Celecoxib prodrug solid dispersion based on electrostatic spraying, its preparation method and uses

Abstract

The present invention relates to a celecoxib prodrug solid dispersion based on electrostatic spraying. The celecoxib raw material drug is structurally modified and covalently combined with polyethylene glycol 2000 (mPEG2000) to obtain a pH-responsive prodrug (CXB-mPEG). On this basis, a celecoxib prodrug solid dispersion (CXB-mPEG-SD) is further prepared. This prodrug solid dispersion can improve the bioavailability of celecoxib, increase the targeting effect of celecoxib on tumor cells, and significantly improve the anti-cancer and anti-inflammatory analgesic efficacy of celecoxib. The present invention discloses its preparation method.

Description

Technical Field

[0001] The present invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, including the synthesis of celecoxib-mPEG prodrug and the construction of celecoxib prodrug solid dispersion based on electrospray, as well as its application in drug delivery. Background Art

[0002] Celecoxib has the chemical name of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide, with a molecular weight of 381.38, and is stored in a dry environment at room temperature. The structural formula is as follows:

[0003] 。

[0004] As a traditional selective non-steroidal anti-inflammatory drug, celecoxib, in addition to its antipyretic, anti-inflammatory and analgesic effects, also has an anti-tumor effect, and can significantly reduce the recurrence rate of sporadic colorectal adenomas. However, it belongs to BSC Ⅱ drugs, and its poor water solubility limits the development of its dosage forms. In addition, poor targeting also leads to relatively large side effects. Therefore, a method is needed to solve its water solubility and targeting problems to broaden the administration routes.

[0005] The pH value of normal tissues is about 7.4, and the microenvironment of cancer cells is at pH 6.0 - 7.0. Therefore, a chemical bond that breaks in an acidic environment can be designed according to the special acidic environment of tumor cells to achieve pH response.

[0006] In the present invention, celecoxib is covalently bonded with polyethylene glycol with characteristics such as non-toxicity and high biosafety to form a pH-responsive prodrug, and further made into a solid dispersion by electrospray technology. On the basis of increasing its solubility, the carbamate bond is used to easily cleave in an acidic environment (pH 6.0) to achieve the targeted effect of celecoxib on colorectal cancer and expand its application in the field of tumors. Summary of the Invention

[0007] One of the purposes of the present invention is to provide a celecoxib-mPEG prodrug and its preparation method.

[0008] Another purpose of the present invention is to provide a celecoxib prodrug solid dispersion and its preparation method.

[0009] The third purpose of the present invention is to provide the application of the celecoxib prodrug solid dispersion in the preparation of anti-inflammatory and analgesic drugs, anti-colorectal cancer drugs and improving the bioavailability of celecoxib.

[0010] The fourth purpose of the present invention is to provide the application of the celecoxib prodrug solid dispersion in an oral or topical drug delivery system of celecoxib.

[0011] The technical solution of the present invention is as follows:

[0012] A celecoxib-mPEG prodrug, which uses celecoxib as the parent drug and is covalently bonded with polyethylene glycol 2000 (mPEG2000) to obtain a pH-responsive prodrug. The catalysts used include: triethylamine and sodium hydride. The structural formula of the celecoxib prodrug is shown as follows:.

[0013] A method for preparing the above-mentioned celecoxib-mPEG prodrug, which comprises the following steps:

[0014] Step 1: Dissolve 5 mmol of p-NPC in 10 mL of dichloromethane solution for later use. Weigh precisely 1 mmol of mPEG2000, add 5 mmol of triethylamine as a catalyst, add 20 mL of dichloromethane solution, and dissolve it thoroughly. Under ice bath and anaerobic conditions, slowly add the p-NPC solution drop by drop. After complete addition, raise the reaction temperature to room temperature and continue the reaction for 24 h. Precipitate with ether and dry under vacuum to obtain 2 g of the product (mPEG-NPC).

[0015] Step 2: Take 2.4 mmol of celecoxib, add 25 mL of DMF to dissolve it. Under ice bath conditions, add 5.1 mmol of sodium hydride in batches. After addition, keep stirring in the ice bath state for 30 minutes, add 1 mmol of mPEG-NPC and react for 20 minutes. Raise the reaction temperature to 50 °C and continue the reaction for 12 h. Prepare a solution by adding 1.7 mmol of citric acid to 100 mL of deionized water, cool it to 0 °C for later use. Under ice bath conditions, pour the reaction solution into 100 mL of ice-citric acid solution, filter off the precipitated celecoxib, and then dialyze (MWCO 1000 Da) in deionized water at 4 °C for 24 h (3 times, 8 hours each time). Lyophilize to obtain the product, a white solid celecoxib-mPEG prodrug (CXB-mPEG).

[0016] The reaction formula is as follows:

[0017] A celecoxib prodrug solid dispersion prepared by electrospray, which is a celecoxib prodrug solid dispersion formed by the above-mentioned celecoxib-mPEG prodrug in a carrier solution. The solubility of the celecoxib prodrug solid dispersion in water is 1.52 mg / mL, and the drug loading is 49.36%.

[0018] A method for preparing the above-mentioned celecoxib prodrug solid dispersion by electrospraying, which dissolves the celecoxib prodrug and the carrier in a mixed solvent (water: methanol: DMF = 2:1:1), stirs for 4 h to obtain a clear and transparent electrospray solution, sucks the electrospray solution into a 10 mL syringe, expels air bubbles, places the syringe on a constant flow injection pump, adjusts the distance, flow rate, voltage, temperature and humidity, connects a positive voltage to the injection needle, connects a negative voltage to the receiving plate, and collects the jet ejected from the needle on the tin foil on the receiving plate, and collects the powder on the tin foil.

[0019] The electrospray voltage of the above-mentioned celecoxib prodrug solid dispersion is 12 - 20 kV.

[0020] The electrospray flow rate of the above-mentioned celecoxib prodrug solid dispersion is 0.02 - 0.08 mm / min.

[0021] The electrospray distance of the above-mentioned celecoxib prodrug solid dispersion is 10 - 20 cm.

[0022] The application of the above-mentioned celecoxib prodrug solid dispersion in the preparation of a nano drug delivery system.

[0023] The application of the above-mentioned celecoxib prodrug solid dispersion in a drug delivery system.

[0024] The application of the above-mentioned celecoxib prodrug solid dispersion in the preparation of anti-inflammatory, analgesic and anti-colorectal cancer drugs.

[0025] The above-mentioned celecoxib prodrug solid dispersion is used in the preparation of anti-inflammatory, analgesic and anti-colorectal cancer drugs, and is characterized in that: the drug is an injection for injection. Beneficial effects

[0026] The solubility of the prepared celecoxib prodrug solid dispersion in water is 1.52 mg / mL, and the drug loading is 49.36%. This prodrug solid dispersion can increase the solubility of celecoxib, significantly improve the bioavailability of celecoxib, and increase the targeting effect of celecoxib on tumor cells. Description of the drawings

[0027] Figure 1 For the celecoxib-mPEG prodrug in Example 1 of the present invention 1 1H-NMR spectrum.

[0028] Figure 2 The infrared spectrum of the celecoxib-mPEG prodrug in Example 1 of the present invention.

[0029] Figure 3 For the cytotoxicity of CXB-mPEG-SD on CT-26 cells under different treatment conditions at 24, 48, and 72 h in Example 3 of the present invention.

[0030] Figure 4 This is the in vivo imaging diagram (4h, 8h) of the mice administered with CXB-mPEG-SD in Example 4 of the present invention.

[0031] Figure 5 This is the evaluation of antitumor effect in Example 5 of the present invention. (A) Tumor morphology of Balb / c mice (B) Tumor volume growth curve (C) Tumor mass of each group of mice and antitumor rate of each group of drugs (n = 5) (D) Body weight change curve of each group of mice. *P < 0.05, **P < 0.01, ***P < 0.001. Detailed implementation manners

[0032] The following listed examples are helpful for those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0033] Dissolve 5 mmol of p-NPC in 10 mL of dichloromethane solution for later use. Weigh 1 mmol of mPEG2000 precisely, add 5 mmol of triethylamine, and add 20 mL of dichloromethane solution to make it dissolve fully. Under ice bath and anaerobic conditions, dropwise add the p-NPC solution. After complete addition, raise the reaction temperature to room temperature and continue the reaction for 24 h. Precipitate with ether and dry under vacuum to obtain 2 g of the product (mPEG-NPC).

[0034] Take 2.4 mmol of celecoxib, add 25 mL of DMF to dissolve it. Under ice bath conditions, add 5.1 mmol of sodium hydride in batches. After addition, keep stirring for 30 minutes under the ice bath state, add 1 mmol of mPEG-NPC and react for 20 minutes. Raise the reaction temperature to 50 °C and continue the reaction for 12 h. Prepare a solution by adding 1.7 mmol of citric acid to 100 mL of deionized water and cool it to 0 °C for later use. Under ice bath conditions, pour the reaction solution into 100 mL of ice-citric acid solution, filter off the precipitated celecoxib, and then dialyze (MWCO 1000 Da) in deionized water at 4 °C for 24 h (3 times, 8 hours each time), and freeze-dry to obtain the product, the off-white solid celecoxib-mPEG prodrug (CXB-mPEG).

[0035] Use nuclear magnetic resonance to determine the structure of the celecoxib-mPEG prodrug in Example 1. Take 20 mg of the monomer component, dissolve it with 0.6 mL of deuterated DMSO, and detect it using a nuclear magnetic resonance analyzer.

[0036] 1 The 1H-NMR spectrum is shown in Figure 1. The infrared spectrum is shown in Figure 2 .

[0037] The preparation process is as follows: Take the celecoxib-mPEG prodrug and the carrier in Example 1 and dissolve them together in a mixed solvent (water: methanol: DMF = 2:1:1), stir for 4 h to obtain a clear and transparent electrospray solution. Aspirate the electrospray solution into a 10 mL syringe, expel the air bubbles, place the syringe on a constant flow injection pump, adjust the distance, flow rate, voltage, temperature and humidity. A positive voltage is connected to the injection needle, and a negative voltage is connected to the receiving plate. The jet ejected from the needle is collected on the tin foil on the receiving plate, and the powder on the tin foil is collected. Among them, the carrier is PVP K30, the positive voltage is +16 kV, the negative voltage is -1.5 kV, the electrospray flow rate is 0.05 mm / min, the receiving distance is 18 cm, the inner diameter of the needle is 0.34 mm, and it is prepared under the conditions of a temperature of 37 °C and a humidity of 25%, and the prodrug solid dispersion is obtained.

[0038] Take CT-26 cells and perform subculture. Discard the culture solution in the culture flask, add PBS buffer to wash the cells 2-3 times, add trypsin to digest the cells. When the cells become round and partially detached, add 1 mL of culture solution to terminate the digestion, pipette repeatedly to suspend the cells that have not detached, transfer the culture solution into a 5 mL EP tube and centrifuge, discard the supernatant, add culture solution and pipette evenly, transfer to 2 culture flasks for subculture, count the cells, and set aside.

[0039] Precisely weigh an appropriate amount of the prodrug solid dispersion (containing 4.5 mg of CXB), add 1% DMSO, and dilute it to 2 mL with serum-free culture solution to obtain a mother liquor of 6 mM. Dilute the mother liquor to obtain prodrug solid dispersions of 5, 10, 15, 30, and 60 μM.

[0040] The biological activity of the prodrug solid dispersion was evaluated by MTT assay. Adjust the cell suspension concentration to 1×10 5 / mL, seed three 96-well plates, measure the pH on the second day after seeding. Replace one of the acidic 96-well plates with DMEM culture solution to ensure that the cell surrounding environment is acidic (pH 6.0, simulating the tumor cell surrounding environment) and neutral (pH 7.4, simulating the in vivo normal cell surrounding environment) respectively. Add 100 μL of 5, 10, 15, 30, 60 μM prodrug solid dispersions respectively, and add 100 μL of culture solution containing DMSO to the remaining wells as the blank group. Measure the absorbance after co-culturing with CT-26 cells for 24, 48, and 72 h, calculate the cell inhibition rate, plot with the drug concentration as the abscissa and the cell inhibition rate as the ordinate, and calculate the IC 50 value. The cell inhibition rate is calculated as in "Equation (1)":

[0041]

[0042]

[0043] The results showed that the IC 50 value of the prodrug solid dispersion under the condition of pH 6.0 was lower than that under the condition of pH 7.4, indicating that the prodrug solid dispersion had a stronger inhibitory effect on CT-26 under the condition of pH 6.0. Therefore, the pH-responsive prodrug was more likely to play a role in the acidic environment of tumors and had a stronger effect on inhibiting tumor growth.

[0044] Example 4. In vivo imaging of mice

[0045] Celecoxib prodrug with amino group was combined with Sulfo-Cyanine-NHS Ester (Sulfo-Cy7). 2 mg of Sulfo-Cy7 was dissolved in 200 μL of DMSO, 1 mL of 2 mg / mL prodrug was taken, 200 μL of Sulfo-Cy7 was added, 30 μL of triethylamine was added, and after stirring in the dark for 12 h, dialysis was carried out in the dark for 6 h, and then freeze-dried to obtain the Sulfo-Cy7-labeled prodrug. The labeling method of celecoxib was the same as above, and the post-treatment method was purification by silica gel column. Further, the celecoxib solid dispersion and the prodrug solid dispersion were prepared according to the method of Example 2, and were respectively denoted as Cy7@CXB and Cy7@CXB-mPEG-SD (mass ratio 1:1).

[0046] A colorectal cancer xenograft model was established. CT-26 was injected into the axilla of Balb / c mice with a cell suspension of 5×10 6 / mL. The mice were randomly divided into the Cy7@CXB group and the Cy7@CXB-mPEG-SD group. When the tumor reached about 100 mm 3 , 0.2 mL of Cy7@CXB and Cy7@CXB-mPEG-SD (Cy7 3 mg / kg) were injected into the tail vein. 4 h and 8 h after administration, in vivo imaging technology was used to observe the accumulation and distribution of the drugs in the body. Before in vivo imaging, 0.1 mL of 10% chloral hydrate solution was injected intraperitoneally for anesthesia, and fluorescence photography was carried out at Ex and Em = 730 nm / 790 nm. After the shooting was completed, the mice were sacrificed by the CO2 method, the tumors and the heart, liver, spleen, lungs, and kidneys were dissected, washed with normal saline, and then the fluorescence images of the organs were taken, and image analysis was carried out with Bruker Molecular Imaging Software.

[0047] As Figure 4, the tumor site is within the red circle. CXB accumulates at the tumor site after 4 h, and the accumulation at the tumor site decreases after 8 h. CXB-mPEG-SD accumulates at the tumor site at 4 h and has less accumulation in other sites. After 8 h, the accumulation amount at the tumor site increases significantly, indicating that after structural modification of celecoxib, the circulation time of the drug in the body is prolonged and the clearance rate of the drug is slowed down.

[0048] Digest CT-26 with trypsin, and after digestion, resuspend it with PBS to 5×10 6 / mL. Take 0.2 mL and inoculate it subcutaneously under the left axilla of mice. Observe the status of each mouse. After 7 days, tumors begin to grow subcutaneously in mice. After 10 days, the tumors grow to about 100 mm 3 , and the mouse colorectal cancer model is successfully established.

[0049] Divide the successfully modeled Balb / c mice into three groups, namely the PBS group, the CXB group, and the CXB-mPEG-SD group, with 5 mice in each group. When the tumor volume reaches 100 mm 3

[0050] Start administering drugs when it is about 100 mm or so, administer drugs once every two days, the administration dose is 20 mg / kg, and the administration method is tail vein injection. Observe the growth status of Balb / c mice every two days, record the body weight of Balb / c mice and the growth of tumors. Sacrifice the mice after 21 days, calculate the tumor volume (V) and the tumor inhibition rate (IR), draw curves. The calculation method of the tumor volume is shown as "Equation (2)", and the calculation method of the tumor inhibition rate is shown as "Equation (3)".

[0051]

[0052]

[0053] Administer drugs on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, and 17th days after successful modeling, record the body weight and tumor volume of each group of mice. On the second day after the drug administration is completed, sacrifice the mice, and dissect the tumors of five mice in each group respectively. As Figure 5 shown in (A) and (B), the tumor volumes of the celecoxib group and the prodrug solid dispersion are significantly smaller than those of the PBS group. The tumor volume of the CXB-mPEG-SD group is significantly smaller than that of the CXB group, and the growth rate is significantly slowed down. Compared with the PBS group, there is a significant difference (P<0.001). As Figure 5 shown in (C), the tumor inhibition rate of the CXB group is 33%, and the tumor inhibition rate of CXB-mPEG-SD is 59%. The above results prove that both CXB and CXB-mPEG-SD have antitumor effects, and the antitumor effect of CXB-mPEG-SD is significantly stronger than that of CXB. As Figure 5As shown in (D), there was no significant difference in the body weight of the mice in the drug intervention group, indicating that the prodrug solid dispersion had good biocompatibility.

Claims

1. A celecoxib prodrug solid dispersion CXB-mPEG-SD prepared by electrostatic spraying, characterized in that: It is a celecoxib prodrug solid dispersion prepared from CXB-mPEG in a carrier solution by electrospray technology. The solubility of the CXB-mPEG-SD in water is 1.52 mg / mL, and the drug loading is 49.36%. The CXB-mPEG is a pH-responsive prodrug obtained by covalently binding celecoxib as the parent drug with polyethylene glycol 2000 mPEG2000. The catalysts used include: triethylamine and sodium hydride. The structural formula of the CXB-mPEG is shown as follows: The celecoxib prodrug solid dispersion CXB-mPEG-SD prepared based on electrospray, wherein the preparation method of the CXB-mPEG includes the following steps: Step 1: Dissolve 5 mmol of p-NPC in 10 mL of dichloromethane solution for later use. Weigh 1 mmol of mPEG2000 precisely, add 5 mmol of triethylamine as a catalyst, and add 20 mL of dichloromethane solution to dissolve it fully. Under ice bath and anaerobic conditions, dropwise add the p-NPC solution. After complete addition, raise the reaction temperature to room temperature and continue the reaction for 24 h. Precipitate with ether and dry in vacuum to obtain 2 g of the product mPEG-NPC. Step 2: Take 2.4 mmol of celecoxib and add 25 mL of DMF to dissolve it. Under ice bath conditions, add 5.1 mmol of sodium hydride in batches. After the addition is completed, keep stirring in the ice bath state for 30 minutes. Add 1 mmol of mPEG-NPC and react for 20 minutes. Raise the reaction temperature to 50 °C and continue the reaction for 12 h. Prepare a solution by adding 1.7 mmol of citric acid to 100 mL of deionized water and cool it to 0 °C for later use. Under ice bath conditions, pour the reaction solution into 100 mL of ice-citric acid solution, filter off the precipitated celecoxib, and then dialyze in deionized water at 4 °C using MWCO 1000 Da for 3 times for 24 h, 8 hours each time, and freeze-dry to obtain the product CXB-mPEG as a white solid.

2. A preparation method of celecoxib prodrug solid dispersion CXB-mPEG-SD prepared by electrostatic spraying according to claim 1, characterized in that: Weigh a certain amount of celecoxib prodrug and carrier, dissolve them in a mixed solvent, stir to obtain a clear and transparent electrospray solution. Absorb the electrospray solution into a syringe, expel air bubbles, place the syringe on a constant flow injection pump, adjust the distance, flow rate, voltage, temperature, and humidity. A positive voltage is connected to the injection needle, and a negative voltage is connected to the receiving plate. The jet ejected from the needle is collected on the tin foil on the receiving plate, and the powder on the tin foil is collected.

3. Use of the celecoxib prodrug solid dispersion CXB-mPEG-SD prepared based on electrospray as claimed in claim 1 in the preparation of a nano drug delivery system.

4. Use of the celecoxib prodrug solid dispersion CXB-mPEG-SD prepared based on electrospray as claimed in claim 1 in the preparation of a drug delivery system.

5. Use of the celecoxib prodrug solid dispersion CXB-mPEG-SD prepared based on electrospray as claimed in claim 1 in the preparation of anti-inflammatory, analgesic, and anti-colorectal cancer drugs.

6. Use of a celecoxib prodrug solid dispersion CXB-mPEG-SD prepared by electrostatic spraying according to claim 5 in the preparation of anti-inflammatory, analgesic and anti-colorectal cancer drugs, characterized in that: The drug is an injectable injection.

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