Metronidazole phospholipid composition as well as preparation method and application thereof

Abstract

The invention discloses a metronidazole phospholipid composition as well as a preparation method and application thereof, and relates to the technical field of biological medicines, and the metronidazole phospholipid composition comprises metronidazole and phospholipid. The metronidazole phospholipid composition prepared by the invention improves the intracellular drug delivery efficiency under the condition of retaining the original chemical structure and biological activity of metronidazole, and a new method is provided for treatment of intracellular infection of anaerobic bacteria. The problem that existing metronidazole is poor in intracellular infection treatment effect is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a metronidazole phospholipid composition, its preparation method, and its application. Background Technology

[0002] Metronidazole is a widely used clinical anti-anaerobic drug, particularly effective against various anaerobic bacteria such as Porphyromonas gingivalis and Bacteroides. It works by interacting with bacterial DNA, inhibiting its synthesis, and ultimately leading to bacterial death. Therefore, it is widely used to treat various infections caused by anaerobic bacteria, including infections of the oral cavity, abdominal cavity, and intestines.

[0003] Although metronidazole is highly effective in treating anaerobic infections, its efficacy against intracellular anaerobes is limited. Metronidazole primarily relies on passive diffusion to enter host cells, a slow and inefficient process that limits its effectiveness against intracellular anaerobes. In many clinical situations, anaerobes can enter host cells through endocytosis or other mechanisms, leading to intracellular infection. Traditional metronidazole treatment often fails to effectively eliminate these intracellular pathogens due to low drug delivery efficiency. Furthermore, there are currently no reports on improving the intracellular delivery efficiency of metronidazole and enhancing its anti-intracellular efficacy through the preparation of metronidazole phospholipid compositions. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a metronidazole phospholipid composition, its preparation method, and its application, thereby resolving the issue of poor efficacy of existing metronidazole treatment for intracellular infections.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a metronidazole phospholipid composition is provided, comprising metronidazole and phospholipid.

[0006] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the molar ratio of metronidazole to phospholipid is 1:(0.1-5).

[0007] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: Furthermore, the molar ratio of metronidazole to phospholipid is 1:(1-3).

[0008] Furthermore, the molar ratio of metronidazole to phospholipid is 1:(0.5-3).

[0009] Furthermore, the molar ratio of metronidazole to phospholipids is 1:1. Furthermore, the molar ratio of metronidazole to phospholipids is 1:2.

[0010] Furthermore, the phospholipids are natural phospholipid components or phospholipids obtained through synthesis.

[0011] Furthermore, the phospholipids are soybean lecithin, egg yolk lecithin, DSPE, DPPE, DSPC, DPPC, DSPG, DPPA, DOTAP, DEPC, DPPG, or DSPE-MPEG2000.

[0012] Furthermore, the phospholipids are soybean lecithin, egg yolk lecithin, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearyl-sn-glycerol-3-phosphate glycerol (DSPG), and 1 2-Dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dierucic acid-sn-glycerol-3-phosphate choline (DEPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate glycerol (DPPG) or 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-MPEG2000).

[0013] The present invention also provides a method for preparing the above-mentioned metronidazole phospholipid composition, comprising the following steps: (1) Dissolve metronidazole and phospholipids in an organic solvent, and then stir at 20-60℃ to obtain a mixed solution; (2) Remove the organic solvent from the mixed solution obtained in step (1) under reduced pressure to obtain the metronidazole phospholipid composition.

[0014] Furthermore, in step (1), the organic solvent is at least one of ethyl acetate, acetone, chloroform, dichloromethane, tetrahydrofuran, n-hexane, ethanol, and methanol. Furthermore, in step (1), the mass-to-volume ratio of metronidazole to organic solvent is 45 mg: 4.5-5.5 mL.

[0015] Furthermore, in step (1), the mass-to-volume ratio of metronidazole to organic solvent is 45 mg: 5 mL.

[0016] Furthermore, in step (1), the mixture is stirred at 20-60℃ for 2-12 hours.

[0017] Furthermore, in step (1), the mixture is stirred at 20-60℃ for 2-4 hours.

[0018] Furthermore, in step (1), the mixture is stirred at 50-60℃ for 2-4 hours.

[0019] Furthermore, in step (1), the mixture is stirred at 55°C for 2 hours.

[0020] Furthermore, in step (1), magnetic stirring is performed.

[0021] Furthermore, in step (2), the pressure is reduced for 30 minutes at 50-55℃.

[0022] Furthermore, in step (2), the pressure is reduced for 30 minutes at 50°C.

[0023] The present invention also provides the use of the above-mentioned metronidazole phospholipid composition in the preparation of antibacterial agents for intracellular anaerobic bacterial infections.

[0024] The present invention also provides an antibacterial agent for intracellular anaerobic bacterial infections, the active ingredient of which includes the above-mentioned metronidazole phospholipid composition.

[0025] Furthermore, anaerobic bacteria include Porphyromonas gingivalis, Bacteroides, Streptococcus lactis, Klebsiella pneumoniae, Forsbothrium fossa, anaerobic streptococci, Helicobacter pylori, or anaerobic Campylobacter.

[0026] The present invention has the following beneficial effects: 1. Metronidazole has a significant bactericidal effect against anaerobic bacteria. However, when anaerobic bacteria enter eukaryotic cells, metronidazole can only passively diffuse into the cell, resulting in extremely low cell entry efficiency and an inability to effectively kill intracellular anaerobic bacteria. The metronidazole phospholipid composition prepared in this invention, while retaining the original chemical structure and biological activity of metronidazole, improves the efficiency of intracellular drug delivery, providing a new method for the treatment of intracellular infections caused by anaerobic bacteria.

[0027] 2. Improved intracellular delivery efficiency: Metronidazole, upon binding with phospholipids, significantly enhances drug delivery efficiency in eukaryotic cells, overcoming the low cell entry efficiency caused by passive diffusion when used alone. This composition also has advantages in cell membrane penetration, effectively enhancing intracellular drug accumulation and improving its bactericidal effect against intracellular anaerobic bacteria.

[0028] 3. Preservation of drug bioactivity: The metronidazole phospholipid composition retains the original chemical structure and bioactivity of metronidazole, enabling the drug to enter target cells more effectively. This design not only ensures the original efficacy of the drug but also improves its intracellular stability and effectiveness, avoiding the loss of drug performance.

[0029] 4. A novel treatment method for intracellular anaerobic bacteria: This metronidazole phospholipid composition provides a new strategy for treating intracellular anaerobic bacterial infections, particularly suitable for anaerobic bacteria capable of invading eukaryotic cells, such as *Porphyromonas gingivalis*. By improving the intracellular delivery capability of the drug, this invention can more effectively eliminate anaerobic pathogens within host cells and reduce recurrence of infection.

[0030] In summary, the metronidazole phospholipid composition provided by this invention can significantly improve the therapeutic effect of the drug and expand its clinical application scope, representing an innovative treatment method for anaerobic bacteria. Attached Figure Description

[0031] Figure 1 This is a comparison chart of the cellular uptake of the composition prepared in Example 2 and metronidazole. Figure 2 The intracellular bacterial inactivation rate of the composition prepared in Example 2 after treatment with metronidazole for 6 hours; Figure 3 The intracellular bacterial inactivation rate is shown for the composition prepared in Example 2 after treatment with metronidazole for 12 hours. Detailed Implementation

[0032] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0033] Example 1: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:1.

[0034] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 199.3mg soybean lecithin, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically at 55℃ for 2h to obtain a transparent clear liquid, thus preparing a mixed solution; (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) at 55°C for 30 min under reduced pressure to obtain metronidazole phospholipid composition.

[0035] Example 2: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:2.

[0036] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 398.6mg soybean lecithin, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically at 55℃ for 12h to obtain a transparent clear liquid, thus preparing a mixed solution; (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) under reduced pressure at 50°C for 30 min to obtain metronidazole phospholipid composition.

[0037] Example 3: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:2.

[0038] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 398.6mg egg yolk lecithin, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically for 4h at 60℃ to obtain a transparent clear liquid and prepare a mixed solution; (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) under reduced pressure at 50°C for 30 min to obtain metronidazole phospholipid composition.

[0039] Example 4: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:1.

[0040] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 207.7mg DSPC, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically for 2h at 50℃ to obtain a transparent clear liquid and prepare a mixed solution; (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) at 55°C for 30 min under reduced pressure to obtain metronidazole phospholipid composition.

[0041] Example 5: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:3.

[0042] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 623.2mg DSPC, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically at 55℃ for 10h to obtain a transparent clear liquid and prepare a mixed solution; (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) at 55°C for 30 min under reduced pressure to obtain metronidazole phospholipid composition.

[0043] Example 6: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:3.

[0044] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45mg metronidazole and 217.1mg DPPA, dissolve them together in 5mL of anhydrous ethanol, and then stir magnetically at 55℃ for 10h to obtain a transparent clear liquid and prepare a mixed solution. (2) Remove anhydrous ethanol from the mixed solution obtained in step (1) at 55°C for 30 min under reduced pressure to obtain metronidazole phospholipid composition.

[0045] Example 7: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:0.1.

[0046] A method for preparing a metronidazole phospholipid composition includes the following steps: (1) Take 45 mg of metronidazole and 19.9 mg of soybean lecithin, dissolve them together in 4.5 mL of ethyl acetate, and then stir magnetically for 12 h at 20 °C to obtain a clear and transparent solution, thus preparing a mixed solution; (2) Remove ethyl acetate from the mixed solution obtained in step (1) under reduced pressure at 55°C for 30 min to obtain metronidazole phospholipid composition.

[0047] Example 8: A metronidazole phospholipid composition comprising metronidazole and phospholipid, wherein the molar ratio of metronidazole to phospholipid is 1:5.

[0048] A metronidazole phospholipid composition containing 996.5 mg of soybean lecithin was dissolved together in 5.5 mL of acetone, and then magnetically stirred at 60 °C for 2 h to obtain a transparent clear liquid, thus preparing a mixed solution. (2) The mixed solution obtained in step (1) was subjected to reduced pressure at 55°C for 30 min to remove acetone, thereby obtaining the metronidazole phospholipid composition.

[0049] Test case I. Cellular drug uptake The metronidazole phospholipid complex (MPC) and metronidazole (MTR) prepared in Example 2 were co-incubated with cells, and the amount of drug taken up by the cells was detected. The specific detection method was as follows: MPC and MTR were dissolved in culture medium and diluted to concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, to obtain drug-containing culture media; HOK cells were cultured in keratinocyte-specific culture medium in a 5% CO2, 37°C incubator; HOK cells were seeded into 12-well plates, and the experiment was started when the cell confluence reached 80%; the original culture medium in the well plates was aspirated, the plates were washed once with PBS, and the prepared drug-containing culture media were added, and co-incubated for 12 h; the supernatant of the culture medium was collected, and the volume was adjusted to the original volume (to eliminate interference from liquid evaporation), diluted with acetonitrile, centrifuged (15000 rcf, 5 min), and the supernatant was collected. The drug concentration in the supernatant was determined by HPLC. The amount of drug entering the cells was calculated using the following formula: Drug penetration (%) = (Pre-treatment concentration of drug - Post-treatment concentration of drug) / Pre-treatment concentration of drug See results Figure 1 .

[0050] Depend on Figure 1 It is evident that the metronidazole phospholipid composition of the present invention can significantly increase the amount of metronidazole entering cells.

[0051] II. Intracellular bacterial inactivation rate 1. After treating intracellular *Porphyromonas gingivalis* with the metronidazole phospholipid composition (MPC) and metronidazole (MTR) prepared in Example 2, the intracellular bacterial inactivation rate was detected. The specific detection method was as follows: MPC and MTR were dissolved in the culture medium and diluted to concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL to obtain the prepared drug-containing culture medium. HOK cells were cultured in a 5% CO2, 37°C incubator using keratinocyte-specific medium. HOK cells were seeded into 12-well plates, and experiments were started when cell confluence reached 80%. The original medium was aspirated from the plates, and the cells were washed with PBS. *Porphyromonas gingivalis* was added at an MOI of 100. After 2 hours of infection, extracellular bacteria were washed away, and 200 μg / mL metronidazole and 300 μg / mL gentamicin were added to remove any remaining extracellular bacteria. The cells were incubated for 1 hour, followed by washing with PBS. Prepared drug-containing medium was added, and the cells were incubated for 6 or 12 hours. The drugs were removed, the cells were washed with PBS, and water was added to lyse the cells and release intracellular bacteria. The cells were then plated on blood agar plates, anaerobically cultured, and CFU counted. The inactivation rate was calculated using the following formula: Inactivation rate = (N0 - N) / N0 × 100% Where N0 is the number of colony-forming units (CFU) in the control group and N is the number of colony-forming units in the experimental group.

[0052] See results Figures 2-3 .

[0053] Depend on Figures 2-3 It is known that the antibacterial effect of metronidazole against intracellular bacteria is very limited at all concentrations; however, the metronidazole phospholipid composition (MPC) prepared by the method of this invention showed an inhibition rate of 29.29% at 50 μg / mL, 46.77% at 100 μg / mL, and 60.02% at 200 μg / mL after 6 hours of treatment with intracellular bacteria; after 12 hours of treatment with intracellular bacteria, the inhibition rate was 37.12% at 50 μg / mL, 83.19% at 100 μg / mL, and 92.14% at 200 μg / mL.

[0054] 2. After treating intracellular *Porphyromonas gingivalis* with the metronidazole phospholipid composition (MPC) prepared in Example 1, the intracellular bacterial inactivation rate was detected. The specific detection method was as follows: MPC was dissolved in the culture medium and diluted to a concentration of 200 μg / mL to obtain the prepared drug-containing culture medium. HOK cells were cultured in a 5% CO2, 37°C incubator using keratinocyte-specific medium. HOK cells were seeded into 12-well plates, and experiments were started when cell confluence reached 80%. The original medium was aspirated from the plates, and the cells were washed with PBS. *Porphyromonas gingivalis* was added at an MOI of 100. After 2 hours of infection, extracellular bacteria were washed away, and 200 μg / mL metronidazole and 300 μg / mL gentamicin were added to remove any remaining extracellular bacteria. The cells were incubated for 1 hour, followed by washing with PBS. Prepared drug-containing medium was added, and the cells were incubated for a total of 6 hours. The drugs were removed, the cells were washed with PBS, and water was added to lyse the cells and release intracellular bacteria. The cells were then plated on blood agar plates, anaerobically cultured, and CFU counted. The inactivation rate was calculated using the following formula: Inactivation rate = (N0 - N) / N0 × 100% Where N0 is the number of colony-forming units (CFU) in the control group and N is the number of colony-forming units in the experimental group.

[0055] When the metronidazole phospholipid composition (MPC) prepared in Example 1 was applied at a concentration of 200 μg / mL to intracellular bacteria for 6 hours, the inactivation rate of the intracellular bacteria was 50.87%.

[0056] In summary, the preparation method of the metronidazole phospholipid composition of the present invention is simple, can significantly improve the cell entry efficiency of metronidazole and the antibacterial effect against intracellular anaerobic bacteria, and can effectively broaden the application range of metronidazole.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A metronidazole phospholipid composition, characterized in that, Including metronidazole and phospholipids.

2. The metronidazole phospholipid composition according to claim 1, characterized in that, The molar ratio of metronidazole to phospholipid is 1:(0.1-5).

3. The metronidazole phospholipid composition according to claim 1, characterized in that, The molar ratio of metronidazole to phospholipid is 1:(0.5-3).

4. The metronidazole phospholipid composition according to claim 1, characterized in that, The molar ratio of metronidazole to phospholipids is 1:

1.

5. The metronidazole phospholipid composition according to claim 1, characterized in that, The phospholipids are natural phospholipid components or phospholipids obtained through synthesis.

6. The metronidazole phospholipid composition according to claim 5, characterized in that, The phospholipids are soybean lecithin, egg yolk lecithin, DSPE, DPPE, DSPC, DPPC, DSPG, DPPA, DOTAP, DEPC, DPPG, or DSPE-MPEG2000.

7. A method for preparing the metronidazole phospholipid composition according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Dissolve metronidazole and phospholipids in an organic solvent, and then stir at 20-60℃ to obtain a mixed solution; (2) Remove the organic solvent from the mixed solution obtained in step (1) under reduced pressure to obtain the metronidazole phospholipid composition.

8. The method for preparing the metronidazole phospholipid composition according to claim 7, characterized in that, In step (1), the organic solvent is at least one of ethyl acetate, acetone, chloroform, dichloromethane, tetrahydrofuran, n-hexane, ethanol and methanol.

9. The use of the metronidazole phospholipid composition according to any one of claims 1-6 in the preparation of an antibacterial agent for intracellular anaerobic bacterial infections.

10. An antibacterial agent for intracellular anaerobic bacterial infections, characterized in that, The active ingredient includes the metronidazole phospholipid composition according to any one of claims 1-6.

Images