CN113616597A

CN113616597A - Meloxicam injection and preparation method and application thereof

Info

Publication number: CN113616597A
Application number: CN202110879176.9A
Authority: CN
Inventors: 周玉玉; 魏晓慧
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-11-09

Abstract

The invention provides a meloxicam injection, a preparation method and application thereof, wherein the injection comprises the following raw material components: the preparation comprises meloxicam, an external aqueous phase and liposomes, wherein the liposomes contain an internal aqueous phase, the internal aqueous phase is a salt solution containing metal ions, and the meloxicam can form an insoluble salt with the metal ions and is stably loaded in the liposome internal aqueous phase. The injection has high drug loading and good storage stability, and can quickly release the drug after intravenous injection into blood circulation to play the role of analgesia; can also be used for local injection of joint cavity, etc., to improve local analgesic and antiinflammatory effects, and reduce systemic administration dosage.

Description

Meloxicam injection and preparation method and application thereof

Technical Field

The invention relates to the field of pharmacy, in particular to a meloxicam injection and a preparation method and application thereof.

Background

Postoperative pain (postsurgical pain) is acute pain that occurs after surgery. The active adoption of effective postoperative analgesia measures to relieve pain is a key link for accelerating recovery and improving the comfort level and the life quality of patients. (clinical pharmacist postoperative pain management guide, guangdong institute of medicine, 2019.1) at present, opioids are the most commonly used clinical drugs for treating moderate and severe pain, and exert analgesic effect by binding with peripheral and central nervous system (spinal cord and brain) opioid receptors. The strong opiates comprise morphine, fentanyl, pethidine, sufentanil and the like, and have the advantages of strong analgesic effect, no organ toxicity and capping effect. But opioids may cause respiratory depression, nausea, vomiting, ileus, etc. At the same time, opioids have potential addiction and dependence. For this reason, non-opioid drugs have been the focus of post-operative analgesia, particularly post-operative acute pain control studies.

Meloxicam (MLX) is a novel nonsteroidal anti-inflammatory drug (NSAIDs) marketed in south africa by the company brigreger, germany in 1996. As a cyclooxygenase-2 (COX-2) selective inhibitor, meloxicam has excellent anti-inflammatory and analgesic effects, and is widely used for treating osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and other muscular or skeletal pains (e.g., lumbago), and also for relieving minor and moderate postoperative pain. Because the inhibitory activity of meloxicam on COX-2 is greater than that on cyclooxygenase-1 (COX-1). Thus, meloxicam has fewer side effects on blood (platelets), gut, kidney, and cardiovascular, etc. than COX-1 inhibitors. Compared with traditional NSAIDs such as naproxen, ibuprofen, piroxicam and the like, the meloxicam has similar anti-inflammatory and analgesic effects, and the gastrointestinal tolerance is obviously improved. The patients have few gastrointestinal perforation, bleeding and ulcer during the administration period, and have less nausea and vomiting. Therefore, meloxicam is expected to be a non-opioid analgesic drug for postoperative moderate and severe pain control, but high lipid solubility (LogP ═ 2.47) and extremely low water solubility lead to long peak reaching time of meloxicam after oral administration, and the peak of blood concentration can be reached after about 4-5 hours, thus limiting the application of meloxicam in acute moderate and severe pain.

In order to accelerate the onset of meloxicam, researchers have developed a number of different formulations. In terms of systemic analgesia, only one meloxicam nanocrystal injection (trade name Anjeso) is currently on the market. Is administered by intravenous injection once daily for postoperative analgesia. The nano-crystal injection can take effect quickly after being injected into veins rapidly, and can obviously relieve moderate to severe pain. But the rapid intravenous injection and the physical instability of the nanocrystal make the nanocrystal potentially threatening. In the aspect of local analgesic preparation, the preparation mainly comprises meloxicam transdermal drug delivery preparation, such as gel, liposome, patch, microemulsion and the like. The transdermal drug delivery preparation can maintain a certain drug concentration and lower plasma concentration at the local drug delivery part, avoid the stimulation to the gastrointestinal tract and reduce the adverse reaction of the whole body. Transdermal delivery, however, has difficulty overcoming the stratum corneum barrier of the skin and the high lipid solubility of meloxicam also affects the amount of drug that reaches the dermis. Only one meloxicam transdermal absorbent was successfully marketed by 2016. (Jianmin Chen, Yunhua Gao. strategies for memory Delivery to and across the skin: a review [ J ]. Drug Delivery,2016,23 (8)).

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a meloxicam injection, a preparation method and a use thereof, which are used to solve the problems of the prior art.

To achieve the above objects and other related objects, the present invention is achieved by the following technical solutions.

The invention provides a liposome for loading meloxicam, wherein an internal water phase is contained in the liposome, the internal water phase is a salt solution containing metal ions, and the meloxicam can be complexed with the metal ions.

Preferably, the metal ion is selected from calcium ion, zinc ion, copper ion, magnesium ion, manganese ion or cobalt ion.

Preferably, the pH value of the salt solution is 6.0-8.0.

Preferably, the liposomes comprise one or both of phospholipids and cholesterol.

The invention provides a meloxicam injection, which comprises the following raw material components: meloxicam, an external aqueous phase and liposomes as described above; the external aqueous phase contains at least water.

Preferably, the concentration of the meloxicam is more than or equal to 0.01mg/mL based on the volume of the injection; and/or the external aqueous phase further comprises one or more of meglumine, sucrose or glucose.

Preferably, the meloxicam is loaded within the liposome.

The invention also discloses a preparation method of the injection, which is to mix the meloxicam, the external aqueous phase and the liposome.

The invention also discloses the application of the injection as an analgesic for injection.

The invention also discloses an anti-inflammatory preparation, which comprises the injection and the cytotoxic drug or the injection and the immune drug.

Preferably, the cytotoxic drug is doxorubicin or doxorubicin hydrochloride.

Preferably, the cytotoxic drug and/or the immuno-drug is loaded within the liposome.

The invention also discloses application of the anti-inflammatory preparation in preparing a medicament for treating tumors.

The injection has high drug loading and good storage stability, and after intravenous injection enters blood circulation, meloxicam can be quickly released to play a role of relieving pain of the whole body; can also be used for local injection of articular cavity, etc., to maintain local drug concentration for a long time, improve local analgesic and anti-inflammatory effects, and reduce the dosage of systemic drug administration.

Drawings

FIG. 1 is a standard curve of an aqueous solution of Meloxicam (MLX).

Figure 2 precipitation of meloxicam and a solution of a metal ion salt formed upon mixing.

Figure 3 cryo-transmission electron micrograph of meloxicam liposomes with calcium acetate as internal aqueous phase.

Figure 4 entrapment efficiency, storage stability and release profile of meloxicam liposomes with calcium acetate as the inner aqueous phase.

Figure 5 entrapment efficiency, storage stability and release profile of meloxicam liposomes with calcium chloride as the inner aqueous phase.

Figure 6 entrapment efficiency, storage stability and release profile of meloxicam liposomes with zinc acetate as the internal aqueous phase.

Figure 7 entrapment efficiency, storage stability and release profile of meloxicam liposomes with copper sulfate as the internal aqueous phase.

Figure 8 entrapment efficiency, storage stability and release profile of meloxicam liposomes with magnesium chloride as the internal aqueous phase.

Figure 9 entrapment efficiency, storage stability and release profile of meloxicam liposomes with cobalt chloride as the internal aqueous phase.

Figure 10 entrapment efficiency, storage stability and release profile of meloxicam liposomes with manganese chloride as the internal aqueous phase.

FIG. 11 shows the encapsulation efficiency of meloxicam and doxorubicin liposome in which manganese chloride is used as the inner aqueous phase.

FIG. 12 shows the encapsulation efficiency of meloxicam-doxorubicin co-entrapped liposomes with manganese chloride as the inner aqueous phase.

Figure 13 inhibition of meloxicam-doxorubicin co-loaded liposomes on (doxorubicin-resistant) K562 cells.

Figure 14 simulates the cumulative release rate of liposomes in aqueous phase for different local injections.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The present invention contemplates providing a liposomal meloxicam formulation that can be used for injection. Different from the prior meloxicam liposome prepared by a film dispersion method, the meloxicam liposome preparation provided by the method adopts an active drug loading method, and combines the meloxicam with metal ions in an inner water phase of the liposome to form insoluble salt (precipitate), so that the meloxicam can effectively enter the inner water phase of the liposome, and the purpose of stable loading is achieved.

The application firstly provides a liposome for loading meloxicam, wherein an inner water phase is contained in the liposome, the inner water phase is a salt solution containing metal ions, and the meloxicam can be complexed with the metal ions to form insoluble salts. Preferably, the metal ion is selected from calcium ion, zinc ion, copper ion, magnesium ion, manganese ion or cobalt ion. Preferably, the pH value of the salt solution is 6.0-8.0. Preferably, the liposomes comprise one or both of phospholipids and cholesterol. The applicant in the application unexpectedly finds that the liposome has higher drug loading capacity of meloxicam and good storage stability.

The application also discloses a meloxicam injection, which comprises the following raw material components: meloxicam, an external aqueous phase and liposomes as described above; the external aqueous phase contains at least water. Preferably, the concentration of meloxicam is greater than or equal to 0.01mg/mL based on the volume of the injection. Preferably, the external aqueous phase further comprises one or more of meglumine, sucrose or glucose.

Preferably, the meloxicam is loaded within the liposome.

The preparation method of the injection comprises the following steps: the meloxicam, the external aqueous phase and the liposomes are mixed.

Upon bulk dilution (e.g., intravenous injection), meloxicam can be rapidly released for the control of acute pain and systemic analgesia of moderate to severe pain; upon local injection, e.g., into the joint cavity, the meloxicam liposomes can be slowly released, maintain local drug concentrations over a longer period of time, improve local analgesic and anti-inflammatory effects, and reduce the amount of systemic administration.

In addition, new studies have shown that inflammatory factors such as tumor necrosis factor (TNF- α) and interleukin-6 (IL-6) in the tumor microenvironment influence the penetration of immune cells such as T cells into tumors (e.g., Jose R.Conejo-Garcia, Breaking barriers for T cells by targeting the EPHA2/TGF-b/COX-2axis in secretory cancer, J Clin invest.2019; 129(9):3521-3523) or influence the resistance of tumor cells to cytotoxic drugs, etc., through complex signaling pathways, thereby reducing the effects of tumor immunotherapy or chemotherapy. Considering that the other main effect of the meloxicam is to play an anti-inflammatory role by inhibiting COX-2, the stably drug-loaded meloxicam liposome provided by the invention is expected to improve the effect of immunotherapy or chemotherapy by correcting the tumor inflammatory microenvironment through the combined use of cytotoxic drugs or immune drugs.

In a specific embodiment, the cytotoxic drug is doxorubicin or doxorubicin hydrochloride.

In a preferred embodiment, the cytotoxic drug and/or the immunological drug is loaded within the liposome.

It is also disclosed in the present application that the specific preparation method of the liposome can be prepared by referring to the conventional coating method in the prior art. Specifically, the following method may be employed: mixing the organic solvent solution of lipid with the internal water phase to obtain liposome suspension; the metal salts in the external aqueous phase of the liposomes were then removed by dialysis. The pH of the general external water phase is 6-7. Thus, the liposome has a pH gradient and a metal salt concentration gradient between the internal and external aqueous phases. In a preferred embodiment, the temperature for preparing the liposomal suspension is 45-85 ℃.

Example 1

This example is a method for detecting meloxicam.

In this example, a standard curve of meloxicam was established, as follows.

An Ultraviolet (UV) detection analysis method is adopted: the detecting instrument is TECAN

200 PRO; the detection wavelength is 362 nm; the detection temperature is 25 ℃; the detection orifice plate is

96well plates, UV-transparent; the detection volume was 200. mu.l.

(1) 2mg of meloxicam is precisely weighed and added into 2mL of sodium hydroxide solution (pH value is 10), and the mixture is swirled until the mixture is completely dissolved, so that 1mg/mL of meloxicam stock solution is prepared.

(2) The meloxicam aqueous solution is mixed with ultrapure water and is subjected to gradient dilution to obtain meloxicam standard solutions with the concentrations of 5 mu l/mL, 10 mu l/mL, 15 mu l/mL, 20 mu l/mL, 30 mu l/mL and 40 mu l/mL. The meloxicam standard solution with the concentration is detected by a UV method, and the detection result is shown in Table 1.

TABLE 1 UV detection values of meloxicam aqueous solutions of different concentrations

A standard curve was established based on the results shown in Table 1. The operating curve is shown in figure 1. The UV standard curve for the aqueous meloxicam solution is 0.0306X +0.0126(n is 6) and R2 is 1, which fit well.

Example 2

This example is the solubilization of meloxicam and the formation of insoluble salts of meloxicam with metal ions.

2.1 solubilization of Meloxicam by hydroxypropyl-. beta. -Cyclodextrin (HPCD)

Dissolving a certain amount of HPCD (Shanghai Fengshi Biotechnology Co., Ltd.) with deionized water to prepare HPCD solutions with the concentrations of 0%, 10%, 20% and 30%. 2mL of HPCD solutions of different concentrations were added to meloxicam (2 mg each), vortexed for 5min and shaken on a 37 ℃ homomixer (ThermoMixer C, Eppendorf) for 24 h. After being taken out, the solution is centrifuged, the ultraviolet absorption value of the meloxicam in the supernatant solution is measured, and the solubility of the meloxicam in HPCD solutions with different concentrations is calculated according to the working curve in the example 1.

2.2 solubilization of Meloxicam by meglumine

Meglumine used in this example was purchased from carbofuran technologies ltd

Appropriate amounts of meglumine and excess meloxicam were weighed out to prepare solutions containing 10mg/mL, 20mg/mL, 30mg/mL of meglumine and shaken at 500rpm for 24h at 37 ℃. After being taken out, the mixture is centrifuged, the ultraviolet absorption value of the meloxicam in the supernatant solution is measured, and the solubility of the meloxicam in meglumine solutions with different concentrations is calculated according to the working curve in the example 1.

TABLE 2 saturated solubilities of meloxicam in solutions of different solubilizers

As shown in table 2, HPCD had some solubilization of meloxicam, and the higher the concentration of HPCD, the stronger the solubilization. The solubilization of meloxicam by meglumine is much greater than that of HPCD.

2.3 Meloxicam solution binds to the metal ion, forming an insoluble precipitate

Preparing meglumine-solubilized meloxicam solution (at a concentration of about 16mg/mL) according to the method of 2.2; preparing 300mM calcium acetate, calcium chloride, zinc acetate, copper sulfate, magnesium chloride, cobalt chloride and manganese chloride solution. Meloxicam solution (50. mu.l) was added to each metal ion solution (1mL) separately. Meloxicam is able to bind to these metal ions to form insoluble precipitates (FIG. 2)

Example 3

This example is the preparation and characterization of meloxicam liposomes with calcium acetate solution as the internal aqueous phase.

The phospholipids used in this example were all purchased from Lipoid, germany. Specifically hydrogenated soybean phospholipids (HSPC, molecular weight 783.8); the PEGylated phospholipid is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG 2000); cholesterol (CHOL, molecular weight 386.7); the meloxicam used was purchased from echiei (shanghai) chemical industry development limited.

In the following examples, the phospholipids used were hydrogenated soybean phospholipids from Lipoid corporation; the used PEGylated phospholipids are distearoyl phosphatidyl ethanolamine-polyethylene glycol of Lipoid company; the cholesterol used is Lipoid cholesterol; the meloxicam used was purchased from Chishiai (Shanghai) chemical industry development Co., Ltd.

3.1 preparation of Meloxicam liposomes with calcium acetate solution as the internal aqueous phase

The preparation method comprises the following steps:

(1) respectively preparing calcium acetate aqueous solutions with the concentrations of 150mM, 300mM and 500mM, and adjusting the pH value to 8.0 by hydrochloric acid;

(2) melting hydrogenated soybean phospholipid, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and cholesterol in absolute ethanol according to a molar ratio of 3:1:1 to obtain an ethanol mixed solution of the lipid;

(3) adding 3mL of the calcium acetate solution prepared in the step (1) into the ethanol mixed solution of the lipid obtained in the step (2), placing the solution in a stirring water bath at 60 ℃ for 30 minutes, and fully hydrating the lipid to obtain a more uniform liposome suspension;

(4) sequentially extruding the liposome suspension obtained in the step (3) through a carbon film with a specific aperture by using a liposome extruder, and further controlling the particle size and uniformity of blank liposomes;

(5) putting the liposome prepared in the step (4) into a dialysis bag with the molecular weight cutoff of 10KDa, taking a 10% sucrose aqueous solution which is isotonic with a human body as a dialysis medium, wherein the volume ratio of a sample to the dialysis medium is 1:1000, dialyzing, removing calcium acetate in an external aqueous phase of the liposome to obtain an external aqueous phase consisting of 10% sucrose, an internal aqueous phase consisting of the calcium acetate solution and a blank liposome consisting of phospholipid bilayers, wherein the internal aqueous phase of the liposome has certain pH and calcium acetate concentration gradient. Specifically, the inner aqueous phase of the lipid bilayer is a calcium acetate aqueous solution, and the outer aqueous phase of the lipid bilayer is a sucrose aqueous solution (pH is 6-7).

(6) An aqueous solution of meglumine containing 8mg/mL of meloxicam was prepared according to the method of meloxicam-meglumine solution (1: 1 molar ratio) prepared in example 2.

(7) Mixing appropriate amount of meloxicam solution with blank liposome according to the drug-lipid ratio of 0.05, 0.1, 0.2, incubating at 60 deg.C for 30min to obtain meloxicam liposome solution.

3.2. Characterization of Meloxicam liposome with calcium acetate solution as internal water phase

3.2.1 particle size of Meloxicam liposomes with calcium acetate solution as internal aqueous phase

The prepared meloxicam liposomes were diluted 100-fold with deionized water, and the particle size of the liposomes was measured using Zetasizer ZS90 (marvens, uk). As shown in Table 3, the meloxicam liposomes had particle sizes of about 100nm and particle size distributions (PDI) of less than 0.1.

3.2.2 morphology of Meloxicam Liposome with calcium acetate solution as internal aqueous phase

Approximately 5. mu.l of meloxicam liposomes (drug to lipid ratio 0.1) prepared with 300mM calcium acetate solution as the internal aqueous phase were added dropwise to 300 mesh Lacy copper mesh (TedPella, USA). Excess solution was sucked off and quickly taken up in liquid ethane. The morphology of the liposomes was observed by cryo-transmission electron microscopy (FEITalos, Thermo Co., USA) at a voltage of 120 kv. The results are shown in FIG. 3 (blank liposomes, C drug-loaded liposomes). The meloxicam liposome is spherical, and the average grain diameter is about 100 nm; some liposomes have "bowl" structures on their inner wall (white arrows in fig. 3), and it is likely that meloxicam, a poorly soluble compound, will precipitate on the inside of the lipid membrane after entering the liposome's inner aqueous phase. Therefore, the high encapsulation efficiency and drug loading capacity can be obtained, and the high-performance composite material is not easy to leak and has high storage stability.

3.2.2 encapsulation efficiency of Meloxicam liposomes with calcium acetate solution as internal aqueous phase

The prepared meloxicam liposome was diluted 50 times with ultrapure water, and an appropriate amount of Dowex resin (Sigma Aldrich) was added thereto, followed by shaking sufficiently to adsorb unencapsulated meloxicam. After standing, 200. mu.l of the supernatant was collected, and absorbance values of meloxicam in the liposome before and after addition of the resin were measured by the UV detection method in example 1, and the content was calculated.

The Entrapment Efficiency (EE) of meloxicam liposomes was calculated according to the following formula:

wherein Minner is the amount of meloxicam in the liposome preparation after the free drug is adsorbed by the resin, i.e. the amount of meloxicam encapsulated by the liposome; mtotal is the total amount of meloxicam in the meloxicam liposome formulation prior to resin adsorption. The results are shown in table 3, where the formulation meloxicam liposomes had a higher encapsulation efficiency. Wherein, when the concentration of the calcium acetate in the inner water phase is increased from 150mM to 300mM, the drug-loading capacity of the liposome is obviously increased along with the increase of the concentration of the calcium acetate, but the entrapment rate is gradually increased after the concentration of the calcium acetate exceeds 300 mM.

TABLE 3 Meloxicam Liposome particle size and encapsulation efficiency with calcium acetate solution as internal aqueous phase

3.3 Meloxicam Liposome storage stability with calcium acetate solution as internal aqueous phase

And (3) storing the meloxicam liposome with the inner water phase as a calcium acetate solution in a refrigerator at 4 ℃, sampling and diluting by 50 times after a certain number of days respectively, measuring the encapsulation rate according to the method in 3.2.2, and inspecting the stability of the meloxicam liposome with the inner water phase as calcium acetate. As shown in FIG. 4(B), the three calcium acetate solutions with different concentrations were formulated in the internal aqueous phase without significant leakage within 30 days, and thus had better storage stability.

3.4 Meloxicam Liposome external release with calcium acetate solution as internal aqueous phase

Taking the meloxicam liposome with the inner water phase of calcium acetate solution with different concentrations, taking normal saline as a release medium, adding sufficient Dowex resin to adsorb free drugs to form a sink condition, and oscillating in a shaking table (THZ-C constant temperature oscillator, China) with the temperature of 37 ℃ and the rpm of 100. Samples were taken at various time points (0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours and 24 hours), after standing, the supernatant was taken, the content of meloxicam entrapped in the liposomes was determined by UV method, and the cumulative release rate of meloxicam at various time points was calculated. As shown in figure 4(C), the cumulative amount of drug released was greater than 80% over 4 hours for the three formulations with different concentrations of calcium acetate as the internal aqueous phase, and the release slowed after 4 hours.

Example 4

This example is the preparation and characterization of meloxicam liposomes with calcium chloride solution as the internal aqueous phase.

4.1 preparation of Meloxicam liposomes with calcium chloride solution as the internal aqueous phase

Preparing 150mM, 300mM and 500mM calcium chloride aqueous solutions respectively, and adjusting the pH value to 7.5 by hydrochloric acid; meloxicam liposomes with calcium chloride as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

4.2 characterization of Meloxicam liposomes with calcium chloride solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with calcium chloride as the internal aqueous phase were determined according to the method of 3.2 in example 3. As a result, the liposome particles obtained were all 100. + -.10 nm as shown in Table 4. The particle size distribution (PDI) is less than 0.1. When the concentration of calcium chloride is increased from 150mM to 300mM, the drug loading capacity of the liposome is obviously increased along with the increase of the concentration of the calcium chloride, but the increase of the entrapment rate is gradually increased after the concentration of the calcium chloride exceeds 300 mM.

TABLE 4 Meloxicam Liposome particle size and encapsulation efficiency with calcium chloride solution as internal aqueous phase

4.3 storage stability of Meloxicam liposomes with calcium chloride solution as internal aqueous phase

The stability of meloxicam liposomes containing calcium chloride as the inner aqueous phase was examined by storing meloxicam liposomes containing 150mM, 300mM and 500mM sodium acetate in the inner aqueous phase at 4 ℃ in a refrigerator according to the method 3.3 of example 3. As shown in FIG. 5(B), under the storage condition of 4 ℃, the formulations with three different concentrations of calcium chloride as the inner water phase have no obvious leakage within 30 days, so the storage stability is better.

4.4 in vitro Release Rate of Meloxicam liposomes with calcium chloride solution as the internal aqueous phase

Taking the meloxicam liposome with the inner water phase of calcium chloride solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the embodiment 3. The results are shown in fig. 5(C), and the cumulative release of the three calcium chloride solutions with different concentrations in the internal water phase is greater than 80% in 4 hours, and the release is slowed after 4 hours.

Example 5

In this example, meloxicam liposomes with zinc acetate solution as the internal aqueous phase were prepared and characterized.

5.1 preparation of Meloxicam liposomes with Zinc acetate solution as the internal aqueous phase

Preparing zinc acetate aqueous solutions with the concentrations of 150mM, 300mM and 500mM respectively, and regulating the pH value to 6.0 by hydrochloric acid; meloxicam liposomes with zinc acetate as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

5.2 characterization of Meloxicam liposomes with Zinc acetate solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with zinc acetate as the internal aqueous phase were determined according to the method 3.2 in example 3. As shown in Table 5, the particle sizes of the meloxicam liposomes were all about 155. + -.5 nm. The particle size distribution (PDI) is less than 0.1. When the concentration of zinc acetate is increased from 150mM to 300mM, the drug loading capacity of the liposome is obviously increased along with the increase of the concentration of the zinc acetate, but the increase of the entrapment rate is gradually increased after the concentration of the zinc acetate is over 300 mM.

TABLE 5 Meloxicam Liposome particle size and encapsulation efficiency with Zinc acetate solution as internal aqueous phase

5.3 storage stability of Meloxicam liposomes with Zinc acetate solution as internal aqueous phase

The meloxicam liposomes in which the internal aqueous phases were zinc acetate solutions of different concentrations were taken and stored in a refrigerator at 4 ℃ according to the method of 3.3 in example 3, and the stability of the meloxicam liposomes in which zinc acetate was used as the internal aqueous phase was examined, and as a result, as shown in fig. 6(B), the formulations in which three kinds of zinc acetate of different concentrations were used as the internal aqueous phases did not leak out in 30 days, and thus had good storage stability.

5.4 in vitro Release Rate of Meloxicam liposomes with Zinc acetate solution as the internal aqueous phase

Taking the meloxicam liposome with the internal water phase of zinc acetate solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the embodiment 3. The results are shown in fig. 6(C), wherein the cumulative amount of zinc acetate released in the three formulations with different concentrations of zinc acetate as the internal aqueous phase is greater than 70% in 4 hours, and the release is slowed down after 4 hours.

Example 6

Example 6 preparation and characterization of meloxicam liposomes with a copper sulfate solution as the internal aqueous phase.

6.1 preparation of Meloxicam liposomes with copper sulfate solution as internal aqueous phase

Preparing copper sulfate aqueous solutions with the concentrations of 150mM, 300mM and 500mM respectively, and adjusting the pH value to 7.0 by hydrochloric acid; meloxicam liposomes with copper sulfate as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

6.2 characterization of Meloxicam liposomes with copper sulfate solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with copper sulfate as the internal aqueous phase were determined according to the method 3.2 in example 3. As shown in Table 6, the particle sizes of the meloxicam liposomes were all around 130. + -.5 nm. The particle size distribution (PDI) is less than 0.1. The loading capacity of the liposomes increased significantly with increasing copper sulfate concentration when the copper sulfate concentration was increased from 150mM to 300mM, but the encapsulation efficiency increased more slowly beyond 300 mM.

TABLE 6 Meloxicam liposome particle size and encapsulation efficiency with copper sulfate solution as internal aqueous phase

6.3 storage stability of Meloxicam liposomes with copper sulfate solution as internal aqueous phase

The meloxicam liposomes having different concentrations of copper sulfate as the internal aqueous phase were stored in a refrigerator at 4 ℃ according to the method of 3.3 in example 3, and the stability of the meloxicam liposomes having copper sulfate as the internal aqueous phase was examined, as shown in fig. 7(B), the formulations having three different concentrations of copper sulfate as the internal aqueous phase did not leak in 30 days, and thus had good storage stability.

6.4 in vitro Release Rate of Meloxicam liposomes with copper sulfate solution as the internal aqueous phase

Taking the meloxicam liposome with the internal water phase of copper sulfate solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the example 3. The results are shown in FIG. 7(C), where the total release of the formulation was greater than 70% over 4 hours for the three different concentrations of copper sulfate in the internal aqueous phase, and the release slowed after 4 hours.

Example 7

This example is the preparation and characterization of meloxicam liposomes with a magnesium chloride solution as the internal aqueous phase.

7.1 preparation of Meloxicam liposomes with magnesium chloride solution as the internal aqueous phase

Preparing 150mM, 300mM and 500mM magnesium chloride aqueous solutions respectively, and adjusting the pH value to 6.5 by hydrochloric acid; meloxicam liposomes with magnesium chloride as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

7.2 characterization of Meloxicam liposomes with magnesium chloride solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with magnesium chloride as the internal aqueous phase were determined according to the method 3.2 in example 3. As shown in Table 7, the particle sizes of the meloxicam liposomes were all around 110. + -.5 nm. The particle size distribution (PDI) is less than 0.1. When the concentration of magnesium chloride is increased from 150mM to 300mM, the drug loading capacity of the liposome is obviously increased along with the increase of the concentration of the magnesium chloride, but the increase of the entrapment rate is gradually increased after the concentration of the magnesium chloride is over 300 mM.

TABLE 7 Meloxicam Liposome particle size and encapsulation efficiency with magnesium chloride solution as internal aqueous phase

7.3 storage stability of Meloxicam liposomes with magnesium chloride solution as internal aqueous phase

The meloxicam liposomes having different concentrations of magnesium chloride in the internal aqueous phase were stored in a refrigerator at 4 ℃ according to the method of 3.3 in example 3, and the stability of the meloxicam liposomes having magnesium chloride in the internal aqueous phase was examined, as shown in fig. 8(B), the formulations having three different concentrations of magnesium chloride in the internal aqueous phase did not leak significantly in 30 days, and thus had good storage stability.

7.4 in vitro Release Rate of Meloxicam liposomes with magnesium chloride solution as the internal aqueous phase

Taking the meloxicam liposome with the internal water phase of magnesium chloride solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the embodiment 3. The results are shown in fig. 8(C), wherein the cumulative amount of drug released was greater than 70% over 4 hours for three formulations with different concentrations of magnesium chloride in the internal aqueous phase, and the release slowed after 4 hours.

Example 8

This example is the preparation and characterization of meloxicam liposomes with a cobalt chloride solution as the internal aqueous phase.

8.1 preparation of Meloxicam liposomes with cobalt chloride solution as the internal aqueous phase

Preparing 150mM, 300mM and 500mM aqueous solutions of cobalt chloride, and adjusting the pH value to 6.0 by hydrochloric acid; meloxicam liposomes with cobalt chloride as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

8.2 characterization of Meloxicam liposomes with cobalt chloride solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with cobalt chloride as the internal aqueous phase were determined according to the method 3.2 in example 3. As shown in Table 8, the particle sizes of the meloxicam liposomes were all around 120. + -.5 nm. The particle size distribution (PDI) is less than 0.1. When the concentration of cobalt chloride is increased from 150mM to 300mM, the drug loading capacity of the liposome is obviously increased along with the increase of the concentration of cobalt chloride, but the increase of the entrapment rate is gradually increased after the concentration of cobalt chloride is over 300 mM.

TABLE 8 Meloxicam Liposome particle size and encapsulation efficiency with cobalt chloride solution as internal aqueous phase

8.3 storage stability of Meloxicam liposomes with cobalt chloride solution as internal aqueous phase

The method 3.3 in example 3 was followed, and the meloxicam liposomes having different concentrations of cobalt chloride in the internal aqueous phase were stored in a refrigerator at 4 ℃ to examine the stability of the meloxicam liposomes having cobalt chloride in the internal aqueous phase, and as shown in fig. 9(B), the formulations having three different concentrations of cobalt chloride in the internal aqueous phase did not leak significantly in 30 days, and thus had good storage stability.

8.4 in vitro Release Rate of Meloxicam liposomes with cobalt chloride solution as the internal aqueous phase

Taking the meloxicam liposome with the internal water phase of cobalt chloride solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the embodiment 3. The results are shown in fig. 9(C), wherein the cumulative amount of the drug released in 4 hours was greater than 70% for each of the three formulations with different concentrations of cobalt chloride as the internal aqueous phase, and the release slowed down after 4 hours.

Example 9

This example is the preparation and characterization of meloxicam liposomes with a manganese chloride solution as the internal aqueous phase.

9.1 preparation of Meloxicam liposomes with manganese chloride solution as internal aqueous phase

Preparing 150mM, 300mM and 500mM aqueous solutions of manganese chloride, and adjusting the pH value to 6.5 by hydrochloric acid; meloxicam liposomes containing manganese chloride as the internal aqueous phase were prepared according to the preparation method 3.1 in example 3.

9.2 characterization of Meloxicam liposomes with manganese chloride solution as the internal aqueous phase

The particle size and encapsulation efficiency of meloxicam liposomes prepared with manganese chloride as the internal aqueous phase were determined according to the method 3.2 in example 3. As shown in Table 9 and FIG. 10, the particle size of the meloxicam liposomes was about 120. + -.5 nm. The particle size distribution (PDI) is less than 0.1. When the concentration of manganese chloride is increased from 150mM to 300mM, the drug loading capacity of the liposome is obviously increased along with the increase of the concentration of manganese chloride, but the increase of the entrapment rate is gradually increased after the concentration of manganese chloride exceeds 300 mM.

TABLE 9 Meloxicam Liposome particle size and encapsulation efficiency with manganese chloride solution as internal aqueous phase

9.3 storage stability of Meloxicam liposomes with manganese chloride solution as internal aqueous phase

According to the method 3.3 in example 3, the meloxicam liposomes with different concentrations of manganese chloride solution as the internal aqueous phase were stored in a refrigerator at 4 ℃, and the stability of the meloxicam liposomes with manganese chloride as the internal aqueous phase was examined, and as a result, as shown in fig. 10(B), the three formulations with different concentrations of manganese chloride as the internal aqueous phase did not leak significantly in 30 days, and thus had good storage stability.

9.4 in vitro Release Rate of Meloxicam liposomes with manganese chloride solution as the internal aqueous phase

Taking the meloxicam liposome with the inner water phase of manganese chloride solution with different concentrations, and calculating the cumulative drug release percentage of meloxicam in the liposome at different time points according to the in vitro drug release rate characterization method of 3.4 in the embodiment 3. The results are shown in fig. 10(C), wherein the cumulative amount of drug released in 4 hours was greater than 70% for all three formulations with different concentrations of manganese chloride in the internal aqueous phase, and the release slowed after 4 hours.

Example 10

This example is the analgesic effect of meloxicam liposomes after intravenous injection.

The analgesic effect of the meloxicam liposome after intravenous injection is evaluated by adopting a mouse writhing model.

Taking 50 SPF-grade Kunming mice, wherein the weight of the mice is 18-22 g. The groups were randomly divided into 4 groups of 10 each, each half of males and females. The test results were divided into a negative control group (physiological saline), a positive control group (meloxicam injection, bringer bergham), a meloxicam suspension group, a meloxicam liposome group and a blank liposome group. Wherein the liposome group is blank liposome and meloxicam drug-loaded liposome (about 2mg/mL) prepared by using calcium acetate as an internal water phase in example 3. Except for the administration of the meloxicam suspension to the abdominal cavity, the other groups were administered to the tail vein. The dose of meloxicam administration is 2 mg/kg.

After each group is injected with the medicine for 20min, 1% glacial acetic acid is injected into the abdominal cavity to prepare an abdominal pain pathological model, the mouse is placed in an inverted measuring cylinder, the twisting times within 15min are observed and recorded, and the inhibition rate is calculated, namely (the average twisting times of an administration group-the average twisting times of a negative control group)/the average twisting times of the negative control group is multiplied by 100%.

Analysis of group-to-group variability was performed using One-way ANOVA. The difference is significant when P is less than 0.05, and the difference is extremely significant when P is less than 0.01. The results are shown in Table 10.

TABLE 10 inhibition of acetic acid induced writhing response in mice (meloxicam dose 2mg/kg)

The results show that compared with the suspension, the meloxicam liposome can quickly release the medicine in the blood after intravenous injection, and shows the similar analgesic effect to the injection.

Example 11

This example is the preparation and characterization of meloxicam-doxorubicin co-loaded liposomes with manganese chloride as the inner aqueous phase.

11.1 preparation of Meloxicam-Adriamycin Co-loaded liposome with manganese chloride solution as internal aqueous phase

(1) Preparing a manganese chloride aqueous solution with the concentration of 300mM, and adjusting the pH value to 7 by hydrochloric acid;

(2) manganese chloride blank liposomes were prepared according to the method of 3.1(1) to (5) in example 3;

(3) an aqueous solution of meloxicam-meglumine (8 mg/mL) was prepared according to the method in example 2;

(4) 10mg of doxorubicin hydrochloride and 10% of sucrose are precisely weighed and dissolved to 1mL to prepare a 10mg/mL doxorubicin hydrochloride solution.

(5) And respectively adding an adriamycin hydrochloride solution and a meloxicam solution into the blank liposome according to the total medicine-lipid ratio of 0.1, 0.2 and 0.3 (the molar ratio of meloxicam to adriamycin hydrochloride is 1: 1). Incubating for 30min at 60 ℃ to prepare the meloxicam-adriamycin co-loaded liposome solution.

And (3) preparing the meloxicam single-carrier liposome or the adriamycin single-carrier liposome by respectively adding meloxicam solution or adriamycin hydrochloride solution into the manganese chloride liposome in the step (2) according to the drug-to-lipid ratios of 0.05, 0.1 and 0.15.

11.2 characterization of Meloxicam-Adriamycin Co-Loading liposomes with manganese chloride solution as internal aqueous phase

The particle size and encapsulation efficiency of meloxicam-doxorubicin co-loaded liposomes prepared with manganese chloride as the internal aqueous phase (table 11 and fig. 12) were determined according to the method of 3.2 in example 3, and compared to the particle size and encapsulation efficiency of meloxicam-or doxorubicin-loaded liposomes alone (table 12 and fig. 11), the co-loaded liposomes had a particle size of around 110 ± 5 nm. The particle size distribution (PDI) is less than 0.1. The adriamycin and the meloxicam are carried together, so that the encapsulation rate of the adriamycin is obviously improved. Wherein, the drug-to-lipid ratio in fig. 11 refers to the molar ratio of a single drug to a lipid. In fig. 12, the drug-to-lipid ratio refers to the ratio of the total molar amount of meloxicam and doxorubicin to the molar amount of lipid.

TABLE 11 Meloxicam-Adriamycin Co-Loading liposomes particle size and encapsulation efficiency with manganese chloride solution as internal aqueous phase

TABLE 12 particle size and encapsulation efficiency of liposomes singly loaded with meloxicam or doxorubicin with manganese chloride solution as the internal aqueous phase

Example 12

This example is a cytotoxicity test of meloxicam-doxorubicin co-loaded liposomes against doxorubicin-resistant K562 cells:

(1) k562 cells at 1X 10 per well⁵Inoculating the mixture into a 96-well plate at different concentrations, adding adriamycin single-carrier liposome, meloxicam single-carrier liposome and meloxicam-adriamycin co-carrier at different concentrationsLiposomes, incubated for 48 h. After incubation, 10ul CCK-8 reagent (Byunyan day) was added to each well and incubated for 4 h. After the incubation was complete, the absorbance of each well was measured at a wavelength of 450 nm.

(2) Adriamycin-resistant K562 cells at 1X 10 per well⁵Inoculating the mixture into a 96-well plate at different concentrations, adding adriamycin unicarrier liposome, meloxicam unicarrier liposome and meloxicam-adriamycin unicarrier liposome at different concentrations, and incubating for 48 h. After incubation, 10ul CCK-8 reagent (Byunyan day) was added to each well and incubated for 4 h. After the incubation was complete, the absorbance of each well was measured at a wavelength of 450 nm. The result is shown in fig. 13, and the result shows that the inhibition effect of the meloxicam and adriamycin co-loaded liposome on the adriamycin-resistant K562 cells is obviously higher than that of the adriamycin single-loaded liposome, and the meloxicam single-loaded liposome has no obvious inhibition effect on the cells. The results suggest that co-loading with meloxicam is beneficial for reducing doxorubicin resistance in tumor cells.

Example 13

This example is to examine the drug release rate at the injection site after local injection of liposomes.

This example uses the dialysis bag method to determine the release rate of liposomal drug: the liposomes are packed into dialysis bags and placed in a small volume of release medium. Since only the drug released from the liposomes can diffuse from the dialysis bag into the release medium, the drug release rate of the liposomes can be determined by measuring the drug concentration in the release medium at different time points. It is to be noted that the method for determining the drug release rate in this example is different from that in example 3.4. Example 3.4 liposomes were added directly to the release medium to simulate the dilution of liposomes by blood after intravenous injection; 3.4 resin is used to adsorb the drug released from the liposome to simulate the drug released from the liposome and immediately carried away by the blood circulation. In the present embodiment, the drug release rate of the liposome is measured by the dialysis bag method, mainly to simulate the situation that the liposome has less release medium at the injection part and the released drug stays at the part for a certain period of time.

The method comprises the following specific steps:

100. mu.l of meloxicam liposomes (drug-lipid ratio 0.1) containing 300mM calcium acetate, copper sulfate, magnesium chloride and manganese chloride as internal aqueous phases in example 3, example 6, example 7 and example 9, respectively, were taken and placed in dialysis bags (Spectrum labs, model 132570) with a cut-off molecular weight of 10000; the dialysis bag containing the liposomes was immersed in 5ml of a release medium (physiological saline containing 0.2% SDS (w/v)) and incubated and stirred in a water bath at 37 ℃. 200. mu.l of release medium was taken at 0 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, respectively, and the same volume of fresh release medium was replenished. The concentration of meloxicam in the solution was determined by the UV method (example 1) and the cumulative release rate of meloxicam was calculated at different time points. Another 100. mu.l of aqueous solution of meloxicam-meglumine with a concentration of 100. mu.g/ml was taken and added to the dialysis bag, and the release rate was determined in the same manner.

The cumulative release rates of meloxicam solution and liposomes are shown in fig. 14. The cumulative drug release of the meloxicam liposome reaches 50% within 12 hours, and then the drug release rate is flat. The meloxicam solution completely enters the release medium within about 2hr, indicating that meloxicam can diffuse through the dialysis bag used relatively quickly and that the dialysis bag does not block the release of meloxicam.

The cumulative release rate of the meloxicam liposome is calculated according to the following formula:

C_nthe concentration of the sample at each nth time point; v_nIs the total volume of the release medium; c_iAnd V_iThe concentration and the volume of the sample at the ith time point are respectively; w is the dosage; EE% is the encapsulation rate of the meloxicam.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A liposome for loading meloxicam, wherein the liposome contains an internal aqueous phase, the internal aqueous phase is a salt solution containing metal ions, and the meloxicam can be complexed with the metal ions.

2. The liposome of claim 1, wherein the metal ion is selected from the group consisting of calcium ion, zinc ion, copper ion, magnesium ion, manganese ion, and cobalt ion.

3. The liposome of claim 1, wherein the salt solution has a pH of 6.0 to 8.0.

4. The liposome of claim 1, wherein the liposome comprises one or both of a phospholipid and cholesterol.

5. The injection of meloxicam is characterized in that the raw material components of the injection comprise: meloxicam, an external aqueous phase and liposomes according to any of claims 1 to 4; the external aqueous phase contains at least water.

6. The injection according to claim 5, wherein the concentration of meloxicam is 0.01mg/mL or more based on the volume of the injection; and/or the external water phase also contains one or more of meglumine, sucrose or glucose; and/or, the meloxicam is loaded within the liposome.

7. A process for the preparation of an injection according to any of claims 5 to 6, wherein meloxicam, an external aqueous phase and liposomes are mixed.

8. Use of the injection according to any one of claims 5 to 6 as an analgesic for injection.

9. An anti-inflammatory agent comprising the injection according to any one of claims 5 to 6 and a cytotoxic drug, or the injection according to any one of claims 5 to 6 and an immunopharmaceutical.

10. Use of an anti-inflammatory preparation according to claim 9 for the preparation of a medicament for the treatment of tumors.