Fulvestrant nanocrystal gel preparation and preparation method thereof

Abstract

The invention discloses fulvestrant nanocrystals as well as a preparation method and application thereof. The fulvestrant nanocrystals are prepared from dispersion liquid of fulvestrant, a stabilizer and water through ultrasonic treatment. According to the fulvestrant nanocrystal hydrogel and the preparation method thereof, the fulvestrant is prepared into nanocrystals and then wrapped with the temperature-sensitive hydrogel, the advantages of the nanocrystals and the temperature-sensitive gel are combined, the fulvestrant nanocrystal hydrogel is prepared, the nanocrystal technology is successfully applied to development of long-acting injections, and the fulvestrant nanocrystal hydrogel is good in preparation treatment effect, low in cost and simple in process; and the pain of a patient caused by injection of an irritant oil solvent is effectively avoided, so that the preparation becomes an effective preparation with good compliance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a fulvestrant nanocrystal and a preparation method and application thereof. Background Art

[0002] Nanomedicines can improve the bioavailability and therapeutic effects of drugs due to their unique physical and chemical properties, such as high specific surface area, enhanced solubility and biocompatibility. Nanomedicines show great potential in targeted therapy, drug release control, and reducing toxic side effects. Nanocrystals are an important form of nanomedicine, mainly referring to the presence of drugs in the form of nanoscale crystals. The preparation of nanocrystals is usually achieved through anti-solvent method, ultrasonic method or high-pressure homogenization and other technologies. Its advantage is that it significantly improves the solubility and bioavailability of poorly soluble drugs, making these drugs easier to absorb in the body. Nanocrystals show broad application prospects in the fields of tumor treatment, anti-infection and chronic disease management. By adjusting the particle size and morphology of nanocrystals, precise control of drug release rate and targeting can be achieved, thereby improving the therapeutic effect and reducing side effects.

[0003] Thermosensitive hydrogel is a polymer material with temperature-responsive properties. Its structure undergoes reversible changes at different temperatures, which makes it have wide application potential in the biomedical field. This material is usually composed of polymer chains that can absorb water to expand or shrink at a specific temperature to form a gel state. This property makes thermosensitive hydrogels show excellent performance in tumor treatment and other aspects. In tumor treatment, thermosensitive hydrogels can be used as drug carriers to control the release rate of drugs by adjusting the temperature. When the hydrogel is at body temperature or the temperature of the tumor microenvironment, the structure of the gel will change, thereby regulating the release of the drug. This temperature-regulated release mechanism not only improves the bioavailability of the drug, but also reduces the side effects on normal tissues and enhances the therapeutic effect. At the same time, thermosensitive hydrogels can also be used for targeted therapy. Encapsulating anti-tumor drugs or gene drugs in thermosensitive hydrogels can achieve precise release of tumor cells. When the local temperature of the tumor rises, the hydrogel will change from liquid phase to solid phase, thereby concentrating its effect on the tumor area and improving the effectiveness of treatment. In addition, the application of thermosensitive hydrogels in tumor treatment is also reflected in their biocompatibility and biodegradability. Thermosensitive hydrogels are compatible with organisms and reduce the risk of immune response and toxicity. At the same time, as the treatment is completed, the degradation of hydrogels in the body also helps to avoid potential problems caused by long-term residues.

[0004] Fulvestrant is a new type of anti-estrogen drug, which is widely used in the treatment of breast cancer. Its mechanism of action is mainly to reduce tumor cell proliferation by inhibiting the activation of estrogen receptors. However, the solubility of fulvestrant in water is extremely low, which limits its clinical application. Patent CN103070871A discloses a pharmaceutical composition of fulvestrant composed of fulvestrant, at least one pharmaceutically acceptable alcohol, medium-chain triglycerides and castor oil matrix. Although the dosage form is sufficient, it still faces the pain caused by oil solution injection to patients. Patent CN1394141A discloses a fulvestrant sustained-release injection containing a ricinoleate excipient (also containing at least one alcohol and a non-aqueous ester solvent miscible in the ricinoleate excipient). Although the dosage form reduces the intramuscular injection volume, it still faces the pain caused by oil solution injection to patients. CN115554269A discloses a fulvestrant microsphere. Although the preparation makes the release of the drug in the body stable and controllable, and avoids the stimulation caused by the use of oil solvents, the microsphere preparation process is complicated and the cost is high, which limits its wide application. CN103221052A discloses an inclusion complex of fulvestrant and cyclodextrin, which uses water-soluble unsubstituted or substituted α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin to include fulvestrant, overcomes the water solubility of fulvestrant, and avoids the pain caused to patients by the use of oil solvents, but cyclodextrin is mainly excreted through the kidneys, and the use of cyclodextrin will increase the burden on the kidneys, and its danger cannot be ignored.

[0005] Therefore, the present application aims to provide a fulvestrant nanocrystal and prepare it into a thermosensitive hydrogel to meet current treatment needs and technical challenges. Summary of the invention

[0006] One of the purposes of the present invention is to provide a fulvestrant nanocrystal to improve the poor water solubility of fulvestrant.

[0007] Another object of the present invention is to provide a fulvestrant nanocrystal thermosensitive hydrogel to achieve long-term sustained release of fulvestrant in tumors.

[0008] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A fulvestrant nanocrystal suspension is obtained by subjecting a dispersion of fulvestrant, a stabilizer and water to ultrasound; the stabilizer is selected from one or more of polyvinyl pyrrolidone, poloxamer 188, Tween-80 and poloxamer 407.

[0010] Furthermore, the mass ratio of fulvestrant to the stabilizer is 1:0.01-1:5. The average particle size of the fulvestrant nanocrystals is between 150-1200 nm.

[0011] The preparation method of the above-mentioned fulvestrant nanocrystal suspension comprises the following steps:

[0012] (1) dissolving a stabilizer in water to form a stabilizer aqueous dispersion medium;

[0013] (2) dissolving fulvestrant in DMSO to form a fulvestrant DMSO solution;

[0014] (3) Under stirring conditions, adding the fulvestrant DMSO solution to the stabilizer aqueous dispersion medium, continuing stirring to disperse the fulvestrant in the stabilizer aqueous dispersion medium, and ultrasonicating to obtain the fulvestrant nanocrystal suspension.

[0015] Furthermore, the ultrasonic treatment time is 5-15 minutes.

[0016] A fulvestrant nanocrystal dry powder is prepared by freeze-drying the fulvestrant nanocrystal suspension.

[0017] In a specific embodiment of the present invention, the fulvestrant nanocrystal suspension is first pre-frozen and then freeze-dried to obtain the fulvestrant nanocrystal freeze-dried powder.

[0018] A fulvestrant nanocrystal thermosensitive hydrogel is obtained by dispersing the fulvestrant nanocrystal dry powder in a gel matrix, wherein the mass ratio of the fulvestrant nanocrystal to the gel matrix is ​​1:200-1:400.

[0019] Furthermore, the gel matrix is ​​poloxamer 407, poloxamer 188, or a mixture of poloxamer 407 and poloxamer 188.

[0020] In one embodiment of the present invention, the method for preparing the fulvestrant nanocrystalline thermosensitive hydrogel comprises the following steps:

[0021] (1) adding the gel matrix into water to swell and obtain a gel matrix solution;

[0022] (2) Mixing the fulvestrant nanocrystal dry powder with the gel matrix solution under ice bath stirring conditions, and continuing to stir to obtain the fulvestrant nanocrystal thermosensitive hydrogel.

[0023] Beneficial effects:

[0024] (1) The fulvestrant nanocrystals prepared by the present invention have a simple prescription, a simple preparation process, an average particle size of 150-1200 nm, a good particle size distribution, and good stability, which is conducive to storage and stability during transportation, and provides a good prerequisite for injection treatment. In addition, the preparation process of the fulvestrant nanocrystal thermosensitive hydrogel described in the present invention is simple, easy to scale up production, and suitable for industrialization.

[0025] (2) Compared with ordinary fulvestrant injection, the fulvestrant nanocrystalline thermosensitive hydrogel prepared by the present invention has a significantly increased tumor retention amount and a significantly prolonged retention time after verification in an in vivo mouse tumor model, which provides a reliable experimental basis for improving the therapeutic effect and application scope of fulvestrant.

[0026] (3) The present invention prepares fulvestrant nanocrystals after preparing them with thermosensitive hydrogels, combining the advantages of nanocrystals and thermosensitive gels to prepare fulvestrant nanocrystal hydrogels. The nanocrystal technology is successfully applied to the development of long-acting injections. The preparation has good therapeutic effects, low cost, simple process, and effectively avoids the pain caused to patients by injections of irritating oil solvents, making it an effective preparation with good compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a transmission electron microscopy image of the fulvestrant nanocrystal suspension in Example 4.

[0028] Figure 2 This is a real picture of Fulvestrant nanocrystal thermosensitive hydrogel under low temperature conditions.

[0029] Figure 3 This is a real picture of the thermosensitive hydrogel of fulvestrant nanocrystals after being in a 37°C water bath for 2 minutes.

[0030] Figure 4 This is the cytotoxicity test curve of fulvestrant nanocrystal suspension.

[0031] Figure 5 This is the in vivo imaging image of the intratumor retention experiment in Example 14. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.2 mg of polyvinyl pyrrolidone and completely dissolve it in 2 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0037] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 238.37 nm, and the PDI value is 0.147.

[0038] Example 2

[0039] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.4 mg of Poloxamer 188 and completely dissolve it in 2 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0040] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 198.18 nm, and the PDI value is 0.161.

[0041] Example 3

[0042] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.2 mg of Tween 80 and completely dissolve it in 4 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0043] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 671.34 nm, and the PDI value is 0.232.

[0044] Example 4

[0045] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.2 mg of Poloxamer 407 and completely dissolve it in 4 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0046] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 228.79 nm, and the PDI value is 0.141.

[0047] Example 5

[0048] Preparation of Fulvestrant nanocrystal suspension: 2 mg of Fulvestrant was accurately weighed and completely dissolved in 50 μL DMSO. The oil phase was slowly added dropwise to 4 ml of water under magnetic stirring, and the magnetic stirring was continued for 5 min to disperse Fulvestrant in the water, and ultrasonicated for 5 min.

[0049] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 527.34 nm, and the PDI value is 0.277.

[0050] Example 6

[0051] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.2 mg of Poloxamer 407 and 0.2 mg of Poloxamer 188 and completely dissolve them in 4 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0052] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 364.28 nm, and the PDI value is 0.178.

[0053] Example 7

[0054] Preparation of Fulvestrant nanocrystal suspension: Accurately weigh 2 mg of Fulvestrant and completely dissolve it in 50 μL DMSO. Take 0.2 mg of Poloxamer 407 and completely dissolve it in 4 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse Fulvestrant in the stabilizer water dispersion medium, and sonicate for 15 minutes.

[0055] The average particle size of the fulvestrant nanocrystal suspension prepared by the method is 224.28 nm, and the PDI value is 0.147.

[0056] Example 8

[0057] Comparative experiment on preparation of fulvestrant nanocrystal suspension

[0058] The preparation method of this embodiment is the same as that of Example 4, except that the ratio of the fulvestrant API to the stabilizer used is different. Two groups of experiments are designed. The mass ratio of the fulvestrant API to the stabilizer used in experimental group a is 1:10, and the mass ratio of the fulvestrant API to the stabilizer used in experimental group b is 1:100.

[0059] Example 9

[0060] Comparative experiment on preparation of fulvestrant nanocrystal suspension

[0061] The preparation method of this embodiment is the same as that of embodiment 4, except that the ultrasonic treatment of the suspension is different. Two groups of experiments are designed. The ultrasonic treatment time of the suspension of experimental group c is 1 min, and the ultrasonic treatment time of experimental group d is 60 min.

[0062] The particle sizes and PDI of the fulvestrant nanocrystals obtained by changing the ratio of the fulvestrant bulk drug to the stabilizer and changing the ultrasonic time in Example 4, Example 8 and Example 9 are compared. The results are shown in Table 1.

[0063] Table 1

[0064]

[0065] It can be seen that the fulvestrant nanocrystals prepared when the ratio of the fulvestrant raw material to the stabilizer and the ultrasonic time used in the preparation process of the fulvestrant nanocrystals are within the scope of the present invention have a particle size of 150-1200 nm and a PDI of 0.005-0.300, which can effectively improve the saturated solubility of fulvestrant, while the preparation operation beyond the scope of the present invention cannot obtain a qualified product.

[0066] Example 10

[0067] Preparation of fulvestrant nanocrystal freeze-dried powder: 10 ml of the fulvestrant suspension prepared in Example 4 was placed in a watch glass, pre-frozen in a -80°C refrigerator for 8 h, and then immediately transferred to a freeze dryer for freeze drying for 48 h to obtain a full and loose fulvestrant nanocrystal freeze-dried powder.

[0068] Embodiment 11

[0069] Preparation of Fulvestrant nanocrystal thermosensitive hydrogel: 0.2 g of poloxamer 407 was dissolved in 2 ml of water and placed in a 4°C refrigerator to fully swell for 48 h. Then, 1 mg of the fulvestrant nanocrystal freeze-dried powder prepared in Example 10 was added under magnetic stirring in an ice bath, and magnetic stirring was continued for 5 min under ice bath conditions.

[0070] Example 12

[0071] Cytotoxicity test of fulvestrant nanocrystal suspension

[0072] 4T1 cells were inoculated into a 96-well plate at a concentration of 5000 per well. After culturing for 24 hours, an equal amount of the Fulvestrant nanocrystal suspension (Ful-NCs) prepared in Example 4 and a Fulvestrant API suspension (Ful) at a concentration of 0.5 mg / ml were added. After culturing for 24 hours, 10 μl of thiazolyl blue solution was added to each well. After further culturing for 4 hours, 100 μl of DMSO was added to each well and shaken on a shaker for 10 minutes. The absorbance of each well was measured at 570 nm using an ELISA reader. The results are shown in FIG. Figure 4As shown, the cytotoxicity of the fulvestrant nanocrystal suspension was lower than that of the fulvestrant API suspension.

[0073] Embodiment 13

[0074] In vivo antitumor efficacy of fulvestrant nanocrystal thermosensitive hydrogel

[0075] Female mice were injected subcutaneously with 4T1 cells in the left axilla. When the tumor volume reached approximately 500 mm 3 At the same time, the mice were divided into 5 groups, each with 8 mice, and injected intratumorally with 200 μl of normal saline, blank gel preparation (25% poloxamer 407 solution), the fulvestrant nanocrystal suspension prepared in Example 3, 0.5 mg / ml fulvestrant castor oil solution, and the fulvestrant nanocrystal thermosensitive hydrogel prepared in Example 11, respectively. Each group of mice received the same dosing regimen. After dosing, the mice were observed for 20 days, and the tumor size was monitored every 2 days. On the 20th day, the mice were quickly decapitated and killed.

[0076] Embodiment 14

[0077] Preparation of hybrid fulvestrant nanocrystal suspension: Accurately weigh 2 mg of fulvestrant and completely dissolve it in 50 μL of DMSO solution containing 10 μL Dir. Take 0.2 mg of poloxamer 407 and completely dissolve it in 4 mL of water. Slowly add the oil phase to the water phase under magnetic stirring, continue magnetic stirring for 5 minutes, disperse fulvestrant in the stabilizer water dispersion medium, and sonicate for 5 minutes.

[0078] Embodiment 15

[0079] Preparation of hybrid fulvestrant nanocrystal thermosensitive hydrogel: The hybrid fulvestrant nanocrystal suspension prepared in Example 14 was used to prepare hybrid fulvestrant nanocrystal freeze-dried powder in the same manner as in Example 10. The hybrid fulvestrant nanocrystal thermosensitive hydrogel was prepared in the same manner as in Example 11.

[0080] Example 16

[0081] Female mice were injected subcutaneously with 4T1 cells. When the tumor volume reached about 500 mm 3 At 14:00, 200 μl of Dir ethanol solution (Dir concentration was 1.25 μl per ml), the hybrid fulvestrant nanocrystal suspension prepared in Example 14, and the hybrid fulvestrant nanocrystal thermosensitive hydrogel prepared in Example 15 were injected intratumorally. The tumors were removed and in vivo imaging was performed on days 0, 1, 3, 9, 9, 12, and 15 after administration.

[0082] like Figure 5As shown, the fluorescence of the nanocrystal suspension and Dir ethanol solution groups disappeared on the 12th and 3rd days, respectively, while the fluorescence of the nanocrystal thermosensitive hydrogel group still existed until the 15th day.

Claims

1. A fulvestrant nanocrystal suspension, characterized in that: The dispersion of fulvestrant, a stabilizer and water is obtained by ultrasonic treatment; the stabilizer is selected from one or more of polyvinyl pyrrolidone, poloxamer 188, Tween-80 and poloxamer 407.

2. The fulvestrant nanocrystal suspension according to claim 1, characterized in that: The mass ratio of fulvestrant to the stabilizer is 1:0.01-1:

5.

3. The method for preparing the fulvestrant nanocrystal suspension according to claim 1, characterized in that: The steps include: (1) Dissolving the stabilizer in water to form a stabilizer aqueous dispersion medium; (2) dissolving fulvestrant in DMSO to form a fulvestrant DMSO solution; (3) Under stirring conditions, adding the fulvestrant DMSO solution to the stabilizer aqueous dispersion medium, continuing stirring to disperse the fulvestrant in the stabilizer aqueous dispersion medium, and ultrasonicating to obtain the fulvestrant nanocrystal suspension.

4. The preparation method according to claim 3, characterized in that: The ultrasonic time is 5-15 minutes.

5. A fulvestrant nanocrystalline dry powder, characterized in that: The fulvestrant nanocrystal suspension is prepared by freeze-drying the fulvestrant nanocrystal suspension as claimed in claim 1.

6. A fulvestrant nanocrystalline thermosensitive hydrogel, characterized in that: The method is obtained by dispersing the fulvestrant nanocrystalline dry powder described in claim 5 in a gel matrix, and the mass ratio of the fulvestrant nanocrystalline dry powder to the gel matrix is ​​1:200-1:

400.

7. The fulvestrant nanocrystalline thermosensitive hydrogel according to claim 6, characterized in that: The gel matrix is ​​poloxamer 407, poloxamer 188, or a mixture of poloxamer 407 and poloxamer 188.

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