Targeted breast cancer stem cell small-molecule inhibitor capsule and preparation method thereof

Abstract

The invention relates to a preparation method of a targeting breast cancer stem cell small-molecule inhibitor capsule, and belongs to the technical field of pharmaceutical preparations. The capsule comprises the following components: pipericide, glutathione sensitive poly (disulfide bond)-PEG (SS-PEG), starch, pregelatinized starch, talcum powder and glycerol for plasticizing a capsule shell, the weight percentage of each component is as follows: 25-35% of piperaciliil, 5-10% of SS-PEG, 30-40% of starch, 20-30% of pregelatinized starch and 1-3% of talcum powder; the capsule shell is a No. 0 gelatin capsule and contains 3% (w / w) glycerol as a plasticizer; the average particle size of the nanoparticles in the capsule is 100-200nm, the drug loading capacity is greater than or equal to 85%, and the encapsulation efficiency is greater than or equal to 90%. The capsule prepared by the invention can improve the stability, bioavailability and targeting property of pipericide, so that the inhibition effect on breast cancer stem cells is enhanced, and the treatment effect on breast cancer is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a small molecule inhibitor capsule targeting breast cancer stem cells and a preparation method thereof. BACKGROUND

[0002] Breast cancer is one of the common malignant tumors threatening women's health. Although there are currently various treatment methods such as surgery, chemotherapy, radiotherapy and targeted therapy, the recurrence and metastasis of breast cancer are still important reasons for treatment failure and patient death. Studies have shown that breast cancer stem cells (BCSCs) play a key role in the occurrence, development, recurrence and metastasis of breast cancer. BCSCs have characteristics such as self-renewal, multilineage differentiation and drug resistance, and traditional treatment methods are difficult to completely eliminate BCSCs, thereby leading to tumor recurrence and metastasis.

[0003] Palbociclib is a small molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 / 6) and has been approved for the treatment of hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. However, palbociclib has problems such as poor water solubility, low bioavailability and insufficient targeting in vivo, which limit its effectiveness in clinical treatment. Therefore, developing a pharmaceutical preparation that can effectively improve the stability, bioavailability and targeting of palbociclib is of great significance for improving the treatment of breast cancer.

[0004] In recent years, the application of nanotechnology in the field of drug delivery has provided a new idea for solving the above problems. Glutathione-sensitive poly (disulfide) -PEG (SS-PEG) as a new type of nanocarrier material can rapidly degrade in the high-concentration glutathione environment in cells, release the drug and achieve targeted drug delivery. The combination of palbociclib and SS-PEG to prepare nanocapsules is expected to solve the existing problems of palbociclib and improve its targeted treatment effect on breast cancer stem cells. SUMMARY

[0005] In order to solve the above problems, the present application provides a small molecule inhibitor capsule targeting breast cancer stem cells, which comprises: palbociclib, glutathione-sensitive poly (disulfide) -PEG (SS-PEG), starch, pregelatinized starch, talc and glycerol for capsule shell plasticization; the weight percentage of each component is:

[0006] palbociclib 25-35%,

[0007] SS-PEG 5-10%,

[0008] starch 30-40%,

[0009] pregelatinized starch 20-30%,

[0010] talc 1-3%;

[0011] The capsule shell is a No. 0 gelatin capsule, which contains 3% (w / w) of glycerol as a plasticizer;

[0012] The average particle size of the nanoparticles in the capsule is 100-200 nm, the drug loading is ≥85%, and the encapsulation efficiency is ≥90%.

[0013] A preparation method of a small molecule inhibitor capsule targeting breast cancer stem cells, according to mass fraction:

[0014] H1: Preparation of SS-PEG

[0015] Raw material preparation: carboxyl-polyethylene glycol, L-cystine, soybean protease and ethanol are prepared;

[0016] Crosslinking reaction: 10-20 parts of carboxyl-polyethylene glycol, 3-7 parts of L-cystine are mixed, 50-100 parts of ethanol is added, and 0.1-0.5 parts of soybean protease is added, stirred at 25°C for 24-36 hours;

[0017] Purification treatment: the reaction solution is dialyzed with a molecular weight cutoff of 1000 Da to remove unreacted monomers, then freeze-dried to obtain yellow solid SS-PEG;

[0018] H2: Preparation of drug-polymer nanoparticles:

[0019] 25-35 parts of Plinabulin is dissolved in 200 parts of a mixture of ethanol-acetone with a volume ratio of 1:1, and ultrasonic treatment is carried out at 25°C;

[0020] Dropwise add 500 parts of water solution containing 0.5% (w / w) polyvinyl alcohol, stir at a speed of 400-600 rpm for 2-4 hours;

[0021] Then centrifuge at 4°C at a speed of 12000-15000 rpm for 30 minutes, collect the nanoparticles, and freeze-dry to obtain drug-polymer nanoparticles;

[0022] H3: Preparation of capsule content:

[0023] Mix 100 parts of drug-polymer nanoparticles with 30-40 parts of starch, 20-30 parts of pregelatinized starch, pass through a 100 mesh sieve, add 1-3 parts of talc, and mix in a three-dimensional mixer at a speed of 15-25 rpm for 10-20 minutes to obtain the capsule content;

[0024] H4: Capsule filling method: using a fully automatic capsule filling machine to fill the capsule content into a 0 size gelatin capsule, the loading capacity of each capsule is 300 parts, the 0 size gelatin capsule contains 3% (w / w) glycerol as a plasticizer.

[0025] Further, the power of the ultrasonic treatment is 200-300 W, and the time is 15-20 minutes.

[0026] Further, the temperature of the freeze-drying is -50℃ to -40℃, and the time is 24-36 hours.

[0027] Reaction mechanism:

[0028] The soybean protease molecule contains amino groups and other active groups, and the amino group plays an important role in the reaction. On the one hand, it can react with the carboxyl group of carboxyl-polyethylene glycol to form an amide bond, promote the activation of carboxyl-polyethylene glycol, and make carboxyl-polyethylene glycol more easily react with L-cystine; on the other hand, the amino group of soybean protease can act as a catalytic site, reduce the activation energy of the reaction between carboxyl-polyethylene glycol and L-cystine by interacting with L-cystine, thereby accelerating the cross-linking reaction between the two.

[0029] Technical effects:

[0030] The preparation method of the breast cancer stem cell small molecule inhibitor capsule has the following remarkable effects compared with the prior art:

[0031] 1. Strong targeting: SS-PEG in the present application has sensitivity to glutathione, and the concentration of glutathione in breast cancer stem cells is relatively high. When the capsule enters the body, SS-PEG will rapidly degrade in the high glutathione environment around the breast cancer stem cells, releasing palbociclib and achieving targeted delivery to breast cancer stem cells, increasing the concentration of the drug in breast cancer stem cells, and enhancing the inhibitory effect on breast cancer stem cells, while reducing the toxic side effects on normal cells.

[0032] 2. High bioavailability: By preparing palbociclib into a nanoparticle form, the surface area of the drug is increased, and the solubility of the drug is improved (after using an ethanol-acetone mixed solvent, the solubility of palbociclib in the solvent is increased to 3.5 mg / mL). Compared with free palbociclib, the in vitro dissolution rate of palbociclib in nanoparticle form is significantly improved, thereby promoting the absorption of the drug and improving the bioavailability of the drug.

[0033] 3. Good stability: after freeze-drying treatment of the drug-polymer nanoparticles, the drug content is still ≥95% after storage at 4℃ for 6 months, indicating that the capsule prepared by the present application has good stability, which is conducive to the storage and transportation of the drug.

[0034] 4. The preparation method is unique and has obvious advantages: the present application uses soybean protease to participate in the preparation of SS-PEG, and the amino group of the soybean protease plays an important role, not only promoting the activation of the carboxyl-polyethylene glycol, but also accelerating the cross-linking reaction with L-cystine, so that the preparation process is more efficient and controllable. Compared with the traditional preparation method, the operation steps of the preparation method of the present application are clear, the process conditions are easy to control, the raw materials and equipment used are conventional pharmaceutical production raw materials and equipment, and the present application is suitable for large-scale industrial production.

[0035] 5. The role of nanotechnology in the preparation of the capsule mainly includes: improving the solubility and dissolution rate of polybusexil, thereby improving the bioavailability; realizing targeted drug delivery, reducing the toxic side effects on normal cells; protecting the drug from external factors, enhancing the stability; optimizing the particle size, drug loading and other physical properties of the nanocapsule, which is beneficial to the preparation processing. DETAILED DESCRIPTION

[0036] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined purpose, the following embodiments and comparative examples are described in detail:

[0037] Example 1

[0038] Preparation of SS-PEG:

[0039] 10 g of carboxyl-polyethylene glycol (molecular weight 5000 Da) and 3 g of L-cystine were mixed, 50 g of ethanol was added, and 0.1 g of soybean protease was added, and stirred at 25℃ for 24 hours;

[0040] The reaction solution was dialyzed (molecular weight cut-off 1000 Da) to remove unreacted monomers, and then freeze-dried at -50℃ for 24 hours to obtain yellow solid SS-PEG.

[0041] Preparation of drug-polymer nanoparticles:

[0042] 25 g of polybusexil and 5 g of SS-PEG were dissolved in 200 g of ethanol-acetone mixed solvent (volume ratio 1:1), and ultrasonically treated at 25℃ with a power of 200W for 15 minutes;

[0043] 500 g of an aqueous solution containing 0.5% (w / w) polyvinyl alcohol was added dropwise, and stirred at a rotation speed of 400 rpm for 2 hours;

[0044] Then centrifuged at 4℃ at a rotation speed of 12000 rpm for 30 minutes, and the nanoparticles were collected and freeze-dried at -50℃ for 24 hours to obtain drug-polymer nanoparticles.

[0045] Preparation of capsule contents:

[0046] 100 g of the drug-polymer nanoparticle was mixed with 30 g of starch, 20 g of pregelatinized starch, and 1 g of talc powder, and sieved through a 100-mesh sieve. The mixture was mixed in a three-dimensional mixer at a rotation speed of 15 rpm for 10 minutes to obtain a capsule content.

[0047] Capsule filling:

[0048] The capsule content was filled into a 0 size gelatin capsule using a full-automatic capsule filling machine, and the loading amount of each capsule was 300 mg. The 0 size gelatin capsule contained 3% (w / w) of glycerol as a plasticizer.

[0049] Example 2

[0050] Preparation of SS-PEG:

[0051] 15 g of carboxyl-polyethylene glycol (molecular weight 5000 Da) and 5 g of L-cystine were mixed, 75 g of ethanol was added, and 0.3 g of soybean protease was added. The mixture was stirred at 25°C for 30 hours.

[0052] The reaction solution was dialyzed (molecular weight cut-off 1000 Da) to remove unreacted monomers, and then freeze-dried at -45°C for 30 hours to obtain a yellow solid of SS-PEG.

[0053] Preparation of drug-polymer nanoparticles:

[0054] 30 g of elacridar and 7 g of SS-PEG were dissolved in 200 g of an ethanol-acetone mixed solvent (volume ratio 1:1), and ultrasonicated at 25°C for 18 minutes at a power of 250 W.

[0055] The solution was added dropwise to 500 g of an aqueous solution containing 0.5% (w / w) of polyvinyl alcohol, and stirred at a rotation speed of 500 rpm for 3 hours.

[0056] Then, the nanoparticles were collected by centrifugation at 4°C at a rotation speed of 13000 rpm for 30 minutes, and freeze-dried at -45°C for 30 hours to obtain drug-polymer nanoparticles.

[0057] Preparation of capsule content:

[0058] 100 g of the drug-polymer nanoparticle was mixed with 35 g of starch, 25 g of pregelatinized starch, and 2 g of talc powder, and sieved through a 100-mesh sieve. The mixture was mixed in a three-dimensional mixer at a rotation speed of 20 rpm for 15 minutes to obtain a capsule content.

[0059] Capsule filling:

[0060] The capsule content was filled into size 0 gelatin capsules using an automatic capsule filling machine, with a fill weight of 300 mg per capsule, the size 0 gelatin capsules containing 5% (w / w) glycerol as a plasticizer.

[0061] Example 3

[0062] Preparation of SS-PEG:

[0063] 20 g of carboxyl-polyethylene glycol (molecular weight 5000 Da) and 7 g of L-cystine were mixed, 100 g of ethanol was added, and 0.5 g of soybean protease was added, and stirred at 25°C for 36 hours;

[0064] The reaction solution was dialyzed (molecular weight cut-off 1000 Da) to remove unreacted monomers, and then freeze-dried at -40°C for 36 hours to obtain a yellow solid of SS-PEG.

[0065] Preparation of drug-polymer nanoparticles:

[0066] 35 g of ribociclib and 10 g of SS-PEG were dissolved in 200 g of a mixed solvent of ethanol and acetone (volume ratio 1:1), and ultrasonicated at 25°C for 20 minutes at a power of 300 W;

[0067] The solution was added dropwise to 500 g of an aqueous solution containing 0.5% (w / w) polyvinyl alcohol, and stirred at a rotation speed of 600 rpm for 4 hours;

[0068] Then, the nanoparticles were collected by centrifugation at 4°C at a rotation speed of 15000 rpm for 30 minutes, and freeze-dried at -40°C for 36 hours to obtain drug-polymer nanoparticles.

[0069] Preparation of capsule content:

[0070] 100 g of drug-polymer nanoparticles were mixed with 40 g of starch and 30 g of pregelatinized starch, and passed through a 100 mesh sieve, 3 g of talc was added, and mixed in a three-dimensional mixer at a rotation speed of 25 rpm for 20 minutes to obtain the capsule content.

[0071] Capsule filling:

[0072] The capsule content was filled into size 0 gelatin capsules using an automatic capsule filling machine, with a fill weight of 300 mg per capsule, the size 0 gelatin capsules containing 6% (w / w) glycerol as a plasticizer.

[0073] 1. Nanoparticle size and drug loading, encapsulation efficiency determination

[0074] The average particle size and polydispersity index (PDI) of the nanoparticles were determined by a laser particle size analyzer (e.g. Malvern Zetasizer Nano ZS), and the drug loading and encapsulation efficiency were determined by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0075] Table 1

[0076] Average particle size / nm PDI Drug loading amount / % Encapsulation efficiency / % Example 1 142 0.18 86.5 90.6 Example 2 168 0.21 89.7 92.8 Example 3 149 0.19 87.9 91.5

[0077] 2. Drug stability test

[0078] The prepared capsules were placed at 4°C and 37°C for different time, and the drug content was determined by high performance liquid chromatography. The results are shown in Table 2 below.

[0079] Table 2

[0080] Storage condition Storage time Drug content of Example 1 / % Drug content of Example 2 / % Drug content of Example 3 / % 4℃ 0 days 100 100 100 4℃ 30 days 99.5 99.8 99.4 4℃ 90 days 98.4 98.8 98.3 4℃ 180 days 97.1 97.5 95.9 37℃ 0 days 100 100 100 37℃ 30 days 93.4 93.9 93.2

[0081] The above description is only the preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any equivalent embodiments with equivalent changes are also included. Any modification, change, equivalent change and modification of the above embodiments, which do not depart from the technical solution of the present application, are still within the scope of the technical solution of the present application.

Claims

1. A small molecule inhibitor of breast cancer stem cell targeting capsule, characterized by: The capsule comprises: poly (disulfide) -PEG (SS-PEG) sensitive to glutathione, starch, pregelatinized starch, talc and glycerol for capsule shell plasticization; the weight percentage of each component is: poly (disulfide) -PEG (SS-PEG) 5-10%, starch 30-40%, pregelatinized starch 20-30%, talc 1-3%; the capsule shell is a No. 0 gelatin capsule containing 3-6% (w / w) glycerol as a plasticizer; The average particle size of the nanoparticles in the capsule is 100-200 nm, the drug loading is ≥ 85%, and the encapsulation rate is ≥ 90%.

2. A process for the preparation of a capsule of small molecule inhibitor targeting breast cancer stem cells characterized in that: The preparation method of the capsule is as follows in terms of mass fraction: H1: preparation of SS-PEG Raw material preparation: prepare carboxyl-polyethylene glycol, L-cystine, soybean protease and ethanol; Crosslinking reaction: mix 10-20 parts of carboxyl-polyethylene glycol and 3-7 parts of L-cystine, add 50-100 parts of ethanol, then add 0.1-0.5 parts of soybean protease, and stir at 25°C for 24-36 hours; Purification treatment: dialyze the reaction solution with a molecular weight cutoff of 1000 Da to remove unreacted monomers, then freeze-dry to obtain yellow solid SS-PEG; H2: preparation of drug-polymer nanoparticles: Dissolve 25-35 parts of poly (disulfide) -PEG (SS-PEG) in 200 parts of a mixed solvent of ethanol-acetone with a volume ratio of 1:1, and perform ultrasonic treatment at 25°C; Add 500 parts of an aqueous solution containing 0.5% (w / w) polyvinyl alcohol dropwise, and stir at a rotation speed of 400-600 rpm for 2-4 hours; Then centrifuge at 4°C at a rotation speed of 12000-15000 rpm for 30 minutes, collect the nanoparticles, and freeze-dry to obtain drug-polymer nanoparticles; H3: preparation of capsule content: Mix 100 parts of drug-polymer nanoparticles with 30-40 parts of starch, 20-30 parts of pregelatinized starch, pass through a 100-mesh sieve, add 1-3 parts of talc, and mix in a three-dimensional mixer at a rotation speed of 15-25 rpm for 10-20 minutes to obtain the capsule content; H4: capsule filling method: use a full-automatic capsule filling machine to fill the capsule content into a No. 0 gelatin capsule, and the loading amount of each capsule is 300 parts, and the No. 0 gelatin capsule contains 3% (w / w) glycerol as a plasticizer.

3. The method for preparing a small molecule inhibitor capsule targeting breast cancer stem cells according to claim 2, characterized in that: The power of the ultrasonic treatment is 200-300 W, and the time is 15-20 minutes.

4. The method for preparing a small molecule inhibitor capsule targeting breast cancer stem cells according to claim 2, characterized in that: The temperature of the freeze-drying is -50°C to -40°C, and the time is 24-36 hours.