Linezolid liposome inhalation preparation and preparation method thereof

Abstract

The invention discloses a linezolid liposome inhalation preparation and a preparation method thereof, and the linezolid liposome inhalation preparation is prepared by dissolving linezolid, phospholipid and cholesterol in an organic phase, then removing an organic solvent through a film dispersion method, adding a 0.9% sodium chloride solution for hydration, and finally homogenizing to obtain the linezolid liposome inhalation preparation. Meanwhile, compared with a linezolid injection, entrapment of the liposome on the linezolid can enable the linezolid to act on the outer membrane of bacteria more accurately and generate an inhibiting or damaging effect on the outer membrane of the bacteria, so that gram-positive bacteria are effectively killed, meanwhile, an inhalation administration mode is adopted, the administration dosage of the medicine is reduced, and the bioavailability of the medicine is improved. The adverse reaction caused by systemic administration is reduced, and meanwhile, the compliance of a patient is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to a linezolid liposome inhalation formulation and its preparation method. Background Technology

[0002] Linezolid belongs to the oxazolidinone class of drugs. Its chemical name is (S)-N[[3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide, and its structural formula is C 16 H 20 FN3O 4， With a relative molecular mass of 337.35, the structure of linezolid is shown in the figure:

[0003] Linezolid is a synthetic oxazolidinone antibiotic commonly used to treat infections caused by Gram-positive bacteria (G+), including suspected or confirmed hospital-acquired pneumonia (HAP) caused by MRSA, community-acquired pneumonia (CAP), complicated skin or skin and soft tissue infections (SSTI), vancomycin-resistant enterococci (VRE) infections, as well as respiratory and bloodstream infections caused by bacteria.

[0004] Linezolid is a bacterial protein synthesis inhibitor that acts on the 50S ribosomal subunit, most closely adjacent to its site of action. Unlike other drugs, linezolid does not affect peptidyl transferase activity; it acts only at the initiation stage of the translation system, inhibiting the binding of mRNA to the ribosome and preventing the formation of the 70S initiation complex, thereby inhibiting bacterial protein synthesis. Due to its unique site and mechanism of action, linezolid is unlikely to exhibit cross-resistance with other antimicrobial agents that inhibit protein synthesis in positive bacteria with intrinsic or acquired resistance characteristics, and it is also unlikely to induce bacterial resistance in vitro.

[0005] Chinese patent CN 112022802A discloses a method for preparing linezolid injection, which can reduce the level of impurities in linezolid injection, thereby improving the safety of linezolid administration. Chinese patent CN 111686072A also discloses a method for preparing linezolid injection, the purpose of which is to reduce the genotoxic impurity (3-fluoro-4-(4-morpholino)aniline) in linezolid to improve the product quality of linezolid injection. Chinese patent CN113995726A discloses a method for preparing linezolid tablets. As the most commonly used dosage form in clinical practice, linezolid tablets require large dosages and large tablet sizes. Therefore, different preparation methods will affect the key quality attributes of the tablets, thereby affecting clinical efficacy and toxic side effects. In existing technologies for preparing linezolid injections, the removal of impurities is difficult to control. The order of addition of raw materials and excipients, pH adjustment, and sterilization processes can all affect the impurity levels in the injection. Furthermore, linezolid has poor solubility and instability in aqueous solutions, thus limiting its clinical use. In existing linezolid tablet preparation processes, the particle size of raw materials needs to be controlled. Due to the poor flowability of powders and granules, it is difficult to obtain tablets that meet the requirements. Currently, linezolid formulations on the market are mainly available in intravenous injection and oral tablet forms. A review of relevant literature on linezolid reveals that it has poor solubility and instability in aqueous solutions. Both dosage forms are for systemic administration, which requires a large dose and is prone to increasing adverse reactions. Moreover, with increased frequency of use, Gram-positive bacteria develop resistance to linezolid. Biofilm formation is one of the mechanisms by which Gram-positive bacteria develop resistance. Simultaneously, biofilm formation increases the difficulty of treating bacterial infections in clinical practice and also leads to bacterial resistance.

[0006] Therefore, finding a drug delivery system that can disrupt biofilm formation, deliver drugs to the site of infection, reduce systemic drug distribution, decrease toxic side effects, increase therapeutic efficacy, and combat bacterial resistance are urgent problems that need to be solved. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a method for preparing linezolid liposome inhaler with high stability, low toxicity, and better therapeutic effect, aiming to achieve the same therapeutic effect while using a smaller drug dose and reducing drug side effects.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention discloses a linezolid liposome inhalation formulation and its preparation method. The liposomes consist of phospholipid bilayer vesicles composed of phospholipids and cholesterol, with linezolid present in the organic phase and embedded within the phospholipid bilayer.

[0010] According to embodiments of the present invention, the phospholipid is selected from one or more of lecithin, DMPC, DPPC, DSPC, and DOPC. The inventors have found that DPPC, being a major component of pulmonary surfactants, can serve as an endogenous molecule, providing unique advantages for linezolid liposome inhalation formulations. Furthermore, it possesses biocompatibility, biodegradability, and high safety profile; therefore, DPPC is preferred as the phospholipid.

[0011] According to embodiments of the present invention, the concentration of linezolid liposome in the organic phase of the linezolid liposome inhalation formulation is 50-600 mg / mL, preferably 200-300 mg / mL. The inventors have found that the encapsulation efficiency of the linezolid liposome inhalation formulation gradually increases with increasing linezolid concentration. When the drug concentration is greater than 300 mg / mL, the encapsulation efficiency of the linezolid liposome inhalation formulation no longer changes, indicating that the drug concentration has reached the maximum limit that the liposomes can encapsulate. The preferred concentration of linezolid in the organic phase is 200-300 mg / mL.

[0012] According to embodiments of the present invention, the phospholipid to cholesterol mass ratio in the linezolid liposome inhalation formulation of the present invention is 3:7-9:1. Studies have shown that when the proportion of cholesterol is low, the encapsulation efficiency of linezolid decreases. Further analysis revealed that a low cholesterol content leads to insufficient filling of the liposome bilayer, easily causing drug leakage and thus reducing the liposome encapsulation efficiency. Therefore, the preferred phospholipid to cholesterol mass ratio is 2:3-4:1.

[0013] According to embodiments of the present invention, the liposome particle size in the linezolid liposome inhalation formulation of the present invention is 50-3000 nm. Studies have found that when the liposome particle size is greater than 1000 nm, the liposome encapsulation efficiency is high, and the pressure used for homogenization has little impact on the liposomes. When the liposome particle size is less than 100 nm, the encapsulation efficiency drops sharply. This particle size requires higher homogenization pressure, which damages the liposome structure, leading to drug leakage and a decrease in encapsulation efficiency. Considering the stability of liposomes and the characteristics of inhaled administration, smaller particle size liposomes have better stability and penetrate deeper into the lungs; therefore, the preferred liposome particle size is 100-300 nm.

[0014] According to an embodiment of the present invention, the linezolid lipid formulation of the present invention has a linezolid loading of 1%-40%.

[0015] According to embodiments of the present invention, the linezolid liposome inhalation formulation of the present invention is preferably prepared by a thin-film dispersion method or an ethanol injection method. The prepared linezolid liposomes are concentrated through a polycarbonate membrane column to obtain the linezolid liposome inhalation formulation. Studies have found that, compared with the ethanol injection method, the liposome inhalation formulation prepared by the thin-film dispersion method has a smaller particle size, higher encapsulation efficiency, and better stability. The thin-film dispersion method involves dissolving linezolid, phospholipids, and cholesterol in an organic phase, then removing the organic solvent by thin-film dispersion, adding 0.9% sodium chloride solution for hydration, and finally homogenizing to obtain the linezolid liposome inhalation formulation.

[0016] According to embodiments of the present invention, the linezolid liposome inhalation formulation of the present invention is used for infections caused by a variety of bacteria.

[0017] The beneficial effects of this invention are as follows: Compared with the prior art, this invention prepares a liposomal inhalation formulation of linezolid. Compared with linezolid injection, the encapsulation of linezolid in liposomes allows linezolid to act more precisely on the bacterial outer membrane, inhibiting or destroying the bacterial outer membrane, thereby effectively killing Gram-positive bacteria. The developed linezolid liposomal inhalation formulation has high stability and few side effects. By using inhalation administration, the dosage of the drug is reduced, the adverse reactions caused by systemic administration are reduced, and patient compliance is improved. Attached Figure Description

[0018] Figure 1 The diagram shows the particle size and encapsulation efficiency of linezolid liposome inhalation formulations prepared by different methods.

[0019] Figure 2 Particle size and encapsulation efficiency of linezolid liposome inhalation formulations prepared for different phospholipids.

[0020] Figure 3 Particle size and encapsulation efficiency of linezolid liposome inhalation formulations prepared for different linezolid concentrations.

[0021] Figure 4 Particle size and encapsulation efficiency of linezolid liposome inhalation formulations prepared with different ratios of phospholipids and cholesterol. Detailed Implementation

[0022] Experimental materials: linezolid, phospholipids (lecithin, DMPC, DPPC, DSPC, DOPC), chloroform, ethanol, cholesterol, 0.9% sodium chloride solution, anhydrous sodium dihydrogen phosphate, sodium hydroxide.

[0023] Experimental equipment: Electronic balance (Mettler-Toledo International Trading (Shanghai) Co., Ltd.), rotary evaporator (Huachen Instruments), high pressure homogenizer (Antos Nanotechnology (Suzhou) Co., Ltd.), water bath heating magnetic stirrer (Shanghai Lingke), pH meter (Mettler-Toledo International Trading (Shanghai) Co., Ltd.).

[0024] Example 1

[0025] prescription

[0026]

[0027] Preparation of linezolid liposomes:

[0028] Two parallel samples of 320 mg phospholipid (DPPC), 160 mg cholesterol, and 800 mg linezolid were weighed and dissolved in 4 mL of chloroform. Liposomes were then prepared using different methods.

[0029] Thin-film dispersion method: Take one portion of the dissolved sample above, rotary evaporate it, and after the organic phase is completely removed, add 5 mL of 0.9% sodium chloride solution at 37 degrees Celsius for hydration. Homogenize the hydrated sample to obtain linezolid liposomes. Concentrate the prepared linezolid liposomes through a polycarbonate membrane column to obtain the linezolid liposome inhalation formulation.

[0030] Ethanol injection method: Prepare a phosphate buffer solution with a pH of 6.8. Take 20 mL of the phosphate buffer solution and place it in a water bath with a magnetic stirrer. Stir the solution and control the temperature at 50 degrees Celsius. Take a 5 mL syringe, draw up a portion of the dissolved sample, and slowly add it to the 5 mL phosphate buffer solution. After the addition is complete, continue stirring until the organic solvent has evaporated completely. This yields linezolid liposomes. Concentrate the prepared linezolid liposomes through a polycarbonate membrane column to obtain the linezolid liposome inhalation formulation.

[0031] Liposome characterization: Particle size and potential were measured using a nanoparticle size-Zeta potential analyzer. Encapsulation efficiency was determined by ultrafiltration centrifugation to separate the encapsulated drug from the free drug. The drug concentration in the liposomes and the encapsulated drug was then measured by high performance liquid chromatography, and the encapsulation efficiency was calculated.

[0032] Determination of liposome release rate: Take 0.5 mL of the prepared liposomes into a 10 mL centrifuge tube, add 4.5 mL of physiological saline, shake gently to mix, place in a shaker, 37°C, 50 rpm, and take samples at 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 5 h, 7 h, 12 h, 17 h, 21 h, and 24 h, and determine the encapsulation efficiency according to the method for determination, and calculate the cumulative release rate.

[0033] Determination of liposome stability: Take 5 mL of the prepared liposome sample into a vial and place it in a 40°C stability test chamber. The encapsulation efficiency and particle size are determined at 0 days, 5 days, 15 days and 30 days.

[0034] Effects of liposomes on bacterial outer membranes: Drug-resistant Staphylococcus aureus was cultured, and the bacterial suspension was subjected to a minimum inhibitory concentration (MIC) experiment. The lower the MIC, the more significant the inhibitory effect of the drug on the bacteria, thus indicating that liposomes have a more significant destructive effect on the bacterial outer membrane.

[0035] As shown in Table 1, the linezolid liposome inhalation formulations prepared by the two methods have particle sizes between 150-250 nm, potentials of around -1 mV, and encapsulation efficiencies between 35% and 65%. It can be seen that the linezolid liposome inhalation formulations prepared by the film dispersion method have smaller particle sizes, higher encapsulation efficiencies, greater cumulative release, better stability, and stronger antibacterial effects. Therefore, the film dispersion method is preferred.

[0036] Table 1. Results of linezolid liposome inhalation formulations prepared by different methods

[0037]

[0038] Example 2

[0039] Selection of different phospholipids:

[0040] The thin-film dispersion method described in Example 1 was used, but the types of phospholipids in the formulation of Example 1 were changed. Lecithin, DMPC, DPPC, DSPC, and DOPC were selected for liposome preparation. The particle size, potential, and encapsulation efficiency of the prepared liposomes were determined according to the characterization methods described in Example 1. The cumulative release rate, stability, and minimum inhibitory concentration were also determined according to the methods described in Example 1. The results are shown in Table 2. It can be seen that, except for lecithin, the other types of phospholipids have little effect on the particle size, potential, and encapsulation efficiency of the linezolid liposome inhalation formulation. In addition, using DPPC as the framework material for preparing linezolid liposomes can achieve the lowest inhibitory concentration and the strongest antibacterial effect. The reason for this is that DPPC is a major component of pulmonary surfactant. As an endogenous molecule, it provides unique advantages for drug formulations. At the same time, it has the characteristics of biocompatibility, biodegradability, and high safety.

[0041] Table 2 Results of linezolid liposome inhalation formulations prepared with different phospholipids

[0042]

[0043] Example 3

[0044] Selection of linezolid concentration in the organic phase:

[0045] The thin-film dispersion method described in Example 1 was used, with the concentration of linezolid in the formulation of Example 1 modified to prepare liposomes at different concentrations. The prepared liposomes were then characterized according to the methods described in Example 1, with particle size, potential, and encapsulation efficiency determined. Cumulative release rate, stability, and minimum inhibitory concentration were also determined according to the methods described in Example 1. The results are shown in Table 3. It can be seen that the encapsulation efficiency of the linezolid liposome inhalation formulation gradually increases with increasing linezolid concentration. When the drug concentration is greater than 300 mg / mL, the encapsulation efficiency of the linezolid liposome inhalation formulation no longer changes. To reduce waste of active pharmaceutical ingredient, a linezolid concentration of 200-300 mg / mL in the organic phase is preferred.

[0046] Table 3 Results of linezolid liposome inhalation formulations at different concentrations

[0047]

[0048] Example 4

[0049] Phospholipid to cholesterol mass ratio:

[0050] The thin-film dispersion method described in Example 1 was used, while the mass ratio of phospholipids to cholesterol in the formulation of Example 1 was changed. The mass ratio of phospholipids to cholesterol was selected as 3:7-9:1 for the preparation of linezolid liposome inhalation formulation. The particle size, potential and encapsulation efficiency of the prepared liposomes were determined according to the characterization method described in Example 1. The results are shown in Table 4. It can be seen that when the proportion of cholesterol is small, the encapsulation efficiency of linezolid decreases. This is mainly because the proportion of cholesterol is small, and the filling of the liposome bilayer is insufficient, resulting in drug leakage and thus a decrease in the encapsulation efficiency of liposomes. Therefore, the preferred mass ratio of phospholipids to cholesterol is 2:3-4:1.

[0051] Table 4 Results for different linezolid liposomes

[0052]

[0053] Example 5

[0054] Linezolid liposomes with different particle size ranges:

[0055] The formulation in Example 1 was used, and liposomes with a particle size of 50-3000 nm were prepared using the thin-film dispersion method described in Example 1. The prepared liposomes were characterized according to the methods described in Example 1, and their potential and encapsulation efficiency were determined. The cumulative release rate, stability, and minimum inhibitory concentration were also determined according to the methods described in Example 1. The results are shown in Table 5. It can be seen that when the liposome particle size is greater than 100 nm, the liposome encapsulation efficiency is high. At this time, the pressure used for homogenization has little effect on the liposomes. When the liposome particle size is less than 100 nm, the encapsulation efficiency drops sharply, mainly because this particle size requires a larger homogenization pressure. The larger homogenization pressure damages the liposome structure, causing drug leakage and reducing the encapsulation efficiency. Considering the stability of liposomes and the characteristics of inhaled administration, smaller particle size liposomes have better stability and can penetrate deeper into the lungs. Therefore, a liposome particle size of 100-300 nm is preferred.

[0056] Table 5 Results of liposomes with different particle sizes

[0057]

[0058] Experimental Example

[0059] I. Comparison of drug stability in different dosage forms

[0060] Experimental methods: The thin film dispersion method in Example 1 was used, and linezolid liposome inhalation formulation was prepared according to the formulation in Example 1. Linezolid injection (comparative example 1) was selected from the commercially available linezolid injection. The two samples were placed under high temperature and light conditions for stability study.

[0061] The results are shown in Tables 6 and 7. As can be seen from the tables, the total impurities of linezolid injection (Comparative Example 1) increased after six months of exposure to high temperature and light, while the total impurities of linezolid liposome inhalation preparation (Example 1) remained almost unchanged. This shows that the stability of linezolid liposome inhalation preparation is better than that of linezolid injection.

[0062] Table 6. Stability study of samples with different dosage forms (high temperature)

[0063]

[0064] Table 7. Stability study of samples with different dosage forms (under light).

[0065]

[0066] II. Pharmacodynamic studies of different routes of administration

[0067] Experimental methods:

[0068] The thin-film dispersion method described in Example 1 was used, and linezolid liposomes were prepared according to the formulation in Example 1 to construct a mouse model of Staphylococcus aureus-infected pneumonia. Twenty-four male mice were randomly divided into four groups: control group, blank group, inhalation administration group (Example 1 group), and tail vein injection group (Comparative Example 1 group). The blank group received no treatment but was given 0.9% NaCl solution. The control group, inhalation administration group, and tail vein injection group were instilled with drug-resistant Staphylococcus aureus via pulmonary cannulation to construct a Staphylococcus aureus-infected pneumonia model. The control group was given 0.9% NaCl solution. The Example 1 group received inhalation administration of linezolid liposome solution. The Comparative Example 1 group received tail vein injection of linezolid injection. The dosage of the Comparative Example 1 group was 10 mg / kg, and the dosage of the Example 1 group was 5 mg / kg. Pharmacodynamic evaluation was performed based on mouse survival rate, weight change, and living status.

[0069] As shown in Table 8, inhalation administration can improve the survival rate of mice with pneumonia and enable them to recover their living conditions and weight more quickly. This indicates that inhalation administration is superior to tail vein injection, so inhalation administration is preferred.

[0070] Table 8. Results of pharmacodynamic studies for different routes of administration

[0071]

[0072] III. Studies on the toxicity and side effects of drugs in different dosage forms

[0073] Experimental Methods: The thin-film dispersion method described in Example 1 was used, and linezolid liposome inhalation formulation was prepared according to the formula in Example 1. Commercially available linezolid injection was selected for use. Thirty-six male mice were randomly divided into three groups: control group, linezolid liposome inhalation formulation group (Example 1 group), and linezolid injection group (Comparative Example 1 group). The control group received no treatment and was given 0.9% NaCl solution. The Example 1 group was given linezolid liposome solution, and the Comparative Example 1 group was given linezolid injection via tail vein injection. The dosage for both groups was 10 mg / kg, and the administration was carried out continuously for seven days. After the administration period, three mice from each group were randomly selected, and blood was collected from their eyes for biochemical analysis. The changes in the mice's condition were observed during the administration period.

[0074] The results are shown in Table 9. As can be seen from the table, the blood count of mice in the linezolid liposome inhalation group (Example 1 group) showed no change except for a slight decrease in peripheral erythrocytes. However, the platelet count and peripheral blood cell count of mice in the linezolid injection group (Comparative Example 1 group) both decreased, indicating that the linezolid liposome inhalation preparation has lower toxicity. At the same time, during the dosing period, mice in the linezolid liposome inhalation group (Example 1 group) did not experience significant diarrhea, but mice in the linezolid injection group (Comparative Example 1 group) experienced diarrhea and insomnia, indicating that the linezolid liposome inhalation group had fewer side effects.

[0075] Table 9. Toxicity and Side Effects Results of Different Dosage Forms

[0076]

Claims

1. A linezolid liposome inhalation formulation, characterized in that, The liposomes are composed of phospholipid bilayer vesicles made up of phospholipids and cholesterol, with linezolid in the organic phase and embedded in the phospholipid bilayer.

2. The linezolid liposome inhalation formulation according to claim 1, characterized in that, The phospholipid is selected from one or more of lecithin, DMPC, DPPC, DSPC, and DOPC, with DPPC being preferred.

3. The linezolid liposome inhalation formulation according to claim 1, characterized in that, The linezolid liposome inhalation formulation for treating Gram-positive bacteria, wherein the concentration of linezolid in the organic phase is 50-600 mg / mL, preferably 200-300 mg / mL.

4. The linezolid liposome inhalation formulation according to claim 1, characterized in that, The mass ratio of phospholipids to cholesterol is 3:7-9:

1.

5. A linezolid liposome inhalation formulation according to claim 4, characterized in that, The mass ratio of phospholipids to cholesterol is 2:3-4:

1.

6. The linezolid liposome inhalation formulation according to claim 1, characterized in that, The liposomes have a particle size of 50-3000 nm.

7. A linezolid liposome inhalation formulation according to claim 6, characterized in that, The liposomes have a particle size of 100-300 nm.

8. The linezolid liposome inhalation formulation according to claim 1, characterized in that, The linezolid loading is 1%-40%.

9. A method for preparing a linezolid liposome inhalation formulation, wherein the preparation method is selected from the thin-film dispersion method or the ethanol injection method, and the method involves concentrating the prepared linezolid liposomes through a polycarbonate membrane column to obtain the linezolid liposome inhalation formulation.

10. The method for preparing a linezolid liposome inhalation formulation according to claim 9, wherein the preparation method is selected from the thin-film dispersion method.

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