Marbofloxacin liposome and preparation method thereof

Abstract

The application provides a marbofloxacin liposome and a preparation method thereof. The marbofloxacin liposome comprises a liposome carrier and marbofloxacin encapsulated in the liposome carrier. The application creatively develops a marbofloxacin liposome, which encapsulates marbofloxacin drugs in a nanometer-level liposome, can prolong the effective action time of marbofloxacin in an animal body, improve the bioavailability of marbofloxacin, reduce the clinical drug use frequency in animal diagnosis and treatment, and improve the animal disease cure rate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a marbofloxacin liposome and its preparation method. Background Technology

[0002] Marbofloxacin is a veterinary fluoroquinolone antibiotic with the chemical name 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyridine[3,2,1-ij][4,1,2-]benzoxadiazine-6-carboxylic acid. It was first developed by Roche and further developed by Vetoquinol in France, and was launched in Europe in 1995. Marbofloxacin is a third-generation quinolone antibiotic following enrofloxacin, danofoxin, and sarafloxacin. It possesses broad-spectrum antibacterial activity and excellent pharmacokinetic characteristics, exhibiting strong bactericidal power, rapid absorption, wide distribution in the body, no cross-resistance with other antibiotics, ease of use, and few adverse reactions. Pharmacokinetic studies have shown that marbofloxacin has a long elimination half-life in animals, a bioavailability of nearly 100%, and leaves almost no residue in the blood, feces, and tissues of animals, making it very suitable for the requirements of veterinary antibiotics in clinical practice.

[0003] Liposomes are a type of targeted drug delivery system. Formulating marbofloxacin into liposomes allows for drug encapsulation within submicron or nanometer-sized liposomes, thereby prolonging the duration of action, improving bioavailability, reducing the frequency of clinical administration, increasing cure rates, and generating economic benefits. However, the encapsulation efficiency and cure rate of marbofloxacin liposomes still require further improvement. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a marbofloxacin liposome and its preparation method.

[0005] In a first aspect, the present invention provides a marbofloxacin liposome, the marbofloxacin liposome comprising a liposome carrier and marbofloxacin encapsulated in the liposome carrier.

[0006] This invention creatively develops a marbofloxacin liposome, which encapsulates marbofloxacin in nanometer-sized liposomes, thereby prolonging the effective duration of marbofloxacin in animals, improving its bioavailability, reducing the number of clinical doses in animal diagnosis and treatment, and increasing the cure rate of animal diseases.

[0007] Preferably, the raw materials for preparing the marbofloxacin liposomes include 2-4 parts cholesterol, 9-15 parts phospholipids, 0.4-1 parts polyethylene glycol-lipids, and 1-3 parts marbofloxacin by weight.

[0008] The raw materials used to prepare the marbofloxacin liposomes within the above-mentioned range result in marbofloxacin liposomes with small hydrated particle size, stable system, and high encapsulation efficiency and drug loading.

[0009] The cholesterol can be in the following weight proportions: 2 parts, 2.3 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, etc.

[0010] The phospholipids can be present in weight parts of 9, 10, 11, 12, 13, 15, etc.

[0011] The weight parts of the polyethylene glycol-lipid can be 0.4 parts, 0.5 parts, 0.6 parts, 0.8 parts, 0.9 parts, 1 part, etc.;

[0012] The weight fractions of the mapofloxacin can be 1 part, 1.3 parts, 1 part, 2 parts, 2.4 parts, 2.8 parts, 3 parts, etc.

[0013] All specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0014] Preferably, the phospholipid is selected from any one of distearylphosphatidylethanolamine, hydrogenated phosphatidylcholine, 1,2-distearyl-sn-glycerol-3-phosphatecholine, or 1,2-dioleoyl-sn-glycerol-3-phosphateethanolamine; hydrogenated phosphatidylcholine is preferred.

[0015] The liposomes made from hydrogenated phosphatidylcholine used in this invention have a higher encapsulation efficiency and a higher drug loading capacity for marbofloxacin compared to other types of phospholipids.

[0016] Preferably, the molecular weight of polyethylene glycol in the polyethylene glycol-lipid is 1000-5000.

[0017] The liposomes made with DSPE-PEG of molecular weight of 1000-5000 have a higher encapsulation efficiency and higher marbofloxacin loading compared with liposomes made with other molecular weights.

[0018] The molecular weight of the polyethylene glycol can be 1000, 1500, 2000, 3000, 3500, 4000, 5000, etc.

[0019] In a second aspect, the present invention provides a method for preparing the marbofloxacin liposomes described in the first aspect, the method comprising the following steps: (1) mixing phospholipids, cholesterol, polyethylene glycol-lipids, marbofloxacin and an organic solvent, and rotary evaporating to obtain a first mixture;

[0020] (2) The first mixture is mixed with deionized water and heated in a water bath to obtain the second mixture;

[0021] (3) Ultrasonic vibration of the second mixture;

[0022] (4) High-pressure homogenization.

[0023] This invention uses a thin-film hydration method to prepare marbofloxacin liposomes. By using specific parameters and process formulations, the encapsulation efficiency and drug loading of marbofloxacin liposomes are improved.

[0024] Preferably, the organic solvent in step (1) includes any one or a combination of at least two of DMF, DCM, methanol, diethyl ether, ethanol or chloroform; preferably, a combination of methanol and chloroform.

[0025] Methanol and chloroform, as organic solvents for preparing marbofloxacin liposomes, have good dissolving power, resulting in marbofloxacin liposomes with better morphology.

[0026] Preferably, the volume ratio of methanol to chloroform is (1-3):(1-3).

[0027] The volume ratio of methanol to chloroform can be 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, etc. Specific values ​​within the above range can be selected, and will not be elaborated further here.

[0028] The liposomes prepared at the above ratio have better morphology.

[0029] Preferably, the water bath conditions in step (2) include a water bath at 50-80℃ for 10-30 minutes.

[0030] The temperature of the water bath can be 50℃, 56℃, 67℃, 68℃, 79℃, 80℃, etc.;

[0031] The water bath time can be 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0032] All specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0033] Preferably, the conditions for ultrasonic oscillation in step (3) include 5-15 minutes of ultrasonication at 20-30℃ and 200-400W power.

[0034] The ultrasonic oscillation time in step (3) can be 20℃, 22℃, 24℃, 26℃, 29℃, 30℃, etc.;

[0035] The ultrasonic power can be 200W, 220W, 240W, 260W, 280W, 300W, etc.;

[0036] The ultrasound duration can be 5 min, 7 min, 8 min, 10 min, 12 min, 14 min, 15 min, etc. Specific values ​​within the above range can be selected, and will not be elaborated further here.

[0037] Preferably, the high-pressure homogenization conditions in step (4) include homogenization 3-5 times at a pressure of 10000-15000 psi.

[0038] The pressure value can be 10000psi, 11000psi, 12000psi, 13000psi, 14000psi, 15000psi, etc.; the number of high-pressure homogenization cycles can be 3, 4, or 5.

[0039] All specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention creatively develops a marbofloxacin liposome, which encapsulates marbofloxacin in nanometer-sized liposomes, thereby prolonging the effective duration of marbofloxacin in animals, improving its bioavailability, reducing the number of clinical doses in animal diagnosis and treatment, and increasing the cure rate of animal diseases. Attached Figure Description

[0042] Figure 1 This is a transmission electron microscope image of the marbofloxacin liposomes prepared in Example 1;

[0043] Figure 2 This is a hydrated particle size diagram of the marbofloxacin liposomes prepared in Example 1;

[0044] Figure 3 This is a Zeta potential diagram of the marbofloxacin liposomes prepared in Example 1. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0046] The sources of some components in the following examples and comparative examples are as follows:

[0047] Hydrogenated phosphatidylcholine is derived from Sigma-Aldrich's product 840058P-1G; DSPE-PEG2000 is derived from Xi'an Ruixi Biotechnology Co., Ltd.'s product CAS number 474922-77-5; distearylphosphatidylcholine is derived from Sigma-Aldrich's product 850365C-200MG; distearylphosphatidylethanolamine is derived from Sigma-Aldrich's product 850715P-200MG; dipalmitoylphosphatidylcholine is derived from Sigma-Aldrich's product 850355C-25MG; vitamin E is derived from Maclean's product D819005; lactic acid is derived from Maclean's product D812427; cholesterol is derived from Sigma-Aldrich's product C8667; and marbofloxacin is derived from Maclean's product M813801-1g.

[0048] Example 1

[0049] This embodiment provides a marbofloxacin liposome, and the preparation steps are as follows:

[0050] (1) Weigh 10mg of hydrogenated phosphatidylcholine, 3mg of cholesterol, 0.6mg of DSPE-PEG2000 and 1.8mg of marbofloxacin and dissolve them in 4mL of a methanol and chloroform mixture (1:1, v / v). Transfer the mixture to a 50mL round-bottom flask and evaporate it for 20min at 37℃ with a rotation speed of 100r / min to remove the solvent. The remaining components form a thin film on the bottom wall of the flask.

[0051] (2) Slowly add 3 mL of deionized water to the round-bottom flask after rotary evaporation, and incubate in a water bath at 60°C for 20 min. Gently shake the flask several times during this period to ensure that the lipid film adhering to the wall is fully hydrated.

[0052] (3) The above solution was sonicated at room temperature for 10 minutes at 300W power to disperse the liposomes.

[0053] (4) A microfluidic high-pressure homogenizer was used to homogenize the solution four times at a pressure of 12000 psi, resulting in a solution that changed from turbid to clear, yielding a liposome solution with uniform particle size. The microstructure was observed using a transmission electron microscope as follows: Figure 1 As shown.

[0054] The zeta potential and hydrated particle size of the mabofloxacin liposomes prepared in Example 1 were measured using a Malvern laser particle size analyzer. The results of the hydrated particle size test are as follows: Figure 2 As shown, the hydrated particle size is 75 nm, and the Zeta test results are as follows. Figure 3 As shown, the Zeta potential is -20.03mV.

[0055] Example 2

[0056] This embodiment provides a marbofloxacin liposome, and the preparation steps are as follows:

[0057] (1) Weigh 10mg of hydrogenated phosphatidylcholine, 1mg of cholesterol, and 0.6mg of DSPE-PEG2000 and dissolve them in 4mL of a methanol and chloroform mixture (3:1, v / v). Add 1μL of vitamin E and transfer the mixture to a 50mL round-bottom flask. Evaporate the solvent in a 37℃ water bath at 150r / min for 30min. The remaining components will form a thin film on the bottom wall of the flask.

[0058] (2) Slowly add 3 mL of deionized water containing 3 mg of marbofloxacin to a rotary-evaporated round-bottom flask and incubate in a water bath at 80°C for 30 min. Gently shake the flask several times during this period to ensure that the lipid film adhering to the wall is fully hydrated.

[0059] (3) The above solution was sonicated at room temperature for 15 minutes at 400W power to disperse the liposomes.

[0060] (4) A microfluidic high-pressure homogenizer was used to homogenize the solution five times at a pressure of 10,000 psi. The solution changed from turbid to clear, and the liposome solution was obtained.

[0061] Example 3

[0062] This embodiment provides a marbofloxacin liposome, and the preparation steps are as follows:

[0063] (1) Weigh 10mg of hydrogenated phosphatidylcholine, 1mg of cholesterol, and 0.6mg of DSPE-PEG2000, dissolve them in 4mL of a methanol and chloroform mixture (1:3, v / v), add 1μL of vitamin E, and transfer the mixture to a 50mL round-bottom flask. Under the conditions of a 37℃ water bath, set the rotation speed to 100r / min, and evaporate for 20min to remove the solvent. The remaining components form a thin film on the bottom wall of the flask.

[0064] (2) Slowly add 3 mL of deionized water containing 1.2 mg of marbofloxacin and 1 μL of lactic acid to a rotary-evaporated round-bottom flask and incubate in a 50°C water bath for 10 min. Gently shake the flask several times during this period to ensure that the lipid film adhering to the wall is fully hydrated.

[0065] (3) The above solution was sonicated at room temperature for 5 minutes at 200W power to disperse the liposomes.

[0066] (4) The solution was homogenized three times at a pressure of 15,000 psi using a micro-jet high-pressure homogenizer. The solution changed from turbid to clear, and the liposome solution was obtained.

[0067] Example 4

[0068] This embodiment provides a marbofloxacin liposome, and the preparation steps are as follows:

[0069] (1) Weigh 10.1 mg of hydrogenated phosphatidylcholine, 5.6 mg of cholesterol, 0.6 mg of DSPE-PEG2000 and 0.9 mg of marbofloxacin and dissolve them in 4 mL of a methanol and chloroform mixture (1:1, v / v). Transfer the mixture to a 50 mL round-bottom flask and evaporate it for 20 min at 37 °C with a rotation speed of 100 r / min to remove the solvent. The remaining components form a thin film on the bottom wall of the flask.

[0070] (2) Slowly add 3 mL of deionized water to the round-bottom flask after rotary evaporation, and incubate in a water bath at 60°C for 20 min. Gently shake the flask several times during this period to ensure that the lipid film adhering to the wall is fully hydrated.

[0071] (3) The above solution was sonicated at room temperature for 10 minutes at 300W power to disperse the liposomes.

[0072] (4) The solution was homogenized four times at a pressure of 12000 psi using a micro-jet high-pressure homogenizer. The solution changed from turbid to clear, and the liposome solution was obtained.

[0073] Example 5

[0074] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in the high-pressure homogenization conditions. The difference is that in step 4, "a microfluidic high-pressure homogenizer is used to homogenize 7 times at a pressure of 12000 psi", while other conditions remain unchanged. The preparation method is the same as in Example 1.

[0075] Example 6

[0076] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in the phospholipid formulation, replacing hydrogenated phosphatidylcholine with an equal amount of distearate phosphatidylcholine. All other conditions remain unchanged, and the preparation method is the same as in Example 1.

[0077] Example 7

[0078] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in the phospholipid formulation, replacing hydrogenated phosphatidylcholine with an equal amount of distearate phosphatidylethanolamine. All other conditions remain unchanged, and the preparation method is the same as in Example 1.

[0079] Example 8

[0080] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in the phospholipid formulation, replacing hydrogenated phosphatidylcholine with an equal amount of dipalmitoyl phosphatidylcholine. All other conditions remain unchanged, and the preparation method is the same as in Example 1.

[0081] Example 9

[0082] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in that the volume ratio of methanol to chloroform in the methanol-chloroform mixture is 4:1, while the volume of the methanol-chloroform mixture remains unchanged. All other conditions remain the same, and the preparation method is the same as in Example 1.

[0083] Example 10

[0084] This embodiment provides a marbofloxacin liposome, which differs from Example 1 only in that the methanol and chloroform mixture is replaced with an ethanol / ether mixture at a volume ratio of 1:1, while the volume of the organic solution remains unchanged. All other conditions remain the same, and the preparation method is the same as in Example 1.

[0085] Test Example 1

[0086] Characterization of Marbofloxacin liposomes:

[0087] The marbofloxacin liposomes prepared in Examples 1-10 were characterized using a Malvern laser particle size analyzer, and the hydrated particle size and dispersion coefficient of the marbofloxacin liposomes were tested.

[0088] The test results are shown in Table 1.

[0089] Table 1

[0090] Group Hydrated particle size PDI Example 1 75nm 0.256 Example 2 74.50nm 0.261 Example 3 48.19nm 0.448 Example 4 52.37nm 0.532 Example 5 42.96nm 0.479 Example 6 54.76nm 0.556 Example 7 58.37nm 0.561 Example 8 50.7nm 0.456 Example 9 72.21nm 0.325 Example 10 69.85nm 0.336

[0091] As shown in Table 1, the preferred raw materials and reaction conditions of this invention can produce better marbofloxacin liposomes, which is reflected in their particle size and polymer dispersion coefficient.

[0092] Test Example 2

[0093] Encapsulation efficiency and drug loading were determined using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0094] (1) Analysis method: Waters ACQUITY was used. HSST3 column (100×2.1mm, 1.8μm); mobile phase A was formic acid:water (0.001:1, v / v), and mobile phase B was formic acid:methanol (0.001:1, v / v). Flow rate was 0.3ml / min; pump pressure was 60MPa; column temperature was 40℃.

[0095] The ultra-high performance liquid chromatography elution program is shown in the table below:

[0096]

[0097]

[0098] Mass spectrometry parameters: positive ion mode (ESI+) was used, quantitative ion pair was 363>320 m / z, qualitative ion pair was 363>345 m / z, and collision energy was -35 V.

[0099] (2) Sample pretreatment: Take 1 mL of the marbofloxacin liposome solution prepared in Examples 1-10, add 4 mL of methanol, shake for 10 min, centrifuge at 8000 rpm for 15 min, and serially dilute by 2, 5, 20, and 50 times. Take 1 mL of the supernatant and detect it by ultra-high performance liquid chromatography-tandem mass spectrometry. Calculate the mass of marbofloxacin loaded into the liposomes.

[0100] The encapsulation ratio formula is:

[0101] Encapsulation rate (%) = (mass of encapsulated marbofloxacin / initial mass of marbofloxacin) × 100%

[0102] Drug loading test method: Drug loading (%) = (mass of encapsulated mabofloxacin drug / total mass of drug-loaded liposomes) × 100%

[0103] The test results are shown in Table 2.

[0104] Table 2

[0105]

[0106]

[0107] As shown in Table 2, the mabofloxacin liposomes exhibit excellent encapsulation efficiency and drug loading in the preferred technical solution and formulation of this invention, resulting in a drug with superior efficacy.

[0108] The applicant declares that the detailed process flow of this invention is illustrated by the above embodiments, but this invention is not limited to the above detailed process flow, that is, it does not mean that this invention must rely on the above detailed process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A method for preparing marbofloxacin liposomes, characterized in that, The preparation method includes the following steps: (1) Phospholipids, cholesterol, polyethylene glycol-lipids, marbofloxacin and organic solvents are mixed and rotary evaporated to obtain the first mixture; (2) The first mixture is mixed with deionized water and heated in a water bath to obtain the second mixture; (3) Ultrasonic oscillation of the second mixture; (4) High-pressure homogenization; The mabofloxacin liposomes include a liposome carrier and mabofloxacin encapsulated in the liposome carrier; The phospholipid is hydrogenated phosphatidylcholine; The organic solvent in step (1) is a combination of methanol and chloroform; The volume ratio of methanol to chloroform is (1-3):(1-3). The conditions for high-pressure homogenization in step (4) are to homogenize 3-5 times at a pressure of 10000-15000 psi; The raw materials for preparing the marbofloxacin liposomes include, by weight, 2-4 parts cholesterol, 9-15 parts phospholipids, 0.4-1 parts polyethylene glycol-lipids, and 1-3 parts marbofloxacin.

2. The method for preparing marbofloxacin liposomes according to claim 1, characterized in that, The polyethylene glycol in the polyethylene glycol-lipid has a molecular weight of 1000-5000.

3. The method for preparing marbofloxacin liposomes according to claim 1, characterized in that, The water bath conditions in step (2) are 50-80℃ for 10-30 minutes.

4. The method for preparing marbofloxacin liposomes according to claim 1, characterized in that, The conditions for ultrasonic oscillation in step (3) are 20-30℃ and 200-400W power for 5-15 minutes.

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