Cholesterol derivative pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and relates to a cholesterol derivative pharmaceutical composition as well as a preparation method and application thereof. The cholesterol derivative pharmaceutical composition comprises a cholesterol derivative, an adriamycin prodrug and a PEG modifier, the cholesterol derivative and the adriamycin prodrug form a pharmaceutical composition through electrostatic interaction and hydrophobic interaction, and then the pharmaceutical composition is modified by PEG. The molar ratio of the adriamycin prodrug to the cholesterol derivative to the PEG modifier is (1: 0.5: 0.1)-(1: 9: 0.5). The doxorubicin prodrug and the cholesterol derivative are simply mixed and stirred to obtain nanoparticles with uniform particle size, but doxorubicin cannot be formed, which proves that the affinity between the doxorubicin prodrug and the cholesterol derivative can be improved by enhancing the hydrophobicity of the positively charged drug. Compared with commercially available adriamycin preparations, the composition disclosed by the invention has a good anti-tumor effect, can effectively reduce the toxic and side effects of adriamycin, and meets the urgent clinical demand on high-efficiency and low-toxicity chemotherapy preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a cholesterol derivative doxorubicin prodrug composition, a preparation method and an application thereof. Background Art

[0002] In recent years, the incidence and mortality of cancer have gradually increased, posing a serious threat to the health of all mankind. At present, chemotherapy is the most commonly used method for cancer treatment. Doxorubicin (DOX) is an antitumor antibiotic drug that acts on DNA topoisomerase II and inhibits the synthesis of nucleic acids by embedding into DNA. As a broad-spectrum antitumor drug, it is widely used in the treatment of breast cancer, soft tissue sarcoma and lymphoma. At present, the marketed preparations of doxorubicin are mainly divided into two categories: doxorubicin hydrochloride injection and doxorubicin liposomes. Doxorubicin hydrochloride injection can cause severe cardiotoxicity and bone marrow suppression, and its clinical usage rate has gradually declined. Although doxorubicin liposomes It can effectively reduce the cardiotoxicity of doxorubicin, but it increases the risk of acral erythema, and its release rate at the tumor site is slow, which seriously affects the treatment effect and the quality of life of patients. Therefore, constructing an efficient and low-toxic delivery system is a difficult problem that needs to be solved in pharmacy.

[0003] Cholesterol derivatives are compounds produced by modification or metabolism of cholesterol molecules and have good biocompatibility. Their structure usually contains a hydrophobic steroid structure and a hydrophilic side chain containing a sulfate group or a carboxylic acid group. Therefore, cholesterol derivatives are usually amphiphilic. In addition, the sulfate group or carboxylic acid group in its structure has the potential to co-assemble with positively charged drugs in a neutral environment. Therefore, cholesterol derivatives are expected to be used as delivery carriers for delivering positively charged drugs (such as doxorubicin). At present, amphotericin B cholesterol sulfate sodium composition based on this strategy It has been successfully marketed. It uses the positively charged amino group in amphotericin B to assemble with the negatively charged cholesterol sodium sulfate to form a nanostructure, and then cholesterol-PEG (CHOL-PEG 2k ) modification to achieve long circulation. However, its preparation method requires the use of a mixing emulsifier for stirring and microfluidization for granulation to obtain a nanocomposite with uniform particle size, and the preparation process is relatively complicated. The reason may be that amphotericin B and sodium cholesterol sulfate are only bound by electrostatic interaction, and the affinity is insufficient, and external force is still required to further granulate to obtain nanoparticles with uniform particle size. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a cholesterol derivative pharmaceutical composition and a preparation method and application thereof. In addition to being negatively charged, cholesterol derivatives also contain four steroid rings and have strong hydrophobicity. From the perspective of enhancing the hydrophobicity of positively charged drugs, the present invention hopes to improve the affinity between cholesterol derivatives and drugs, thereby improving the stability of the pharmaceutical composition, thereby simplifying the preparation method. The doxorubicin prodrug utilizes a tumor site intelligent response chemical bond to couple the positively charged drug doxorubicin with a hydrophobic modified chain fatty acid, which can enhance the hydrophobicity of the drug molecule and promote interaction with cholesterol derivatives. In addition, the introduced intelligent response sensitive bond can also enable the drug to reach a specific part of the tumor, respond to activation and release active drugs to exert therapeutic effects.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a cholesterol derivative pharmaceutical composition, the pharmaceutical composition comprising: a cholesterol derivative, a doxorubicin prodrug, and a PEG modifier;

[0007] The cholesterol derivative and the doxorubicin prodrug form a drug composition through electrostatic and hydrophobic interactions, and then are modified by PEG; the molar ratio of the doxorubicin prodrug, the cholesterol derivative and the PEG modifier is 1:0.5:0.1-1:9:0.5.

[0008] In the above technical solution, further, the cholesterol derivative is sodium cholesterol sulfate, deoxycholic acid, chenodeoxycholic acid, sodium deoxycholate, cholic acid, sodium dehydrocholate; preferably sodium cholesterol sulfate.

[0009] In the above technical solution, further, the doxorubicin prodrug is selected from doxorubicin-acylhydrazone bond-fatty acid prodrug; preferably doxorubicin-acylhydrazone bond-myristic acid (DOX-C14) prodrug;

[0010]

[0011] In the above technical solution, further, the PEG modifier is selected from DSPE-PEG 2k or CHOL-PEG 2k .

[0012] In the above technical solution, further, the molar ratio of doxorubicin prodrug, cholesterol derivative and PEG modifier is 1:0.5:0.1-1:9:0.2; preferably 1:1:0.2-1:3:0.2.

[0013] The second aspect of the present invention provides a method for preparing the aforementioned cholesterol derivative pharmaceutical composition, comprising the following steps:

[0014] 1) dissolving the doxorubicin prodrug, cholesterol derivative and PEG modifier in an organic solvent according to a molar ratio and mixing them thoroughly;

[0015] 2) slowly adding the mixture obtained in step 1) dropwise into water under stirring;

[0016] 3) removing the organic solvent from the solution obtained in step 2) to obtain a cholesterol derivative pharmaceutical composition; preferably, the method for removing the organic solvent includes reduced pressure rotary evaporation, ultrafiltration or dialysis.

[0017] In the above technical solution, further, the volume ratio of the organic solvent to water is 1:5-1:20; preferably 1:5-1:10.

[0018] In the above technical solution, further, the organic solvent is one or more of acetonitrile, dichloromethane, methanol, ethanol and dimethyl sulfoxide; preferably methanol and dimethyl sulfoxide.

[0019] The present invention can obtain nanoparticles with uniform particle size by a simple mixing and stirring method with doxorubicin prodrug and cholesterol derivative, while doxorubicin cannot be formed, which proves that enhancing the hydrophobicity of positively charged drugs can improve the affinity with cholesterol derivatives. According to an embodiment of the present invention, the particle size of the pharmaceutical composition formed by doxorubicin and sodium cholesterol sulfate is 400-600nm, while the particle size of the pharmaceutical composition formed by doxorubicin prodrug and sodium cholesterol sulfate is 80-130nm. In addition, the type and proportion of cholesterol derivatives affect the pharmaceutical composition's pharmaceutics properties and in vivo and in vitro fate.

[0020] In the above technical solution, further, the particle size of the cholesterol derivative pharmaceutical composition is not greater than 200 nm.

[0021] The third aspect of the present invention provides the use of the aforementioned pharmaceutical composition, wherein the use comprises:

[0022] Use of the above cholesterol derivative pharmaceutical composition in the preparation of a drug for treating or preventing cancer, including breast cancer, prostate cancer, acute leukemia, malignant lymphoma, osteosarcoma, ovarian cancer and soft tissue sarcoma;

[0023] The cholesterol derivative pharmaceutical composition is used in the preparation of a drug delivery system or a drug administration system; the drug administration system is injection administration or local administration.

[0024] The present invention utilizes the intelligent response chemical bond of the tumor site to connect doxorubicin (containing an amino group that can be protonated into a positive charge) with hydrophobic myristic acid to construct a doxorubicin prodrug, so as to improve the hydrophobicity of doxorubicin. The constructed doxorubicin prodrug can form a nanostructure with a cholesterol derivative with a negative charge and strong hydrophobicity through electrostatic and hydrophobic effects, and then be modified by PEG to achieve long circulation. A nanoparticle pharmaceutical composition with uniform particle size can be constructed by a simple mixing and stirring method using a negatively charged cholesterol derivative and a positively charged doxorubicin prodrug. By optimizing the type and ratio of cholesterol derivatives and the type of PEG modifier, the best cholesterol derivative pharmaceutical composition is screened out, the toxic and side effects of doxorubicin are further reduced, and the delivery efficiency of doxorubicin is improved.

[0025] The present invention has the following beneficial effects:

[0026] (1) The hydrophobicity of doxorubicin (containing an amino group that can be protonated to a positive charge) is enhanced through a prodrug strategy, and the affinity between doxorubicin and cholesterol derivatives that are negatively charged and highly hydrophobic is improved by using doxorubicin prodrugs;

[0027] (2) The preparation method of the pharmaceutical composition of the present invention is simple. Due to the improvement of affinity, a nanopharmaceutical composition with uniform particle size can be obtained by a simple mixing and stirring method. The particle size is between 100-120 nm and the particle size distribution is uniform.

[0028] (3) The nano drug composition of the present invention has a good anti-tumor effect compared with commercially available doxorubicin preparations and can effectively reduce the toxic side effects of doxorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The structure of the doxorubicin fatty acid (DOX-C14) prodrug of Example 1 of the present invention is confirmed;

[0030] A: Mass spectrum of DOX-C14;

[0031] B:DOX-C14 1 H-NMR spectrum;

[0032] C: HPLC purity chart of DOX-C14.

[0033] Figure 2 This is a prescription screening diagram for the cholesterol derivative doxorubicin prodrug composition prepared in Example 2 of the present invention.

[0034] Figure 3 This is the potential diagram of the sodium cholesterol sulfate doxorubicin prodrug composition prepared in Example 2 of the present invention.

[0035] Figure 4This is a stability diagram of the cholesterol sodium sulfate doxorubicin prodrug composition without PEG modification in Example 3 of the present invention.

[0036] Figure 5 The particle size diagram and transmission electron microscope diagram of the PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition prepared in Example 5 of the present invention;

[0037] A: Particle size distribution diagram of the sodium cholesterol sulfate doxorubicin prodrug composition;

[0038] B: Transmission electron microscopy image of the sodium cholesterol sulfate doxorubicin prodrug composition.

[0039] Figure 6 This is a stability diagram of the PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition in Example 6 of the present invention;

[0040] A: Stability of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition in PBS;

[0041] B: Stability of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition in PBS containing 10% FBS;

[0042] C: Storage stability of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition at 4°C;

[0043] D: Storage stability of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition at 25°C.

[0044] Figure 7 This is a graph showing the in vitro release test of the sodium cholesterol sulfate doxorubicin prodrug composition in Example 7 of the present invention;

[0045] A: In vitro release test diagram of the sodium cholesterol sulfate doxorubicin prodrug composition at pH 7.4;

[0046] B: In vitro release test of the sodium cholesterol sulfate doxorubicin prodrug composition at pH 6.5;

[0047] C: In vitro release test diagram of the sodium cholesterol sulfate doxorubicin prodrug composition at pH 5.0.

[0048] Figure 8 This is a cytotoxicity graph of the sodium cholesterol sulfate doxorubicin prodrug composition in Example 8 of the present invention;

[0049] A: half inhibitory concentration of sodium cholesterol sulfate doxorubicin prodrug composition on 4T1 cells;

[0050] B: half inhibitory concentration of the sodium cholesterol sulfate doxorubicin prodrug composition on B16-F10 cells;

[0051] C: half inhibitory concentration of the sodium cholesterol sulfate doxorubicin prodrug composition on RM-1 cells;

[0052] D: half inhibitory concentration of the sodium cholesterol sulfate doxorubicin prodrug composition on L02 cells;

[0053] ns: P≥0.05; **: P<0.01; ****: P<0.0001.

[0054] Fig. 9 This is a blood drug concentration-time curve of the sodium cholesterol sulfate doxorubicin prodrug composition in Example 9 of the present invention;

[0055] A: blood drug concentration-time curve of the prodrug in the sodium cholesterol sulfate doxorubicin prodrug composition;

[0056] B: blood drug concentration-time curve of doxorubicin in the sodium cholesterol sulfate doxorubicin prodrug composition;

[0057] C: Blood drug concentration-time curve of the total amount of prodrug and doxorubicin in the sodium cholesterol sulfate doxorubicin prodrug composition.

[0058] Fig.10 This is a graph showing the in vivo anti-tumor experiment of the sodium cholesterol sulfate doxorubicin prodrug composition of Example 10 of the present invention (2.5 mg / kg);

[0059] A: A graph showing the change in tumor volume of the sodium cholesterol sulfate doxorubicin prodrug composition in an in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0060] B: Body weight change diagram of the sodium cholesterol sulfate doxorubicin prodrug composition in the anti-tumor experiment on subcutaneous breast cancer tumors of Balb / C mice;

[0061] C: Tumor-bearing rate of the sodium cholesterol sulfate doxorubicin prodrug composition in the in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0062] D: Tumor image of the sodium cholesterol sulfate doxorubicin prodrug composition in the in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0063] ns: P≥0.05; **: P<0.01; ****: P<0.0001.

[0064] Fig.11 This is a graph showing the in vivo anti-tumor experiment of the cholesterol sulfate sodium doxorubicin prodrug composition of Example 10 of the present invention (5 mg / kg and 10 mg / kg);

[0065] A: A graph showing the change in tumor volume of the sodium cholesterol sulfate doxorubicin prodrug composition in an in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0066] B: Body weight change diagram of the sodium cholesterol sulfate doxorubicin prodrug composition in the anti-tumor experiment on subcutaneous breast cancer tumors of Balb / C mice;

[0067] C: Tumor-bearing rate of the sodium cholesterol sulfate doxorubicin prodrug composition in the in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0068] D: Tumor image of the sodium cholesterol sulfate doxorubicin prodrug composition in the in vivo anti-tumor experiment on subcutaneous breast cancer tumors in Balb / C mice;

[0069] ns: P≥0.05; **: P<0.01; ****: P<0.0001. DETAILED DESCRIPTION

[0070] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0071] Example 1 Synthesis of doxorubicin fatty acid (DOX-C14) prodrug

[0072] 6mmol myristic acid and 5mmol benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were dissolved in 15-20mL N,N-dimethylformamide (DMF), stirred in an ice bath for 1 hour, and then 5mmol tert-butyloxycarbonylhydrazine and 5mmol N,N-diisopropylethylamine (DIPEA) dissolved in DMF were slowly added, and reacted at 25°C for 24 hours. The product was spin-dried and separated and purified by chromatographic column to obtain the product tert-butyloxycarbonyl-protected myristic acid hydrazide.

[0073] 3 mmol of tert-butyloxycarbonyl-protected myristic acid hydrazide was dissolved in dichloromethane, and then 8.5 mmol of trifluoroacetic acid (TFA) was added. After reacting at 25°C for 3 hours, glacial ether was added to precipitate to obtain the deprotected myristic acid hydrazide.

[0074] Then, 0.35 mmol of doxorubicin hydrochloride was dissolved in anhydrous methanol, 1 mmol of triethylamine was slowly added, and the mixture was reacted at 25°C in the dark for 12 hours. The mixture was spin-dried, 0.35 mmol of desalted doxorubicin and 1 mmol of deprotected myristic acid hydrazide were dissolved in anhydrous methanol, 4 mmol of glacial acetic acid was slowly added, and the mixture was reacted at 50°C under condensation reflux for 12 hours. The product was separated and purified by preparative liquid phase separation to obtain DOX-C14.

[0075] The structure of DOX-C14 prepared in Example 1 was determined by mass spectrometry and nuclear magnetic resonance spectroscopy. Figure 1 shown.

[0076] The results of NMR spectroscopy analysis are as follows: DOX-C14

[0077] 1H NMR (600MHz, DMSO-d6) δ10.29(s,1H),7.94-7.87(m,2H),7.66(dd,J=6.7,3.0Hz,1H),5.78(t,J=4.9Hz,1H),5.54(s,1H),5.43(d,J=6 .0Hz,1H),5.32-5.26(m,1H),4.91(t,J=7.0Hz,1H),4.45-4.36(m,2H),4.03(q,J=6.6Hz,1H),3.97(s,3H),3.58(s,1H),3.37-3.34(m, 2H),3.31(s,1H),2.73(d,J=17.3Hz,1H),2.49-2.45(m,1H),2.21-2.11(m,2H),2.06(ddd,J=15.2,8.3,6.7Hz,1H),1.90-1.84(m,1H) ,1.73(dd,J=12.5,4.6Hz,1H),1.30-1.18(m,12H),1.17(d,J=6.6Hz,3H),1.12-1.07(m,2H),1.06-0.92(m,8H),0.84(t,J=7.0Hz,3H).

[0078] The mass spectrometry results are (ESI) m / z [[M+H] + =768.40531, C 41 H 58 N3O 11 The purity results showed that the purity of DOX-C14 was 99.23%.

[0079] Example 2 Prescription Screening of Cholesterol Derivatives and Doxorubicin Fatty Acid Prodrugs

[0080] Accurately weigh cholesterol derivatives (sodium cholesterol sulfate (SCS), deoxycholic acid (DA), chenodeoxycholic acid (CDA), sodium deoxycholate (SDA), cholic acid (CA), sodium dehydrocholate (SDHA)) and dissolve them in 0.1 mL DMSO. Accurately weigh 2 mg of doxorubicin fatty acid prodrug and dissolve it in 0.1 mL of methanol. After thoroughly mixing doxorubicin fatty acid prodrug and cholesterol derivative at a molar ratio of 1:0.5-1:9, slowly add the solution dropwise to 1.8 mL of deionized water solution under stirring, and remove the organic solvent in the preparation using a rotary evaporator at 25°C. The results are as follows Figure 2As shown, the particle size does not exceed 500 and PDI < 0.3 is applicable. The particle size of the nanocomposite formed by the co-assembly of doxorubicin fatty acid prodrug and sodium cholesterol sulfate is the best. When the molar ratio of doxorubicin fatty acid prodrug to sodium cholesterol sulfate is 1:2 and 1:3, the formed nanocomposite is about 100nm, the particle size distribution is uniform, the stability is optimal, and the surface charge is between -20 and -30mV ( Figure 3 ).

[0081] Example 3 Preparation of cholesterol sodium sulfate doxorubicin prodrug composition without PEG modification

[0082] Accurately weigh 2.458 mg and 3.687 mg of sodium cholesterol sulfate and dissolve them in 0.1 mL of DMSO respectively. Accurately weigh 2 mg of doxorubicin fatty acid prodrug and dissolve it in 0.1 mL of methanol. Thoroughly mix the doxorubicin fatty acid prodrug and the sodium cholesterol sulfate solution and add dropwise to 1.8 mL of deionized water under stirring. After stirring for 5 minutes, use a rotary evaporator to remove the organic solvent in the preparation to obtain nanocomposites with molar ratios of 1:2 and 1:3, named DOX-C14-SCS (1:2) and DOX-C14-SCS (1:3). The results are as follows: Figure 4 As shown, DOX-C14-SCS (1:2) and DOX-C14-SCS (1:3) have uniform particle sizes and good storage stability, but poor stability in phosphate buffered saline (PBS, pH 7.4), so further surface modification is required to improve their stability.

[0083] Example 4 Preparation of cholesterol sodium sulfate doxorubicin composition without PEG modification

[0084] Accurately weigh 2.458 mg and 3.687 mg of sodium cholesterol sulfate and dissolve them in 0.1 mL of DMSO, respectively. Accurately weigh 2 mg of doxorubicin and dissolve them in 0.1 mL of methanol. Thoroughly mix the doxorubicin and sodium cholesterol sulfate solutions and add them dropwise to 1.8 mL of deionized water under stirring. After stirring for 5 minutes, use a rotary evaporator to remove the organic solvent in the preparation to obtain nanocomposites with molar ratios of 1:2 and 1:3, named DOX-SCS (1:2) and DOX-SCS (1:3). The particle sizes of DOX-SCS (1:2) and DOX-SCS (1:3) are about 400 nm and 600 nm, respectively, and the PDI is greater than 0.2. The particle size distribution is uneven and the stability is poor.

[0085] Example 5 Preparation of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0086] DSPE-PEG 2k or CHOL-PEG 2KModified nanocomposites (DOX-C14-SCS (1:2) and DOX-C14-SCS (1:3)). Accurately weigh 2.458 mg and 3.687 mg of sodium cholesterol sulfate and dissolve them in 0.1 mL of DMSO, respectively. Accurately weigh 2 mg of doxorubicin fatty acid prodrug and dissolve it in 0.1 mL of methanol. Then, the above solution was mixed with 0.4 mg of DSPE-PEG 2k or CHOL-PEG 2K Mix. Add dropwise into 1.8 mL of deionized water while stirring. After stirring for 5 minutes, use a rotary evaporator to remove the organic solvent in the preparation, and the co-assembled nanocomposites obtained are named DOX-C14-SCS (1:2) @ DSPE, DOX-C14-SCS (1:3) @ DSPE, DOX-C14-SCS (1:2) @ CHOL and DOX-C14-SCS (1:3) @ CHOL. The results are shown in Table 1. The particle size of the nanoparticles is about 100 nm, the particle size distribution is less than 0.2, the surface charge is about -20 mV, and the encapsulation efficiency is above 95%. The particle size and morphology of the above-mentioned PEG-modified sodium cholesterol sulfate doxorubicin prodrug composition were measured by transmission electron microscopy, and the results are as shown in Table 1. Figure 5 The transmission electron microscopy image shows that the sodium cholesterol sulfate doxorubicin prodrug composition is a uniform sphere with an average particle size of 100 nm.

[0087] Table 1. Particle size, particle size distribution, surface charge and encapsulation efficiency of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0088]

[0089] Encapsulation efficiency determination method: agarose gel molecular sieve is used to separate the prodrug composition and free drug, and then quantitative analysis is performed using liquid phase.

[0090] Example 6 Stability test of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0091] The PEG-modified sodium cholesterol sulfate adriamycin prodrug composition prepared in Example 5 was subjected to colloidal stability investigation. The long-term storage stability of the sodium cholesterol sulfate adriamycin prodrug composition at 4°C and 25°C was investigated using the change in particle size as an indicator. The results are shown in FIG. Figure 6 As shown in C and D, the particle size of the prepared PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition does not change significantly when placed at 4°C and 25°C, and has good long-term storage stability.

[0092] 1 mL of the PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition was taken out and added to 20 mL of phosphate buffer (PBS, pH 7.4), incubated at 37°C for 24 hours, and the particle size change was measured by dynamic light scattering at predetermined time points (0, 2, 4, 8, 12 and 24 hours). The results are as follows: Figure 6 As shown in A, the stability of the sodium cholesterol sulfate doxorubicin prodrug composition is good, and the particle size does not change significantly within 24 hours.

[0093] 1 mL of the PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition was taken out and added to 20 mL of phosphate buffered saline (PBS, pH 7.4) containing 10% FBS, and incubated at 37°C for 24 hours, and the particle size change was measured by dynamic light scattering at predetermined time points (0, 2, 4, 8, 12 and 24 hours). The results are as follows Figure 6 As shown in B, the stability of the sodium cholesterol sulfate doxorubicin prodrug composition is good, and the particle size does not change significantly within 24 hours.

[0094] Example 7 In vitro release test of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0095] The in vitro release of the sodium cholesterol sulfate doxorubicin prodrug composition was investigated using PBS containing 30% ethanol (v / v) as the release medium with pH values ​​of 7.4, 6.5, and 5.0, respectively. 1 mL of the PEG-modified nanocomposition prepared in Example 5 (100 nmol, 0.2 mL) was fully mixed with the release medium (30 mL). The above solution was quickly placed in an oscillator (37°C, 100 r / min). At a predetermined time point, 0.2 mL of the sample was taken and the released doxorubicin was determined by high performance liquid chromatography. The release of the nanocomposition under neutral and acidic conditions was investigated, respectively. The results are shown in FIG. Figure 7 As shown, the sodium cholesterol sulfate doxorubicin prodrug composition can release the parent drug in an acidic environment (pH 5.0 and 6.5), and the stronger the acidity, the faster the release rate. This is mainly because the acylhydrazone bond in the doxorubicin fatty acid prodrug structure is unstable under acidic conditions and will hydrolyze rapidly. Under neutral conditions (pH 7.4), the acylhydrazone bond remains stable and difficult to hydrolyze, and the release amount of the prodrug is very low.

[0096] Example 8 Cytotoxicity of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0097] The MTT method was used to investigate the cytotoxicity of the PEG-modified sodium cholesterol sulfate doxorubicin prodrug composition to three tumor cells and one normal cell: mouse breast cancer (4T1) cells, mouse melanoma (B16-F10) cells, mouse prostate cancer cells (RM-1) cells, and human normal liver cells (L02) cells. First, the cells in good condition were digested and diluted to 2×10 4 After cells / mL cells are blown evenly, 100 μL of cell suspension is added to each well of a 96-well plate, and the plates are placed in an incubator at 37°C and 5% CO2 for 24 hours to allow them to adhere to the wall. After the cells adhere to the wall, add doxorubicin hydrochloride solution or the PEG-modified sodium cholesterol sulfate doxorubicin prodrug composition prepared in Example 5. In this experiment, the preparation and dilution of the drug solution and the PEG-modified sodium cholesterol sulfate doxorubicin prodrug composition preparation were both prepared with the corresponding culture fluid and aseptically filtered with a 0.22 μm filter membrane. 100 μL of the test solution was added to each well, with 3 parallel wells for each concentration. For the control group, no test solution was added, and 100 μL of culture fluid was added alone, and the cells were incubated together in an incubator. 48 hours after drug addition, the 96-well plate was taken out, 20 μL of 5 mg / mL MTT solution was added to each well, and the medium was discarded after incubation in an incubator for 4 hours. The 96-well plate was inverted on filter paper to fully absorb the residual liquid, and 200 μL DMSO was added to each well and oscillated on an oscillator for 10 minutes to fully dissolve the blue-purple crystals. Set A1 well (containing only 200 μL DMSO) as the zeroing well. Use an ELISA reader to measure the absorbance value of each well after zeroing at 570 nm.

[0098] The results of the cytotoxicity test were analyzed and the half inhibitory concentration (IC 50 ). The result is Figure 8 As shown, compared with the hydrochloric acid doxorubicin solution, the cytotoxicity of the cholesterol sulfate sodium doxorubicin prodrug composition is reduced. This is because it takes a certain amount of time for doxorubicin to be released from the prodrug composition. There is no significant difference in the cytotoxicity of the four prodrug compositions, mainly because the release rates and pharmaceutical properties of the four prodrug compositions are similar.

[0099] Table 2. Cytotoxicity of PEG-modified cholesterol sodium sulfate doxorubicin prodrug compositions

[0100]

[0101] Example 9 Pharmacokinetic Study of PEG-modified Cholesterol Sodium Sulfate Doxorubicin Prodrug Composition

[0102] SD rats weighing 180-220g were selected for pharmacokinetic study. The rats were randomly divided into groups and fasted for 12 hours before administration. Water was freely available. and the PEGylated cholesterol sodium sulfate doxorubicin prodrug composition prepared in Example 5, the doxorubicin equivalent dose is 10 mg / kg. Blood was collected from the eye sockets at the specified time points, and plasma was separated. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0103] The experimental results are as follows Fig. 9 As shown in the figure, due to the short half-life, the doxorubicin hydrochloride solution is quickly cleared from the blood. In contrast, the circulation time of the sodium cholesterol sulfate doxorubicin prodrug composition is significantly prolonged, and the area under the curve is significantly improved. This is mainly due to the fact that the sodium cholesterol sulfate doxorubicin prodrug composition has a strong colloidal stability, which prolongs its retention in the body. There is no significant difference in the doxorubicin released by the four sodium cholesterol sulfate doxorubicin prodrug compositions, which may be due to similar pharmaceutics properties and stability.

[0104] Example 10 In vivo anti-tumor experiment of PEG-modified cholesterol sodium sulfate doxorubicin prodrug composition

[0105] Using 4T1 tumor-bearing BALB / c mice as a model, mouse breast cancer cell suspension was inoculated into the right back of female BALB / c mice. When the tumor volume grew to 100 mm 3 The tumor-bearing mice were randomly divided into 11 groups, 5 mice in each group: saline, doxorubicin hydrochloride solution 2.5 mg / kg, 2.5mg / kg, DOX-C14-SCS(1:2)@DSPE 2.5mg / kg, DOX-C14-SCS(1:3)@DSPE 2.5mg / kg, doxorubicin hydrochloride solution 5mg / kg, 5mg / kg, DOX-C14-SCS(1:2)@DSPE 5mg / kg, DOX-C14-SCS(1:3)@DSPE 5mg / kg, DOX-C14-SCS(1:2)@DSPE10mg / kg, DOX-C14-SCS(1:3)@DSPE 10mg / kg (equivalent dose of doxorubicin). The nanoparticles used for administration are the PEG-modified sodium cholesterol sulfate doxorubicin prodrug composition prepared in Example 5. The drug was administered once every 2 days for 4 consecutive times. After administration, the survival status and weight changes of the mice were detected every day, and the tumor volume was measured. The mice were killed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation. The results are as follows Fig.10 As shown in the figure, in the saline group, the tumor volume increased rapidly. In contrast, the sodium cholesterol sulfate doxorubicin prodrug composition significantly inhibited the growth of the tumor (<800mm 3), but there was no significant difference in tumor volume between the groups at a dose of 2.5 mg / kg, and there was no significant change in body weight. When the dose was 5 mg / kg and 10 mg / kg, there was no significant difference in tumor load between the sodium cholesterol sulfate doxorubicin prodrug composition group and the doxorubicin solution group. The tumor burden of 5 mg / kg was smaller, but the weight of mice decreased significantly after administration ( Fig.11 ). In summary, the sodium cholesterol sulfate doxorubicin prodrug composition has a strong anti-tumor effect without causing obvious systemic toxicity, and is a safe and effective chemotherapy drug delivery system.

Claims

1. A cholesterol derivative pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: a cholesterol derivative, an adriamycin prodrug, and a PEG modifier; The cholesterol derivative and the doxorubicin prodrug form a drug composition through electrostatic and hydrophobic interactions, and then are modified by PEG; the molar ratio of the doxorubicin prodrug, the cholesterol derivative and the PEG modifier is 1:0.5:0.1-1:9:0.

5.

2. The cholesterol derivative pharmaceutical composition according to claim 1, characterized in that: The cholesterol derivatives are sodium cholesterol sulfate, deoxycholic acid, chenodeoxycholic acid, sodium deoxycholate, cholic acid, sodium dehydrocholate; preferably, sodium cholesterol sulfate.

3. The cholesterol derivative pharmaceutical composition according to claim 1, characterized in that: The doxorubicin prodrug is selected from doxorubicin-acylhydrazone bond-fatty acid prodrug; preferably doxorubicin-acylhydrazone bond-myristic acid (DOX-C14) prodrug.

4. The cholesterol derivative pharmaceutical composition according to claim 1, characterized in that: PEG modifier is selected from DSPE-PEG 2k or CHOL-PEG 2k .

5. The cholesterol derivative pharmaceutical composition according to claim 1, characterized in that: The molar ratio of doxorubicin prodrug, cholesterol derivative and PEG modifier is 1:0.5:0.1-1:9:0.2; preferably 1:1:0.2-1:3:0.

2.

6. The method for preparing the cholesterol derivative pharmaceutical composition according to claim 1, characterized in that: The steps include: 1) dissolving the doxorubicin prodrug, cholesterol derivative and PEG modifier in an organic solvent according to a molar ratio and mixing them thoroughly; 2) adding the mixture obtained in step 1) dropwise into water under stirring; 3) removing the organic solvent from the solution obtained in step 2) to obtain a cholesterol derivative pharmaceutical composition; preferably, the method for removing the organic solvent includes reduced pressure rotary evaporation, ultrafiltration or dialysis.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the organic solvent to water is 1:5-1:20; preferably 1:5-1:

10.

8. The preparation method according to claim 6, characterized in that: The organic solvent is one or more of acetonitrile, dichloromethane, methanol, ethanol and dimethyl sulfoxide; preferably methanol and dimethyl sulfoxide.

9. Use of the cholesterol derivative pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing cancer, wherein the cancer includes breast cancer, prostate cancer, acute leukemia, malignant lymphoma, osteosarcoma, ovarian cancer and soft tissue sarcoma.

10. Use of the cholesterol derivative pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug delivery system or a drug administration system; the drug administration system is injection administration or local administration.

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