A water-soluble gamma-polyglutamic acid-tilmicosin complex and preparation method thereof

Abstract

The invention discloses a water-soluble γ-polyglutamic acid-tilmicosin complex. γ-polyglutamic acid is used as a raw material, tilmicosin is connected to a γ-polyglutamic acid molecule through a salt-forming reaction between tilmicosin and γ-polyglutamic acid, and the γ-polyglutamic acid-tilmicosin complex is prepared. The obtained complex is easily soluble in water, has good palatability, has sustained release performance, low toxicity, and good antibacterial activity.

Description

Technical Field

[0001] The present invention relates to a water-soluble gamma-polyglutamic acid-tilmicosin complex and a preparation method thereof, belonging to the field of preparation of antibacterial drugs. Background Art

[0002] Tilmicosin is a macrolide antibiotic for livestock and poultry. It has the advantages of strong antibacterial activity, broad antibacterial spectrum, and no cross-resistance. It is the first choice for the treatment of bacterial infectious diseases in livestock and poultry. It has been approved for marketing in Australia, Brazil, France, Malaysia, Italy, Spain, the United States and other countries. However, the poor water solubility of Tilmicosin limits its clinical application.

[0003] Polyglutamic acid is a water-soluble, biodegradable, non-toxic biopolymer, which is widely used in the medical field. Because of its good bioaffinity and biodegradability, it can be used as a drug carrier to achieve sustained release and targeting of drugs, improve the water solubility of drugs, and reduce the toxic side effects of drugs. Ye Haifeng et al. used γ-polyglutamic acid as a carrier to connect cisplatin to prepare a polyglutamic acid cisplatin complex (Biomaterials, 2006, 27 (35): 5958-5965). Li Wenjuan et al. prepared a γ-polyglutamic acid aspartic acid-cisplatin complex (China Pharmaceutical Biotechnology, 2010, 5 (2): 98-104), and the water solubility and toxicity of these complexes were greatly improved. The present invention connects water-insoluble tilmicosin to a γ-polyglutamic acid carrier through a salt-forming reaction to prepare a water-soluble polyglutamic acid-tilmicosin complex. The complex has good antibacterial activity. Summary of the invention

[0004] The purpose of the present invention is to provide a water-soluble gamma-polyglutamic acid-tilmicosin complex, which has low toxicity, high drug loading, good animal palatability and sustained release performance.

[0005] Another object of the present invention is to provide a method for preparing a water-soluble γ-polyglutamic acid-tilmicosin complex.

[0006] Another object of the present invention is to provide a water-soluble γ-polyglutamic acid-tilmicosin complex for use in the antibacterial field.

[0007] The technical solution adopted by the present invention is:

[0008] (1) Dissolve γ-polyglutamic acid in distilled water to prepare a solution of about 2%, add concentrated hydrochloric acid to adjust the pH to 2-3, hydrolyze at 121°C, 0.1 MPa for 20 min, cool in an ice bath, adjust the pH to 7-8 with 1 mol / L NaOH solution, dialysis with distilled water for 24 h, and freeze-dry to obtain small molecule γ-polyglutamic acid.

[0009] (2) Dissolve an appropriate amount of small molecule γ-polyglutamic acid in distilled water, and dissolve an appropriate amount of tilmicosin in ethanol. At room temperature, slowly add the ethanol solution of tilmicosin to the γ-polyglutamic acid aqueous solution, stir for 12 hours, and evaporate the ethanol under reduced pressure. The remaining solution is dialyzed with distilled water for 24 hours to remove unreacted tilmicosin and other small molecule compounds, and freeze-dried to obtain a γ-polyglutamic acid-tilmicosin complex.

[0010] The molecular weight of the complex is 40-70 KDa.

[0011] The water-soluble γ-polyglutamic acid-tilmicosin complex connects γ-polyglutamic acid and tilmicosin in the form of an ionic bond.

[0012] The complex is soluble in water, has a high drug loading capacity, good animal palatability, sustained release performance, and has an antibacterial activity superior to that of tilmicosin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : The structural formula of the γ-polyglutamic acid-tilmicosin complex of the present invention;

[0014] Figure 2 : Release curve of the γ-polyglutamic acid-tilmicosin complex of the present invention in PBS buffer;

[0015] Figure 3 : Release curve of the γ-polyglutamic acid-tilmicosin complex of the present invention in artificial gastric juice. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments.

[0017] Example 1

[0018] Dissolve 10 g of γ-polyglutamic acid in 490 mL of distilled water to prepare a 2% solution, add concentrated hydrochloric acid to adjust the pH to 2-3, hydrolyze at 121° C., 0.1 MPa for 20 min, cool in an ice bath, adjust the pH to 7-8 with 1 mol / L NaOH solution, dialysis with distilled water for 24 h, and freeze-dry to obtain small molecule γ-polyglutamic acid.

[0019] 1g of small molecule γ-polyglutamic acid was dissolved in 30mL of distilled water, and 0.15g of tilmicosin was dissolved in 5mL of ethanol. At room temperature, the ethanol solution of tilmicosin was slowly added to the γ-polyglutamic acid aqueous solution, stirred for 12h, and ethanol was evaporated under reduced pressure. The remaining solution was dialyzed with distilled water for 24h to remove unreacted tilmicosin, and freeze-dried to obtain the γ-polyglutamic acid-tilmicosin complex. The tilmicosin loading was 32.1%.

[0020] Example 2

[0021] 1g of small molecule γ-polyglutamic acid was dissolved in 30mL of distilled water, and 0.2g of tilmicosin was dissolved in 5mL of ethanol. At room temperature, the ethanol solution of tilmicosin was slowly added to the γ-polyglutamic acid aqueous solution, stirred for 12h, and ethanol was evaporated under reduced pressure. The remaining solution was dialyzed with distilled water for 24h to remove unreacted tilmicosin, and freeze-dried to obtain the γ-polyglutamic acid-tilmicosin complex. The tilmicosin loading was 33.3%.

[0022] Example 3

[0023] 1g of small molecule γ-polyglutamic acid was dissolved in 30mL of distilled water, and 0.25g of tilmicosin was dissolved in 5mL of ethanol. At room temperature, the ethanol solution of tilmicosin was slowly added to the γ-polyglutamic acid aqueous solution, stirred for 12h, and ethanol was evaporated under reduced pressure. The remaining solution was dialyzed with distilled water for 24h to remove unreacted tilmicosin, and freeze-dried to obtain the γ-polyglutamic acid-tilmicosin complex. The tilmicosin drug loading was 33.7%.

[0024] Example 4

[0025] In vitro release study of the complex

[0026] The γ-polyglutamic acid-tilmicosin complex prepared in Example 1 was formulated into a solution containing 1 mg / mL of tilmicosin, placed in a dialysis bag, and mixed with 5 times the amount of PBS buffer (pH 7.4) or 5 times the amount of artificial gastric juice (pH 1.5), and placed in a constant temperature shaking box at 37°C. 1 mL of sample was taken at different time periods (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36, 48, 60, 72, 84 h) and stored for later use. At the same time, 1 mL of new PBS solution was added to the system. The amount of free tilmicosin released therefrom was detected by HPLC [Waters sysmmtry C18 chromatographic column (4.6 mm × 250 mm), flow rate 1.0 mL / min, detection wavelength 280 nm, column temperature 30°C, mobile phase acetonitrile:tetrahydrofuran:dibutylamine phosphate buffer: water 110:55:25:800 (volume ratio)], and the cumulative release rate ( Figure 2-3 ). The results showed that in PBS buffer, the γ-polyglutamic acid-tilmicosin complex released slowly, with a cumulative release rate of about 50% in 48 hours. In artificial gastric juice, the γ-polyglutamic acid-tilmicosin complex released quickly in the first 9 hours, and then the release tended to be slow, with a cumulative release rate of about 50% in 12 hours.

[0027] Example 5

[0028] In vitro antibacterial assay

[0029] Staphylococcus aureus, Streptococcus, Escherichia coli, Salmonella and Mycoplasma hyopneumoniae were used as test strains, and the bacterial solution in the logarithmic growth phase was cultured, and tilmicosin and the γ-polyglutamic acid-tilmicosin complex prepared in Example 1 were dissolved to a certain concentration, and the minimum inhibitory concentration (MIC) was determined by the two-fold dilution method (Table 1). The results showed that the inhibitory activity of the γ-polyglutamic acid-tilmicosin complex against Staphylococcus aureus, Streptococcus, Escherichia coli and Salmonella was better than that of tilmicosin.

[0030] Table 1 In vitro antibacterial activity of γ-polyglutamic acid-tilmicosin complex

[0031]

[0032] Example 6

[0033] 30 healthy pigs weighing 40-50 kg were selected and randomly divided into 3 groups, each with 10 pigs. The first two groups were fed with feed mixed with the γ-polyglutamic acid-tilmicosin complex prepared in Example 3 of the present invention and tilmicosin, respectively, and the dosage was 200 g / ton of feed in terms of tilmicosin. The third group was the control group, and no drug was added to the feed. The trial period was 1 week, and the average daily feed intake of each group was recorded (Table 2). The results showed that the average daily feed intake of the pigs in the group fed with the γ-polyglutamic acid-tilmicosin complex feed was equivalent to that of the pigs in the control group, indicating that the γ-polyglutamic acid-tilmicosin complex had good palatability and did not affect the pigs' feeding.

[0034] Table 2 Effect of feed intake

[0035]

Claims

1. A water-soluble γ-polyglutamic acid-tilmicosin complex, wherein the complex is obtained by a salt-forming reaction of γ-polyglutamic acid and tilmicosin, characterized in that: The preparation method of the water-soluble γ-polyglutamic acid-tilmicosin complex comprises the following steps: (1) Dissolve γ-polyglutamic acid in distilled water to prepare a 2% solution, add concentrated hydrochloric acid to adjust the pH to 2-3, hydrolyze at 121°C, 0.1 MPa for 20 min, cool in an ice bath, adjust the pH to 7-8 with 1 mol / L NaOH solution, dialyze and purify with distilled water for 24 h, and freeze-dry to obtain small molecule γ-polyglutamic acid; (2) Dissolve an appropriate amount of small molecule γ-polyglutamic acid in distilled water, and then dissolve an appropriate amount of tilmicosin in ethanol. At room temperature, slowly add the tilmicosin ethanol solution to the γ-polyglutamic acid aqueous solution, stir for 12 hours, and evaporate the ethanol under reduced pressure. The remaining reaction solution is dialyzed with distilled water for 24 hours to remove unreacted small molecule compounds, and freeze-dried to obtain a γ-polyglutamic acid-tilmicosin complex.

2. The water-soluble γ-polyglutamic acid-tilmicosin complex according to claim 1, characterized in that: The complex is soluble in water, has a sustained release property, and has better antibacterial activity than tilmicosin.

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