Tosufloxacin tosylate antibacterial external preparation and preparation method thereof

Abstract

The invention discloses a tosufloxacin tosylate antibacterial external preparation and a preparation method thereof. According to the invention, the raw material tosufloxacin mesylate forms the phospholipid complex to improve the solubility of the tosufloxacin mesylate in the external preparation, and the particle size of the external preparation is controlled (the maximum particle size is less than 100 [mu] m, and the release rate of in-vitro release is good) by investigating different matrix dispersions (the phospholipid complex is difficult to disperse well in a single matrix and disperse well in three matrixes); the dispersibility of the phospholipid complex is improved, the viscosity is reduced, and the dissolution of the drug is improved (the in-vitro permeation drug release amount is greater than 25 mu g / cm < 2 > and can be kept for 24 hours). The antibacterial external preparation is an ointment, can be used for bacterial infection of wound surfaces such as skin cut wounds, bruises, small-area burns, surgical wounds and the like or infection of ulcer surfaces, and has better stability and better treatment effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a tosufloxacin tosylate antibacterial external preparation and a preparation method thereof. Background Art

[0002] Skin and soft tissue infections usually refer to infections of the skin and subcutaneous tissues, which are generally caused by hemolytic streptococci and Staphylococcus aureus. When bacterial skin infections occur, the range can range from a small spot to the entire skin surface, and their severity varies, ranging from harmless to life-threatening. Skin ointment preparations are mainly used to lubricate the skin, protect wounds and treat them locally. They are popular with patients because of their convenience, rapid efficacy and few side effects. There are many types of pathogens that cause skin and soft tissue infections, with bacteria being the most common. However, with the widespread use of antibiotics, the problem of bacterial resistance has become increasingly prominent. Changes in bacterial species and resistance have brought huge challenges to the clinical treatment of skin and soft tissue infections.

[0003] Tosufloxacin tosylate is a fourth-generation quinolone broad-spectrum antibacterial drug developed by Toyama Chemical Industry Co., Ltd. of Japan. It achieves antibacterial or bactericidal effects by inhibiting bacterial DNA gyrase. It has a strong antibacterial effect on Gram-positive and negative bacteria, and its effect on Gram-negative bacteria is the strongest among quinolones. Its anti-Staphylococcus aureus effect is 4 to 8 times that of norfloxacin, ciprofloxacin and ofloxacin, and its anti-mycoplasma, chlamydia and gonococcus effect is 8 to 10 times that of ciprofloxacin and ofloxacin. It is also effective against anaerobic bacteria including peptostreptococcus and Propionibacterium acnes. This product is a potent bactericide. When the blood drug concentration exceeds the bacterial MIC (the minimum drug concentration that inhibits 90% bacterial growth), the bacteria can be quickly killed when in contact with it. The frequency of natural drug resistance to various bacteria is low, and even if the bacteria are repeatedly passaged in the drug solution for 14 days, the drug resistance rate is slow. Currently, the dosage forms of tosufloxacin tosylate on the market include tablets and capsules, which are mainly used for upper respiratory tract infections caused by sensitive bacteria, acute and chronic bronchitis, bronchiectasis, pneumonia, urogenital tract infections, biliary tract infections, etc.; in May 2006, the eye drops under the trade name "OAEX" of Toyama Chemical Industry Co., Ltd. were launched on the market as a local medication for blepharitis, dacryocystitis, sty, and conjunctivitis.

[0004] Tosufloxacin tosylate is almost insoluble in water, and the absorption of oral preparations is relatively poor. However, Tosufloxacin tosylate has good cell permeability. Developing Tosufloxacin tosylate into topical preparations can directly penetrate the dermal cells and act on the medication site, which has a better effect than oral administration and avoids the irritation of the gastrointestinal tract by oral preparations. However, Tosufloxacin tosylate is a poorly soluble compound. The preparation of topical preparations will cause the raw materials to be poorly dispersed in the matrix and precipitate, resulting in poor in vitro release data and failure to achieve the ideal pharmacological effect. Summary of the invention

[0005] In view of the above problems, the present invention provides a tosufloxacin tosylate antibacterial external preparation and a preparation method. Compared with the prior art, the present invention forms a phospholipid complex from the raw material tosufloxacin mesylate to improve its solubility in the external preparation, and at the same time examines different matrix dispersions, controls the particle size of the external preparation, increases the dispersibility of the phospholipid complex, reduces the viscosity, and improves the dissolution of the drug. The antibacterial external preparation is an ointment, which can be used for bacterial infection of wounds such as skin cuts, abrasions, small-area burns, surgical wounds, or infection of ulcer surfaces, and the product has better stability and better therapeutic effect.

[0006] The technical scheme of the present invention is: a tosufloxacin tosylate antibacterial external preparation, characterized in that its raw materials and weight ratio are: 2.5-3.0% of tosufloxacin tosylate / soybean lecithin complex, 20-30% of liquid paraffin, 20-30% of silicone oil or squalane, and the balance of white vaseline, lanolin or glyceryl stearate (making up 100%), and the maximum particle size of the product is less than 100 μm.

[0007] The mass ratio of tosufloxacin tosylate and soybean lecithin in the tosufloxacin tosylate / soybean lecithin complex is 3.5-4.5:1, preferably 4:1. The preparation method is as follows: dissolving soybean lecithin in anhydrous ethanol, then adding tosufloxacin tosylate for ultrasonic dispersion, then concentrating under reduced pressure, adding dichloromethane to the concentrate for stirring and dissolving, and concentrating and drying the filtrate to obtain the tosufloxacin tosylate / soybean lecithin complex.

[0008] The preparation method of the tosufloxacin tosylate antibacterial external preparation of the present invention is:

[0009] ① Heat the matrix to above 120°C and stir until the matrix is ​​melted and mixed evenly;

[0010] ② Add the tosufloxacin tosylate / soy lecithin complex to the matrix of step ①, and heat in a water bath to 40-50° C. with stirring;

[0011] ③Control the temperature at 45-50℃, start homogenization, and make the maximum particle size of the product less than 100μm at a homogenization speed of more than 3000rmp, and then disperse it. After dispersion, cool it down to 40-45℃;

[0012] ④ Maintain the temperature at 40-45℃ for filling, and seal the bottle after filling. The average filling volume per bottle is 5g.

[0013] In view of the problem that "Tosufloxacin tosylate is a poorly soluble compound, and the preparation of an external preparation will cause the raw material to be poorly dispersed in the matrix and precipitate", the present invention forms a phospholipid complex with tosufloxacin tosylate according to the structure of tosufloxacin (the active ingredient containing active hydrogen atoms, -COOH and -NH2, can form a hydrogen bond with N-(CH3) of the phospholipid molecule and form a complex), which not only improves the hydrophilicity and lipophilicity of the compound, but also increases stability and improves bioavailability. However, the viscosity of the phospholipid complex will affect the dissolution of the drug. The present invention examines different matrix dispersions and controls the particle size of the external preparation at the same time, so as to increase the dispersibility of the phospholipid complex and reduce the viscosity without changing the complexation of tosufloxacin tosylate and the phospholipid complex, thereby solving the problem of the viscosity of phospholipids affecting the dissolution of the drug.

[0014] The beneficial effects of the present invention are:

[0015] 1. Compared with the prior art, the present invention forms a phospholipid complex from the raw material tosufloxacin mesylate to improve its solubility in the topical preparation, and examines different matrix dispersions (the phospholipid complex is difficult to disperse well in a single matrix, and has better dispersibility in three matrices), controls the particle size of the topical preparation (the maximum particle size is less than 100 μm, and the release rate in vitro is better), increases the dispersibility of the phospholipid complex, reduces the viscosity, and improves the dissolution of the drug (the drug release amount of in vitro penetration is greater than 25 μg / cm 2 , and can be maintained for 24 hours). The external preparation is an ointment, which is easy to apply, has good uniformity, simple process, and is suitable for industrial production.

[0016] 2. The topical preparation has good antibacterial properties and is easy to apply. It can be used for bacterial infections of wounds such as skin cuts, abrasions, small burns, surgical wounds, or infections of ulcer surfaces. The product has better stability and better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures are the particle size distribution and particle size distribution report of Examples 6-9; wherein Figures A and B are the particle size distribution and particle size distribution report of Example 6; wherein Figures C and D are the particle size distribution and particle size distribution report of Example 7; wherein Figures E and F are the particle size distribution and particle size distribution report of Example 8; wherein Figures G and H are the particle size distribution and particle size distribution report of Example 9;

[0018] Figure 2 The linear equation diagrams of the cumulative drug release and the square root of time of Examples 6-9 (A, B, C, and D, respectively);

[0019] Figure 3The time curves of the flux and cumulative permeation of Examples 7, 8, and 9; wherein Figures A and B are time curves of the flux and cumulative permeation of Example 7; Figures C and D are time curves of the flux and cumulative permeation of Example 8; Figures E and F are time curves of the flux and cumulative permeation of Example 9;

[0020] Figure 4 The inhibition zones of the three ointments of Examples 7, 8 and 9 against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa (in order from left to right in the figure), respectively. In the figure, a is the ointment of Example 7, b is the ointment of Example 8, and c is the ointment of Example 9. DETAILED DESCRIPTION

[0021] The effects are described below in conjunction with the embodiments and drawings.

[0022] Embodiment 1-6:

[0023] Prescription screening: The prescriptions of the antibacterial external preparations of Examples 1-6 are shown in Table 1 below.

[0024] Table 1: Antibacterial external preparation prescriptions of Examples 1-6

[0025]

[0026] The specific preparation method is as follows:

[0027] (1) Preparation method of tosufloxacin tosylate / soybean lecithin complex:

[0028] ① Dissolve 200 g of soybean lecithin in 500 mL of anhydrous ethanol, stir for 1 h, filter, and collect the filtrate;

[0029] ② Add 50 g of tosufloxacin tosylate to the filtrate, sonicate for 30 min, and then concentrate under reduced pressure to remove the solvent;

[0030] ③ The concentrate was added with dichloromethane and stirred to dissolve, and filtered to obtain a filtrate. The operation (dissolving the filter residue with dichloromethane and filtering) was repeated until a clear filtrate was obtained. The filtrates were combined and concentrated again under reduced pressure to remove the solvent. The obtained concentrate was placed in a vacuum drying oven and dried to obtain a tosufloxacin besylate / soybean lecithin complex with an encapsulation efficiency of 85% and a drug loading of 18.7%.

[0031] (2) Preparation method of external preparation:

[0032] ① Take the prescribed amount of matrix and add it to a beaker, turn on heating and stirring, heat the matrix temperature to above 120℃, and keep stirring for 2h;

[0033] ② Add the prescribed amount of tosufloxacin tosylate / soy lecithin complex to a beaker and heat in a water bath to 40-50°C while stirring;

[0034] ③ Start homogenization at a temperature of 45-50°C, a homogenization speed of 2000 rpm, and disperse for 20 minutes (disperse for 15 minutes, stop for 5 minutes). After dispersion, cool down to 40-45°C;

[0035] ④Filling, maintain the temperature at 40-45℃, and seal the tubes after filling. The average filling volume of each tube is 5g.

[0036] According to the above experiment, the results were preliminarily counted, and the results are shown in Table 2. From the test results, it can be seen that the phospholipid complex is difficult to disperse well in a single matrix, but has good dispersibility in three matrices, and the obtained ointment has good properties (Example 6), so the conditions of Example 6 are selected as the final prescription. The prescription of Example 6 is a mixture of a matrix in a solid state at room temperature and a liquid matrix at room temperature, so the optimal conditions are selected according to this prescription.

[0037] Table 2: Appearance of the finished antibacterial external preparations of Examples 1-6

[0038]

[0039] Embodiment 7:

[0040] Antibacterial topical preparation formula:

[0041]

[0042] Specific preparation method: Step ③ The homogenization speed is 3000 rpm, and the rest is the same as Example 6.

[0043] Embodiment 8:

[0044] Antibacterial topical preparation formula:

[0045]

[0046] Specific preparation method: Step ③ The homogenization speed is 3500 rpm, and the rest is the same as Example 6.

[0047] Embodiment 9:

[0048] Antibacterial topical preparation formula:

[0049]

[0050] The test results of the finished products of Examples 6-9 are as follows

[0051] 1. Appearance inspection of finished products of Examples 6-9

[0052] The appearance test results of the finished products of Examples 6-9 are shown in Table 3 below. It can be seen from Table 3 that the finished products are white / yellow-white pastes with suitable consistency, good spreadability, uniform color, and good fineness.

[0053] Table 3: Appearance of finished antibacterial external preparations of Examples 6-9

[0054] Sample name Finished product appearance Example 6 White paste, suitable consistency, good spreadability, uniform color, good delicate feeling Example 7 Yellow-white paste, suitable consistency, good spreadability, uniform color, good delicate feeling Example 8 Yellow-white paste, suitable consistency, good spreadability, uniform color, good delicate feeling Example 9 White paste, suitable consistency, good spreadability, uniform color, good delicate feeling

[0055] 2. Detection of related substances in Examples 6-9

[0056] HPLC chromatographic conditions: Agilent ZORBAX SB-C18 (4.6×250mm, 5μm) column; acetonitrile-water (50:30) as mobile phase; flow rate of 1.0 ml per minute; detection wavelength of 250nm; column temperature of 30℃; injection volume of 10μl.

[0057] The test results of related substances in Example 6-9 are shown in Table 4. It can be seen from the table that the purity of Example 6-9 is about 92%, and the total impurity content is about 0.080%.

[0058] Table 4: Test results of related substances in Examples 6-9

[0059]

[0060]

[0061] 3. Particle size test data of Examples 6-9

[0062] According to the Chinese Pharmacopoeia test method, the ointment was tested for particle size using a microscope. The results are as follows Figure 1 The results show that: in Example 6, the homogenization rate was 2000rmp, and the particle size D 90 95.26μm, D max 154.81μm, increasing the homogenization rate to more than 3000rmp, the maximum particle size of the obtained products is less than 100μm. Later, the in vitro release test was carried out on the products with different particle sizes.

[0063] 4. In vitro release determination of Examples 6-9

[0064] Device: vertical diffusion cell; temperature: 32°C; rotation speed: 600rpm;

[0065] Release medium: pH 6.8 phosphate buffer solution;

[0066] Sample volume: 300 mg (2 mm quantitative loop);

[0067] Artificial membrane: PES polyethersulfone (Defilter), pore size: 0.45 μm;

[0068] Medium volume: 10 ml; sampling time: 1h, 2h, 3h, 4h, 5h, 6h.

[0069] Sampling volume: 1ml; Refill volume: 1ml; Diffusion cell orifice diameter: 15mm; Diffusion cell orifice area: 1.77cm 2 ;

[0070] The HPLC chromatographic conditions used an Agilent ZORBAX SB-C18 (4.6×250mm, 5μm) column; acetonitrile-water (50:30) as the mobile phase; a flow rate of 1.0 ml per minute; a detection wavelength of 250nm; a column temperature of 30℃; and an injection volume of 10μl.

[0071] The in vitro release data of Examples 6-9 are shown in Table 5, and the linear equation diagram of the cumulative drug release and the square root of time is shown in Figure 2 As shown in the results, it can be seen that the particle size of Example 6 is relatively large, and the in vitro release rate is relatively low, while the in vitro release rates of Examples 7, 8 and 9 are relatively consistent, and the duration is up to 6 hours, indicating that the particle size has an effect on the release rate, and the particle size needs to be controlled to be less than 100 μm.

[0072] Table 5: In vitro release data of Examples 6-9

[0073]

[0074]

[0075] 5. In vitro permeation assay of Examples 7-9

[0076] Device: vertical diffusion cell; temperature: 32°C; rotation speed: 600rpm;

[0077] Sample volume: 75 mg (0.5 mm quantitative loop); Release medium: pH 6.8 phosphate buffer solution;

[0078] Skin: pig skin (abdomen);

[0079] Medium volume: 10ml; Sampling volume: 1.5ml; Rehydration volume: 1.5ml;

[0080] Diffusion cell orifice diameter: 15mm; Diffusion cell orifice area: 1.77cm 2 ;

[0081] Sampling time: 1h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, 24h.

[0082] The HPLC chromatographic conditions used an Agilent ZORBAX SB-C18 (4.6×250mm, 5μm) column; acetonitrile-water (50:30) as the mobile phase; a flow rate of 1.0 ml per minute; a detection wavelength of 250nm; a column temperature of 30℃; and an injection volume of 10μl.

[0083] The results of in vitro permeation measurements of Examples 7-9 are shown in Table 6. The time curves of the flux and cumulative permeation of Examples 7, 8, and 9 are as follows: Figure 3 As shown in the figure, it can be seen that the in vitro permeation drug release of Example 7, Example 8 and Example 9 is greater than 25 μg / cm 2 , and can be maintained for 24 hours.

[0084] Table 6: In vitro permeation assay results for Examples 7-9

[0085]

[0086]

[0087] 6. Evaluation of in vitro antibacterial activity of Examples 7-9

[0088] The in vitro antibacterial activities of Example 7, Example 8 and Example 9 against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa were determined by agar diffusion method.

[0089] Experimental process:

[0090] (1) Bottom layer: Use a sterile large-mouth pipette (20 ml) to draw about 20 ml of melted agar medium into the double dishes. After all the double dishes have been filled with the bottom layer, cover them with a ceramic tile lid and wait for them to solidify before use;

[0091] (2) Bacterial layer: Take out the pre-tested Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa suspensions (5×10 6 CFU / mL), use a sterile pipette (or sterile syringe) to absorb the bacterial suspension in an appropriate amount and add it to the culture medium that has been melted and kept warm in a water bath (48°C), shake it well, and use it as the bacterial layer. Use a sterile large-mouth pipette to absorb the culture medium with bacteria, spread the culture medium evenly on the bottom culture medium, place it on a water platform, cover it with a ceramic tile cover, and set it aside after solidification;

[0092] (3) Placing the steel pipe: Place the steel pipe in the stainless steel cup of the steel pipe placer, turn on the switch, place the double disc on the operating table of the steel pipe placer, align the position interface, press the handle, and let the steel pipe fall steadily on the culture medium. After the steel pipe is properly placed, let it stand until the steel pipe sinks slightly in the culture medium and stabilizes, then add the ointments of Example 7, Example 8, and Example 9 respectively;

[0093] (4) Sample addition: Add the ointments of Example 7, Example 8 and Example 9 into a petri dish, and place the ointments in the small steel tube until they are flush with the upper edge of the tube.

[0094] The inhibition zones of the three ointments in Examples 7, 8 and 9 against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa (in order from left to right in the figure) are as follows: Figure 4 As shown, it can be seen that the diameters of the inhibition zones of Example 7, Example 8 and Example 9 are almost the same, indicating that the antibacterial effects of the three examples are relatively consistent and the prescription process is stable.

Claims

1. A tosufloxacin tosylate antibacterial external preparation, characterized in that: The raw materials and weight ratio are: 2.5-3.0% of tosufloxacin tosylate / soybean lecithin complex, 20-30% of liquid paraffin, 20-30% of silicone oil or squalane, and the balance of white vaseline, lanolin or glyceryl stearate. The maximum particle size of the product is less than 100 micrometers.

2. The antibacterial external preparation of tosufloxacin tosylate according to claim 1, characterized in that: The mass ratio of tosufloxacin tosylate to soybean lecithin in the tosufloxacin tosylate / soybean lecithin complex is 3.5-4.5:

1.

3. The antibacterial external preparation of tosufloxacin tosylate according to claim 2, characterized in that: The preparation method of the tosufloxacin tosylate / soybean lecithin complex comprises: dissolving soybean lecithin in anhydrous ethanol, then adding tosufloxacin tosylate for ultrasonic dispersion, and then concentrating under reduced pressure, adding dichloromethane to the concentrate for stirring and dissolving, and concentrating and drying the filtrate to obtain the tosufloxacin tosylate / soybean lecithin complex.

4. The method for preparing the antibacterial external preparation of tosufloxacin tosylate according to any one of claims 1 to 3, characterized in that: The following steps are involved: ① Heat the matrix to above 120°C and stir until the matrix is ​​melted and mixed evenly; ② Add the tosufloxacin tosylate / soy lecithin complex to the matrix of step ①, and heat in a water bath to 40-50° C. with stirring; ③Control the temperature at 45-50℃, start homogenization, make the maximum particle size of the product less than 100μm, and then disperse it. After dispersion, cool it down to 40-45℃; ④ Maintain the temperature at 40-45℃ for filling, and seal the bottle after filling.

5. The preparation method according to claim 4, characterized in that: The homogenizing speed is above 3000 rpm.

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