or 2 wt%.

[0018] In one embodiment, the isotonic agent is selected from one or more combinations of glycerol, xylitol, or glucose, and the amount of the isotonic agent used is 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%.

[0019] In one embodiment, the oil for injection is selected from one or more of soybean oil, peanut oil, tea oil, sesame oil, medium- and long-chain fatty acid esters, or olive oil, and the amount of the oil for injection is preferably 10 wt%, 15 wt%, 20 wt%, 25 wt%, or 30 wt%.

[0020] This invention also provides a method for preparing a lincomycin hydrochloride and spectinomycin hydrochloride microemulsion, comprising the following steps:

[0021] 1) Under nitrogen protection at 50-60℃, the emulsifier is completely dissolved in injection oil, and then the antioxidant is added under nitrogen protection. The mixture is stirred until it is completely dissolved to obtain the oil phase.

[0022] 2) Dissolve lincomycin hydrochloride, spectinomycin hydrochloride, isotonic agent, complexing agent and co-emulsifier in water for injection, and stir until completely dissolved to obtain an aqueous phase;

[0023] 3) Mix the above oil phase and water phase, bring to a constant volume, and then stir and shear at high speed to form a primary emulsion.

[0024] 4) Homogenize the above primary emulsion under high pressure 5-8 times to obtain a uniform emulsion. Then filter the homogenized emulsion through a microporous membrane, fill it with nitrogen, fill and seal it, and sterilize it at 115℃ for 30 minutes to obtain the final product.

[0025] In this invention, because the water solubility of antioxidants is different, the way they are added is also different: oil-soluble antioxidants are added to the oil phase, and water-soluble antioxidants are added to the aqueous phase.

[0026] Specifically, the preparation method of the lincomycin hydrochloride and spectinomycin hydrochloride microemulsion includes the following steps:

[0027] 1) Under nitrogen protection at 50-60℃, the emulsifier is completely dissolved in injection oil, and then an antioxidant (vitamin E, tert-butyl-p-hydroxyanisole or propyl gallate) is added under nitrogen protection. The mixture is stirred until it is completely dissolved to obtain the oil phase.

[0028] 2) Dissolve lincomycin hydrochloride, spectinomycin hydrochloride, isotonic agent, complexing agent and co-emulsifier in water for injection, and stir until completely dissolved to obtain an aqueous phase;

[0029] 3) Mix the above oil phase and water phase, bring to a constant volume, and then stir and shear at high speed to form a primary emulsion.

[0030] 4) Homogenize the above primary emulsion under high pressure 5-8 times to obtain a uniform emulsion. Then filter the homogenized emulsion through a microporous membrane, fill it with nitrogen, fill and seal it, and sterilize it at 115℃ for 30 minutes to obtain the final product.

[0031] Specifically, the preparation method of the lincomycin hydrochloride and spectinomycin hydrochloride microemulsion includes the following steps:

[0032] 1) Under nitrogen protection at 50-60℃, the emulsifier is completely dissolved in injection oil, and the mixture is stirred until it is completely dissolved to obtain the oil phase;

[0033] 2) Dissolve lincomycin hydrochloride, spectinomycin hydrochloride, isotonic agent, antioxidant (sodium bisulfite, sodium metabisulfite, ascorbic acid), complexing agent and co-emulsifier in water for injection, stir until completely dissolved, and obtain aqueous phase;

[0034] 3) Mix the above oil phase and water phase, bring to a constant volume, and then stir and shear at high speed to form a primary emulsion.

[0035] 4) Homogenize the above primary emulsion under high pressure 5-8 times to obtain a uniform emulsion. Then filter the homogenized emulsion through a microporous membrane, fill it with nitrogen, fill and seal it, and sterilize it at 115℃ for 30 minutes to obtain the final product.

[0036] This invention improves the stability of the injection, increases the encapsulation rate, and yields microemulsions with small and uniform particle size, extending the shelf life of the drug and ensuring drug safety. The preparation process of this invention is simple, saves time and cost, and is suitable for industrialization. In addition, the formulation composition and preparation process of the formulation of this invention have been scientifically screened and demonstrated.

[0037] By adopting the above technical solution, the present invention has the following beneficial effects:

[0038] 1) The microemulsion formulation prepared by this invention has high stability and can achieve a very high encapsulation rate (above 98.5%).

[0039] 2) The microemulsion formulation of the present invention, by introducing complexing agents and co-emulsifiers, produces a microemulsion formulation with small average particle size, uniform distribution and high stability;

[0040] 3) The preparation process of the present invention is simple, saves time and cost, and is suitable for industrialization. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] The present invention will be further explained below with reference to specific embodiments.

[0043] Example 1

[0044] prescription:

[0045] Spectinomycin hydrochloride 10g

[0046] Lincomycin hydrochloride 5g

[0047] 1.5g of soybean lecithin

[0048] 15g of soybean oil for injection

[0049] PEG4000.6g

[0050] 2.5g of glycerin

[0051] Vitamin E 0.02g

[0052] Disodium EDTA 0.01g

[0053] Add water for injection to 100 mL

[0054] Preparation process:

[0055] 1) Under nitrogen protection, 1.5g of soybean lecithin was completely dissolved in 15g of soybean oil for injection. Then, under nitrogen protection, 0.02g of vitamin E was added and stirred until the mixture was completely dissolved to obtain the oil phase.

[0056] 2) Dissolve 10g spectinomycin hydrochloride, 5g lincomycin hydrochloride, 2.5g glycerol, 0.01g disodium EDTA and 0.6g PEG400 in 70mL of water for injection and stir until completely dissolved to obtain the aqueous phase;

[0057] 3) Mix the above oil phase and water phase, bring the volume to 100 mL, and then transfer it to a high-speed shear emulsifier. Stir and shear at high speed (10000 r, 5 min) to obtain the primary emulsion.

[0058] 4) The prepared primary emulsion was homogenized 6 times under high pressure (800 Bar), and the homogenized emulsion was filtered through a microporous membrane, filled with nitrogen, sealed, and sterilized at 115°C for 30 minutes to obtain the final product.

[0059] Examples 2-4

[0060] Examples 2-4 were prepared according to the method of Example 1, and the specific components and amounts are shown in Table 1.

[0061] Table 1

[0062]

[0063] In Table 1, "-" indicates that no addition was made;

[0064] Comparative Example 1

[0065] prescription:

[0066] Spectinomycin hydrochloride 10g

[0067] Lincomycin hydrochloride 5g

[0068] 1.5g of soybean lecithin

[0069] 15g of soybean oil for injection

[0070] 2.5g of glycerin

[0071] PEG400 0.6g

[0072] Vitamin E 0.02g

[0073] Add water for injection to 100 mL

[0074] Preparation process:

[0075] 1) Under nitrogen protection, 1.5g of soybean lecithin was completely dissolved in 15g of soybean oil for injection. Then, under nitrogen protection, 0.02g of vitamin E was added and stirred until the mixture was completely dissolved to obtain the oil phase.

[0076] 2) Dissolve 10g spectinomycin hydrochloride, 5g lincomycin hydrochloride, 2.5g glycerol and 0.6g PEG400 in 70mL of water for injection, and stir until completely dissolved to obtain the aqueous phase;

[0077] 3) Mix the above oil phase and water phase, bring the volume to 100 mL, and then transfer it to a high-speed shear emulsifier. Stir and shear at high speed (10000 r, 5 min) to obtain the primary emulsion.

[0078] 4) The prepared primary emulsion was homogenized 6 times under high pressure (800 Bar), and the homogenized emulsion was filtered through a microporous membrane, filled with nitrogen, sealed, and sterilized at 115°C for 30 min to obtain the final product.

[0079] Comparative Examples 2-4

[0080] Comparative Examples 2-4 were prepared using the same method as Comparative Example 1. For details of each component and its dosage, please refer to Table 2.

[0081] Table 2

[0082]

[0083] In Table 2, "-" indicates that no addition was made;

[0084] Particle size determination:

[0085] Take 200 μL of each of the microemulsions for injection from Examples 1-4 and Comparative Examples 1-4 and add them to a 10 mL volumetric flask. Dilute to the mark with distilled water, shake gently, and measure the particle size and polydispersity index (PDI) using a Malvern Nano-ZS90 particle size analyzer. The results are shown in Table 3.

[0086] Table 3

[0087] Example 1 154.3±1.25 0.183±0.005 Example 2 162.1±1.32 0.191±0.006 Example 3 167.4±1.43 0.198±0.007 Example 4 150.1±1.09 0.180±0.003 Comparative Example 1 156.3±1.31 0.187±0.006 Comparative Example 2 354.7±1.48 0.278±0.010 Comparative Example 3 368.1±1.51 0.284±0.009 Comparative Example 4 442.4±1.62 0.313±0.012

[0088] As can be seen from Table 3, without the addition of a co-emulsifier, the microemulsions prepared have a larger particle size and a relatively uneven distribution, while the microemulsions prepared after the addition of a co-emulsifier in Examples 1-4 of this application have a smaller average particle size and a more uniform distribution.

[0089] Encapsulation efficiency experiment:

[0090] The encapsulation efficiency of spectinomycin hydrochloride and lincomycin hydrochloride in the injection microemulsions prepared in Examples 1-4 and Comparative Examples 1-4 was determined.

[0091] The injectable microemulsion formulations prepared in Examples 1-4 and Comparative Examples 1-4 were centrifuged at 5000 r / min for 30 min. 1 ml of the supernatant was collected, dissolved in ethanol, and the contents of spectinomycin hydrochloride and lincomycin hydrochloride were determined by high-performance liquid chromatography (HPLC). The encapsulated content M1 was determined, the total amount of spectinomycin hydrochloride and lincomycin hydrochloride in the injectable microemulsion was M0, and the encapsulation efficiency N was:

[0092] N = M1 / M0 × 100%, the results are shown in Table 4.

[0093] Table 4

[0094] Example 1 99.4 Example 2 99.0 Example 3 98.5 Example 4 99.2 Comparative Example 1 98.9 Comparative Example 2 82.6 Comparative Example 3 81.5 Comparative Example 4 76.7

[0095] As can be seen from the results in Table 4, the encapsulation efficiency of the microemulsion prepared without the addition of co-emulsifier PEG400 was significantly reduced. However, the encapsulation efficiency of the microemulsion prepared by adding the co-emulsifier was significantly improved. In addition, when other emulsifiers, such as hydrogenated soybean lecithin, were used, the encapsulation efficiency was reduced. The encapsulation efficiency of the injection microemulsion prepared using soybean lecithin as the emulsifier was the best.

[0096] Stability test:

[0097] The microemulsions prepared in Examples 1-4 and Comparative Examples 1-4 were subjected to accelerated stability tests for 6 months at a high temperature of 40±2℃ and a relative humidity of 75%±5%. Samples were taken at months 1, 2, 3, and 6, and the tests were performed according to the key stability test items. The results are shown in Table 5. The microemulsions were subjected to long-term stability tests for 12 months at a high temperature of 25±2℃ and a relative humidity of 60%±5%. Samples were taken at months 3, 6, 9, and 12, and the tests were performed according to the key stability test items. The results are shown in Table 6.

[0098] Table 5 Results of Accelerated Experiments

[0099]

[0100]

[0101] Table 6. Results of Long-Term Stability Tests

[0102]

[0103] As can be seen from the experimental data in Tables 5 and 6, in Comparative Example 2 without the addition of co-emulsifier PEG400, and in Comparative Example 3 without the addition of co-emulsifier PEG400 and complexing agent disodium EDTA, the pH value and drug content of the microemulsions of both decreased significantly with the extension of storage time. This indicates that the addition of co-emulsifiers and complexing agents has a significant impact on the stability of microemulsions. The microemulsion of the present invention contains co-emulsifiers and complexing agents, and its pH value and drug content are relatively stable under both accelerated and long-term stability test conditions. This shows that the injectable microemulsion of the present invention has good long-term storage stability.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microemulsion of lincomycin hydrochloride and spectinomycin hydrochloride, characterized in that, It comprises the following components: lincomycin hydrochloride, spectinomycin hydrochloride, emulsifier, co-emulsifier, antioxidant, complexing agent, isotonic agent, oil for injection, and water for injection; wherein, the weight ratio of each component is as follows: The dosage of lincomycin hydrochloride is 5 wt%; the dosage of spectinomycin hydrochloride is 10 wt%; the dosage of emulsifier is 1.5 wt%; the dosage of co-emulsifier is 0.6 wt%; the dosage of disodium EDTA is 0.01 wt%; the dosage of antioxidant is 0.02 wt%; the dosage of isotonic agent is 2.5 wt%; the dosage of oil for injection is 15 wt%; the remainder is water for injection. The emulsifier is selected from soybean lecithin; the co-emulsifier is selected from PEG400; the complexing agent is selected from disodium EDTA; the antioxidant is selected from vitamin E; the isotonic agent is selected from glycerin; and the oil for injection is selected from soybean oil.

2. A method for preparing the lincomycin hydrochloride and spectinomycin hydrochloride microemulsion according to claim 1, characterized in that, Includes the following steps: 1) Under nitrogen protection at 50-60℃, the emulsifier is completely dissolved in injection oil, and then an antioxidant is added under nitrogen protection. The mixture is stirred until it is completely dissolved to obtain an oil phase. The antioxidant is selected from vitamin E. 2) Dissolve lincomycin hydrochloride, spectinomycin hydrochloride, isotonic agent, complexing agent and co-emulsifier in water for injection, and stir until completely dissolved to obtain an aqueous phase; 3) Mix the above oil phase and water phase, bring to a constant volume, and then stir and shear at high speed to form a primary emulsion; 4) Homogenize the above primary emulsion under high pressure 5-8 times to obtain a uniform emulsion. Then filter the homogenized emulsion through a microporous membrane, fill it with nitrogen, fill and seal it, and sterilize it at 115℃ for 30 minutes to obtain the final product.

3. The use of the microemulsion according to claim 1 in the preparation of a medicament for treating diarrhea or porcine mycoplasmal pneumonia-related diseases in piglets.

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