Pharmaceutical compositions containing mixed polymer micelles

Abstract

A pharmaceutical composition comprises a mixed polymer micelle and a drug encapsulated in the micelle, wherein the mixed polymer micelle has a particle size of 1 to 1000 nm and comprises an amphiphilic block copolymer and a lipid polymer. Also disclosed are the preparation of the pharmaceutical composition and its use in treating cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition containing mixed micelles. Background Art

[0002] Mixed polymer micelles have been widely studied as effective carriers of low water-soluble drugs. Through site-specific drug delivery, the biopharmaceutical and pharmacokinetic properties of drugs can be improved, thereby enhancing drug efficacy.

[0003] While conventional microparticles or nanoparticles are formed through a complex emulsification process and require the use of surfactants for stabilization, mixed polymer micelles with a particle size of less than 1 μm are usually prepared through a simplified self-assembly process and do not require surfactants or other agents for stability.

[0004] At present, mixed polymer micelles are formed by various types of polymers, including copolymers and lipid polymers. For example, Lee et al., Journal of Controlled Release, 2003, 91, 103-113 (Lee et al., published in Journal of Controlled Release Vol. 91, pp. 103-113 in 2003) and, Vakil et al., Langmuir, 2006, 22, 9723-9729 (Vakil et al. published in Langmuir Vol. 22, pp. 9723-9729 in 2006). Compositions containing these polymer micelles still need to be improved in terms of stability, drug loading and encapsulation efficiency.

[0005] There is a need to develop compositions comprising mixed polymer micelles having improved properties. Summary of the invention

[0006] One aspect of the present invention provides a method for preparing a pharmaceutical composition having unobvious high stability, high drug loading and high encapsulation efficiency.

[0007] The method comprises: (1) preparing starting materials, wherein the starting materials include an amphiphilic block copolymer, a lipid polymer and a drug; (2) mixing the materials in (1) with a solvent; (3) removing the solvent to obtain a dry film or a dry cake; (4) adding water to dissolve the dry film or the dry cake in (3) to form a solution containing mixed polymer micelles; and (5) filtering the solution, wherein the filtrate is the drug composition.

[0008] In one embodiment of the method, the lipid polymer is conjugated to a ligand and the starting material further comprises additional lipid polymer that is not conjugated to a ligand.

[0009] There are three related pharmaceutical compositions within the scope of the present invention, each comprising mixed polymer micelles and a drug encapsulated in the micelles.

[0010] The first pharmaceutical composition is prepared by the above method.

[0011] The second pharmaceutical composition comprises a mixed polymer micelle with a particle size of 1 to 1000 nm and a drug encapsulated in the micelle. The mixed polymer micelle comprises an amphiphilic block copolymer and a lipid polymer conjugated with a ligand. The amphiphilic block copolymer has a hydrophilic segment and a hydrophobic segment, the lipid polymer has a hydrophilic polymer chain and a hydrophobic part covalently linked thereto, and the ligand is a targeting part conjugated with the hydrophilic polymer chain. Optionally, the mixed polymer micelle also comprises an additional lipid polymer that is not conjugated with a ligand.

[0012] The third pharmaceutical composition comprises mixed polymer micelles with a particle size of 1 to 1000 nm and drugs encapsulated in the micelles. The mixed polymer micelles comprise amphiphilic block copolymers and lipid polymers, and the drug loading is 10% to 45%.

[0013] The present invention further encompasses a method of treating a subject in need thereof by administering an effective amount of one of the three pharmaceutical compositions described above.

[0014] The details of the present invention are set forth in the following drawings and description. Other features, objectives and advantages of the present invention can be obtained from the following detailed description of several embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following descriptions are for the accompanying pictures, where:

[0016] Figure 1 The bar graph is a graph showing the survival rates of human oral epidermoid carcinoma cells KB after incubation in a culture medium containing four different pharmaceutical compositions of the present invention for 4 hours and 24 hours.

[0017] Figure 2 Figure 2 is a graph showing the tumor volume of tumor-bearing mice during 22 days after administration of the pharmaceutical composition of the present invention or Jevtana.

[0018] Figure 3 The figure is a graph showing the change of relative fluorescence of mouse tumors over time after treatment with the pharmaceutical composition of the present invention co-loaded with Cy5.5. DETAILED DESCRIPTION

[0019] This section introduces in detail the first, second and third pharmaceutical compositions described in the Summary of the Invention section.

[0020] For ease of discussion, the second pharmaceutical composition is first described. It comprises a mixed polymer micelle and a drug encapsulated in the micelle, wherein the mixed polymer micelle comprises an amphiphilic block copolymer and a lipid polymer conjugated with a ligand. The particle size of the mixed polymer micelle is 1 to 1000 nanometers (e.g., 30 to 500 nanometers).

[0021] The amphiphilic block copolymer has a hydrophilic segment and a hydrophobic segment. The hydrophilic segment includes, but is not limited to, polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), hyaluronic acid, and poly-γ-glutamic acid. The hydrophobic segment includes, but is not limited to, polycaprolactone (PCL), polylactic acid, polyglycolide, polylactic-co-glycolic acid, polyvalerolactone, polybutyrolactone, polypropiolactone, polycarboxylate, and polydioxanone. The hydrophilic segment of an exemplary amphiphilic block copolymer is mPEG, and the hydrophobic segment is PCL.

[0022] The lipopolymer includes a hydrophilic polymer chain and a hydrophobic portion covalently linked thereto. The high hydrophobicity of the hydrophobic portion can greatly improve the stability, drug loading and encapsulation efficiency of the mixed polymer micelle. The lipopolymer includes but is not limited to PEG-cholesterol, PEG-phospholipids and PEG-diaceryl. An exemplary PEG-phospholipid is 1,2-distearoyl-sn-glycerotrioxy-3-phosphoethanolamine-N-methoxypolyethylene glycol (PEG-DSPE).

[0023] The ligand is a targeting moiety conjugated to a lipid polymer hydrophilic polymer chain. Typically, the ligand is a macromolecule or a small molecule with a molecular weight below 2000 Daltons. The small molecule ligand, such as bombesin and bradykinin, can recognize functional cell surface plasminogen activators. An exemplary small molecule ligand is folic acid, N-acetylhistidine, or a peptide (such as an arginine-glycine-aspartic acid peptide and a peptide consisting of 10-15 amino acids). The macromolecular ligand includes, but is not limited to, antibodies, antibody fragments, nucleic acid aptamers, prostate-specific membrane antigen ligands, and growth factors (e.g., epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor, etc.).

[0024] The drug contained in the pharmaceutical composition can be used as a therapeutic agent to treat cancer. The drug includes but is not limited to cabazitaxel, paclitaxel, docetaxel, lalotaxel, doxorubicin, doxorubicin hydrochloride, epirubicin, gemcitabine, letrozole, curcumin, temsirolimus, voriconazole, posaconazole, sirolimus, everolimus, ixabepilone, camptothecin, camptothecin derivatives and photosensitizers. In particular, cabazitaxel is the only known paclitaxel that is effective for docetaxel-resistant cancers to date. It is usually associated with severe dose-limiting toxicity (such as neutropenia). A composition containing mixed polymer micelles encapsulated with cabazitaxel greatly enhances the biophysical properties (such as water solubility) and bioavailability of cabazitaxel. Note that camptothecin derivatives refer to compounds derived from camptothecin and structurally close to camptothecin, which have improved drug properties, such as solubility, metabolic stability and bioavailability. For example, Zunino et al., Current Pharmaceutical Design, 2002, 8(27), 2505-2520.

[0025] Typically, the polydispersity index of the mixed polymer micelles is 0.08-1.0, preferably <0.33, more preferably <0.2, and has a drug encapsulation efficiency greater than 80%, preferably >90%, more preferably >95%.

[0026] In one embodiment, the mixed polymer micelle further comprises an additional lipid polymer that is not bound to a ligand. The lipid polymer may be PEG-cholesterol, PEG-phospholipid, PEG-vitamin E, PEG-diacylglycerol, etc. For example, the PEG-phospholipid is PEG-DSPE.

[0027] In another embodiment, the amphiphilic block copolymer is mPEG-PCL, the lipid polymer is PEG-DSPE, the ligand is folic acid or N-acetylhistidine, and the drug is cabazitaxel. The mixed polymer micelle particle size in the composition is 30 to 500 nm, the polydispersity index is less than 0.33, and the drug encapsulation efficiency is greater than 90%.

[0028] In another embodiment, the mixed polymer micelles include amphiphilic block copolymer mPEG-PCL; ligand-bound lipid polymer ligand-PEG-DSPE, wherein the ligand is folic acid or N-acetyl histidine; lipid polymer PEG-DSPE; and a drug called cabazitaxel. The mixed polymer micelles in the composition have a particle size of 30 to 500 nm, a polydispersity index of less than 0.33, and a drug encapsulation efficiency of greater than 90%.

[0029] In another embodiment, the mixed polymer micelle includes a lipid polymer conjugated to a ligand. The lipid polymer accounts for 4-25% (e.g., 5-15%) of the weight of the mixed polymer micelle. For mixed polymer micelles containing both a lipid polymer conjugated to a ligand and a lipid polymer without a ligand, the two lipid polymers together account for 4-25% (e.g., 5-15%) of the weight of the mixed polymer micelle.

[0030] Turning to the third pharmaceutical composition, it comprises a mixed polymer micelle with a particle size of 1 to 1000 nm (e.g., 30 to 500 nm) and a drug encapsulated in the micelle, wherein the mixed polymer micelle includes an amphiphilic block copolymer and a lipopolymer that can be selectively conjugated to a ligand. The mixed polymer micelle has a high drug loading of 10 to 45% (e.g., 15% to 40%). For example, the amphiphilic block copolymer is mPEG-PCL, the lipopolymer is PEG-DSPE, the drug is cabazitaxel, and the ligand is folic acid or N-acetylhistidine. It is worth noting that when the lipopolymer is conjugated to a ligand, the mixed polymer micelle can also include additional lipopolymers that are not conjugated to a ligand.

[0031] Finally, the first pharmaceutical composition is prepared according to the method described in the above summary of the invention and is described in more detail below.

[0032] It needs to be reiterated that the method includes providing starting materials, which include amphiphilic block copolymers, lipid polymers, and drugs; mixing the starting materials in a solvent; removing the solvent to obtain a dry film or a dry cake; adding water to dissolve the dry film or the dry cake to form mixed polymer micelles encapsulating the drug; optionally ultrasonically treating the solution containing the mixed polymer micelles; filtering the solution, and the filtrate is the pharmaceutical composition.

[0033] In an exemplary method, the lipopolymer comprises a hydrophilic polymer chain and a hydrophobic portion covalently attached thereto. The lipopolymer also comprises a ligand conjugated to the hydrophilic polymer chain. It is noteworthy that the starting material may further comprise another lipopolymer. For example, the starting material comprises DSPE-PEG (except mPEG-PCL), ligand-PEG-DSPE (the ligand is folic acid or N-acetyl histidine) and cabazitaxel.

[0034] In another exemplary method, the lipopolymer is not conjugated to a ligand. Preferably, the lipopolymer is PEG-DSPE, the amphiphilic block copolymer is mPEG-PCL, and the drug is cabazitaxel.

[0035] Still within the scope of the present invention is a method of treating cancer using one of the three pharmaceutical compositions described above. The method comprises administering an effective amount of one of the three pharmaceutical compositions of the present invention to a subject in need thereof.

[0036] In certain embodiments of the method, the anticancer drug is cabazitaxel, the amphiphilic block copolymer is mPEG-PCL, and the lipid polymer is PEG-DSPE or folic acid-PEG-DSPE, wherein the particle size of the mixed polymer micelle is 30 to 500 nm, the polydispersity index is less than 0.33, and the drug encapsulation efficiency is greater than 90%. In another embodiment, the mixed polymer micelle comprises PEG-DSPE and folic acid-PEG-DSPE.

[0037] Cancer can be a solid tumor that overexpresses folate receptors. The solid tumor includes, but is not limited to, lung cancer, ovarian tumors, breast cancer (e.g., metastatic breast cancer and triple-negative breast cancer), endometrial cancer, cervical cancer, kidney cancer, brain cancer, head and neck cancer, hormone-refractory metastatic prostate cancer, bladder cancer, gastric cancer (e.g., metastatic gastric cancer), transitional cell carcinoma, and liposarcoma.

[0038] The term "treatment" as used herein refers to the administration of the pharmaceutical composition to a person who has the above-mentioned disease, such as cancer, symptoms of the above-mentioned disease, or is prone to the above-mentioned disease, to achieve a therapeutic or preventive effect. The term "effective amount" refers to the amount of active drug required to achieve such an effect. It is well known to those skilled in the art that the effective dose will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the use in combination with other treatment methods.

[0039] The pharmaceutical composition of the present invention can be administered by various routes, such as parenteral administration, such as subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection.

[0040] Sterile injectable pharmaceutical compositions can be solutions or suspensions in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Carriers and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are commonly used as solvents or suspension media (e.g., synthetic monoglycerides or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives; natural pharmaceutically acceptable oils, such as olive oil and castor oil, especially their polyoxyethylated forms, can be used to prepare injections. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersants. Other surfactants commonly used to prepare pharmaceutically acceptable solid, liquid or other dosage forms, such as Tween and Spans or other similar emulsifiers or bioavailability enhancers, may also be used for preparation.

[0041] Without further elaboration, it is believed that those skilled in the art can make the best use of the present invention on the basis of the above description. Therefore, the following specific embodiments will be interpreted as merely illustrative and not limiting the disclosed content in any way. All publications cited are incorporated into this application by reference.

[0042] Example 1: Preparation of folic acid conjugated lipid polymer

[0043] The folic acid-conjugated lipid polymer was prepared according to the method described in Cho et al., Journal of Nanomaterials, 2015, 16(1), Article No.36.

[0044] More specifically, to prepare folate-conjugated lipopolymers, 25 mg of folate was dissolved in 1 mL of dimethyl sulfoxide (DMSO), 100 mg of amino-substituted 1,2-distearoyl-sn-glycerotrioxy-3-phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG 5k -NH2) was dissolved in 0.5 mL of pyridine containing 32.5 mg of N, N′-dicyclohexylcarbodiimide (DCC). The two solutions were mixed and allowed to stand at room temperature for 4 hours. The pyridine was then removed by rotary vacuum evaporation. 3 mL of water was added and the mixture was centrifuged at 36000 g for 15 min to remove the insoluble matter. The resulting supernatant was dialyzed against saline for 24 hours in Spectra / Por CE and then dialyzed against water for 24 hours. The dialyzed product was freeze-dried folic acid-PEG 5k -DSPE, stored at -20°C before use. Note that the subscript "5k" indicates the molecular weight of the polymer, i.e., the average M n is 5000.

[0045] The obtained folic acid-conjugated lipid polymer was used to prepare a lipid polymer containing cabazitaxel and mPEG 5k -b-PCL 2k of mixed polymer micelles.

[0046] Example 2: Preparation and characterization of folic acid-conjugated cabazitaxel-loaded mixed polymer micelles

[0047] Three pharmaceutical compositions were prepared, each containing different folate-conjugated cabazitaxel mixed polymer micelles (CBZ-mPMs), according to the methods described in Cho et al., Journal of Nanomaterial, 2015, 16(1), Article No. 36 and Vakil et al., Langmuir, 2006, 22, 9723-9729.

[0048] First, block copolymer mPEG was heated at 60°C. 5k -b-PCL 2k 10mg, folic acid-PEG 5k -DSPE (0.46, 0.97 or 2.20 mg) and 5 mg of cabazitaxel were dissolved in 1 mL of tetrahydrofuran (THF) to prepare a mixture. Subsequently, THF was removed by rotary evaporation to obtain a dry film or a dry cake. Water was then added to dissolve the dry film or the dry cake at room temperature, and folic acid-conjugated CBZ-mPM was spontaneously formed during the dissolution process. Further, the solution was ultrasonicated at room temperature for 5 minutes to reduce the particle size of the generated micelles and narrow the particle size distribution. Finally, a 0.22 μm polyvinylidene fluoride (PVDF) filter membrane was used to filter out the unencapsulated cabazitaxel to obtain a pharmaceutical composition.

[0049] Table 1 shows the characterization data of three exemplary folate-conjugated CBZ-mPMs, namely DFB001, DFB002 and DFB003, and two folate-free CBZ-mPMs, namely DMB001 and DMB002. It is worth noting that DMB001 and DMB002 were prepared according to the above method, wherein folate-PEG 5k -DSPE is conjugated with a lipid polymer (i.e., PEG 5k -DSPE) was used as a replacement. Table 1 also includes non-mixed polymer micelles loaded with cabazitaxel (CBZ-PM), i.e., DB003, as a comparison.

[0050] The five CBZ-mPMs and one CBZ-PM were characterized by particle size, polydispersity index, cabazitaxel concentration, drug loading and encapsulation efficiency. TM Nano S) was used to measure the particle size and polydispersity index (PDI). The amount of cabazitaxel encapsulated in each CBZ-mPM was determined by HPLC. The drug-to-polymer weight percentage (D / P weight %), drug loading (DL), and encapsulation efficiency (EE) were calculated according to the following formulas:

[0051] D / P weight % = drug mass / total polymer mass (mPEG-PCL+lipid polymer) x 100%

[0052] DL (%) = drug mass in micelles / (total polymer mass + drug mass in micelles) × 100%

[0053] EE (%) = drug mass after filtration / drug mass before filtration × 100%

[0054] Referring to Table 1, "A" represents mPEG 5k -b-PCL 2k "B" stands for PEG5k -DSPE, "C" stands for folic acid-PEG 5k -DSPE, "particle size" refers to particle size. "CBZ" and "PDI" refer to cabazitaxel and polydispersity index, respectively. Further expansion includes PEG 5k -The two CBZ-mPMs containing DSPE, i.e., DMB001 and DMB002, were denoted as “AB”, while the CBZ-mPMs containing folic acid, i.e., DFB001, DFB002, and DFB003, were denoted as “AC”.

[0055] Table 1. Characterization of CBZ-mPM

[0056]

[0057]

[0058] As shown in Table 1, the five CBZ-mPMs all showed a drug loading of about 50% and an encapsulation efficiency greater than 90%. On the other hand, the drug loading of CBZ-PM, i.e., DB003, was 50% and the encapsulation efficiency was only 72.5%. In other words, the five CBZ-mPMs all showed a higher encapsulation efficiency than CBZ-PM, while the drug loading of the six micelles was comparable.

[0059] These results indicate that the CBZ-mPM of the present invention unexpectedly has a high encapsulation efficiency.

[0060] Example 3: Study on the antitumor activity of CBZ-mPM in vitro

[0061] In this study, the in vitro antitumor activities of three of the five CBZ-mPMs described in Example 2, namely DMB001, DFB001, and DFB002, were evaluated.

[0062] MTT assay (MTT refers to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed according to the method of Chan et al., Biomaterials, 2009, 30, 1627-1634 and Kumar et al., Biomaterials, 2012, 33, 1180-1189.

[0063] Specifically, human oral epidermoid carcinoma cells KB were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100U / mL penicillin, and 100mg / mL streptomycin at 37°C and 5% CO2. KB cells were seeded in a 96-well plate at a density of 15,000 / well and cultured in 200mL of culture medium per well for 24h. Then 200mL of culture medium containing 4 different concentrations (i.e., 1.5625, 3.125, 6.25, and 12.5ng / ml) of DMB001, DFB001, or DFB002 was replaced and tested after incubation for 4h and 24h at 37°C and 5% CO2. The culture medium containing CBZ-mPM was then removed to avoid interference with the experiment. A culture medium containing 0.5mg / mL of MTT solution was added and incubated for another 4 hours. The culture medium containing MTT was removed and the cells were rinsed three times with phosphate buffer. Then, 200 mL of DMSO was added to lyse the cells, and the resulting mixture was incubated at room temperature for 30 minutes.

[0064] The absorbance of each well was measured at 570 nm on an enzyme-linked immunosorbent assay. The survival rate of untreated control cells was calculated as 100%. The cell survival rate (%) was calculated according to the following formula ("OD" means optical density):

[0065] Cell survival rate (%) = [(OD value of sample at 570 nm - OD value of blank sample) / OD value of control group at 570 nm] × 100%

[0066] like Figure 1 As shown, the three tested CBZ-mPMs all exhibited strong in vitro antitumor activity after 24 h of treatment at the four tested doses.

[0067] Unexpectedly, compared with AB CBZ-mPM, DMB001, both AC CBZ-mPM, DFB001 and DFB002, showed higher antitumor activity against KB tumor cells overexpressing folate receptors in both 4-hour and 24-hour treatments. More specifically, in 4h treatment, the cell survival rate of KB cells treated with 4 different concentrations (i.e., 1.5625, 3.125, 6.25, and 12.5ng / ml) of DMB001 was about 90%, while the cell survival rate shown by DFB001 or DFB002 was about 70%; in 24h treatment, at the same concentration, the survival rate of KB cells under DMB001 treatment was 35-45%, while the survival rate of KB cells under DFB001 or DFB002 treatment was about 20%. Apparently, the enhanced antitumor activity was due to the use of folic acid as a ligand in DFB001 and DFB002, whereas folic acid was not present in DMB001.

[0068] These results indicate that the CBZ-mPM of the present invention has a good therapeutic effect on tumors at a low dose.

[0069] Example 4: PEG-containing 5k -DSPE, folic acid-PEG 5k Preparation and characterization of CBZ-mPMs containing DSPE or both

[0070] Eight exemplary CBZ-mPMs were prepared using the method in Example 2, wherein the CBZ-mPMs contained PEG 5k -DSPE, folic acid-PEG 5k -DSPE or PEG 5k -DSPE and folic acid-PEG 5k -DSPE.

[0071] More specifically, mPEG 5k -b-PCL 2k 20mg, folic acid-PEG 5k -DSPE 0~2mg, PEG 5k -DSPE 0-3 mg and cabazitaxel 4 mg were dissolved in 1 mL of chloroform / methanol (9:1 v / v) at 60°C. The solvent was then removed by rotary evaporation to obtain a dry film or dry cake. Water was then added at room temperature to dissolve the dry film or dry cake, thereby spontaneously forming CBZ-mPM during the dissolution process. In addition, ultrasonic treatment was performed at room temperature for 5 minutes to reduce the particle size of the resulting micelles and narrow their particle size distribution. Finally, unencapsulated cabazitaxel was filtered out using a 0.22 μm PVDF membrane filter to obtain a pharmaceutical composition.

[0072] Table 2 shows the characterization data of 8 exemplary CBZ-mPMs and 1 reference CBZ-PM, namely DB005. Among the 8 exemplary CBZ-mPMs, DMB022, DMB023, and DMB025 contain PEG. 5k -DSPE; DFB008 and DFB012 contain folic acid-PEG 5k -DSPE; DFB009, DFB010, and DFB014 also contain PEG 5k -DSPE and folic acid PEG 5k -DSPE.

[0073] The eight exemplary CBZ-mPM and CBZ-PM were characterized by five parameters: particle size, polydispersity index, cabazitaxel concentration, drug loading, and encapsulation efficiency. TMThe particle size and PDI (polydispersity index) were determined by HPLC. The amount of cabazitaxel encapsulated in each micelle was determined by HPLC. The encapsulation efficiency and drug loading were calculated using the formula provided in Example 2.

[0074] In Table 2, PEG 5k -DSPE and folic acid-PEG 5k -The three CBZ-mPMs of DSPE, namely DFB009, DFB010 and DFB014, are denoted as "ABC". For the definitions of "A", "B", "C", "CBZ", "particle size", "PDI", "DL", "EE", "AB" and "AC", please refer to Example 2.

[0075] Table 2. Characterization of CBZ-mPMAB, AC, and ABC

[0076]

[0077] As shown in Table 2, except for DFB012 with a particle size of 56.5 nm, the particle sizes of CBZ-mPM and CBZ-PM are similar (all in the range of 41-46 nm). In addition, the polydispersity index of the nine micelles is less than 0.3, and the encapsulation efficiency is approximately equal to or greater than 90%.

[0078] These results indicate that the CBZ-mPM of the present invention unexpectedly has a high encapsulation efficiency and a narrow particle size distribution.

[0079] Example 5: Storage stability of CBZ-mPM

[0080] The eight CBZ-mPM and CBZ-PM described in Example 4 were stored in water at 25°C for 6 days (144 hours). TM Nano S) to measure the particle size to monitor storage stability. The stability data obtained are shown in Table 3.

[0081] Table 3. Stability of CBZ-mPM stored in water at 25°C

[0082]

[0083] *p=Precipitation

[0084] As shown in Table 3, CBZ-PM, i.e., DB005, is stable in water at 25°C for less than 24 hours. In contrast, the eight exemplary CBZ-mPMs are all stable for more than 96 hours under the same storage conditions. In particular, DFB012, DFB010, and DFB014, all containing 15 wt% lipid polymers (see column 6 of Table 2), are the most stable.

[0085] These results indicate that the storage stability of the CBZ-mPM of the present invention is unexpectedly high. They also show that increasing the amount of lipid polymer contained in the micelles can enhance the storage stability of CBZ-mPM.

[0086] Furthermore, the change in turbidity within 96 h was measured to evaluate the serum stability of the eight exemplary CBZ-mPMs and the reference sample DB005. The increase in turbidity indicates particle aggregation caused by nonspecific interactions with serum proteins (Z.-X. Zhao et al., Biomaterials, 2012, 33, 6793-6807).

[0087] Specifically, 100 μl of each of the eight CBZ-mPM aqueous solutions and the DB005 aqueous solution were mixed with an equal volume of FBS (100 μl) in a centrifuge tube, and the resulting mixed solution was then incubated at 37°C.

[0088] Using phosphate buffered saline (PBS) as a blank solution, serum-induced aggregation was measured by measuring the turbidity reflected by the absorbance values ​​at different time points (i.e., 0 hours, 24 hours, and 96 hours) at a wavelength of 630 nm. The results obtained are shown in Table 4 below.

[0089] Table 4. Serum stability of CBZ-mPM at 37°C

[0090]

[0091] *p=Precipitation

[0092] As shown in Table 4, the turbidity of the solution containing CBZ-PM, i.e., DB005, increased significantly after incubation at 37°C for 24 hours, and CBZ-PM precipitated between 24 and 96 hours. On the other hand, the turbidity of the solution containing CBZ-mPM did not increase like DB005 after 24 hours of incubation. In addition, among the 8 CBZ-mPMs tested, 6 of them, including DMB023, DMB025, DFB008, DFB009, DFB010, and DFB014, were significantly more stable than CBZ-mPM. They did not precipitate after 96 hours of incubation. Unexpectedly, the solution containing PEG 5k -DSPE and folic acid PEG 5k -DSPE-CBZ-mPM relative to folic acid-PEG-containing 5k -DSPE-derived CBZ-mPMs had higher serum stability.

[0093] These results indicate that the CBZ-mPM of the present invention unexpectedly has improved storage stability and serum stability.

[0094] Example 6: In vivo anti-tumor activity of DFB014

[0095] Female nude mice (nu / nu, 20-25 g) were used to study the in vivo antitumor activity of DFB014. The mice were subcutaneously implanted with human epidermal carcinoma xenograft cell line, KB cells (2x10 cells per animal). 7 cells), which overexpressed the folate receptor. After implantation, the tumor grew to 350 mm in 28 days. 3 The volume of the mouse was then given a single dose of Jevtana (trade name of cabazitaxel) or DFB014 (equivalent dose of cabazitaxel = 10 mg / kg) PBS suspension via the tail vein, which was recorded as day 0. At the predetermined time point, the long and short axes of the tumor were measured using a caliper. Then, the formula (3 / 4)πa was used to calculate the long and short axes of the tumor. 2 b Calculate the tumor volume, where a and b are the lengths of the short and long axes of the tumor, respectively.

[0096] The tumor volume was then used to calculate the % tumor growth inhibition (TGI) of Jevtana and DFB014 according to the formula:

[0097] TGI=(V Tm -V T0 )*100% / V T0 ,

[0098] Where V Tm is the tumor volume at a predetermined time point, V T0 The tumor volume on day 0, i.e. the day of Jevtana / DFB014 administration.

[0099] Figure 2 The changes in tumor volume over 22 days of treatment with Jevtana and DFB014 are shown. For tumors treated with Jevtana, the tumor volume remained roughly the same from day 0 to day 11, but increased significantly from day 12. The TGI for Jevtana treatment was 52% on day 15, 131% on day 18, and 228% on day 22.

[0100] In contrast, the volume of DFB014-treated tumors remained consistent from day 0 to day 9, decreased from day 10 to day 15, and then expanded. The TGI of DFB014 treatment was -40% on day 15 (negative values ​​indicate tumor shrinkage), -26% on day 18, and 13% on day 22, which was significantly lower than the TGI of Jevtana at the same time points. The results showed that the anti-tumor activity of DFB014 was significantly higher than that of Jevtana.

[0101] Example 7: In vivo tumor targeting of CBZ-mPM

[0102] Fluorescence imaging was used to study the in vivo tumor targeting of CBZ-mPM. Near infrared dye Cy5.5 was co-loaded into two exemplary CBZ-mPMs, DMB025 (which does not contain folic acid) and DFB014 (which contains folic acid), to prepare two CBZ-mPMs containing Cy5.5, namely CyDMB025 and CyDFB014. Specifically, the two CBZ-mPMs containing Cy5.5 were prepared by adding Cy5.5 to the starting materials according to the method described in Example 4.

[0103] Table 5 below shows the characterization data of two CBZ-mPMs containing Cy5.5. Both CBZ-mPMs were characterized by four parameters: particle size, polydispersity index, Cy5.5 concentration, and cabazitaxel concentration. TM Nano S) was used to measure the particle size and polydispersity index. The amount of encapsulated Cy5.5 and encapsulated cabazitaxel in each CBZ-mPM was determined by HPLC. For the definitions of "A", "B", "C", "CBZ", "particle size" and "PDI", see Example 2.

[0104] Table 5. Characterization of CBZ-mPM co-loaded with Cy5.5

[0105]

[0106] The tumor targeting of two CBZ-mPMs containing Cy5.5 was studied in female nude mice (nu / nu, 20-25 g in weight). The mice were implanted subcutaneously with KB cells, a human epidermal carcinoma xenograft cell line (2 × 10 cells per animal). 7 The tumor grew to 350 mm in size 28 days after implantation. 3 The mice were injected with CyDMB025 or CyDFB014 via the tail vein, and a total of 2 nmol of Cy5.5 was injected. Subsequently, the mice were anesthetized with 2% isoflurane before fluorescence imaging at each time point. In vivo fluorescence imaging was performed using the IVIS 3D imaging system.

[0107] Figure 3 The relative fluorescence of KB tumors in mice treated with CyDMB025 and CyDFB014 changes over time. As shown in the figure, both Cy5.5-containing CBZ-mPMs gradually enter and accumulate in KB tumors within 4 hours. Importantly, the relative tumor fluorescence of mice injected with CyDFB014 is always higher than that of mice injected with CyDMB025, indicating that CyDFB014 accumulates more effectively in KB tumors. This advantage is attributed to the folate ligand in CyDFB14, which enables CBZ-mPM to target tumors with overexpression of folate receptors.

[0108] These findings suggest that CyDFB14 can enhance tumor targeting.

[0109] Example 8: Preparation of N-acetylhistidine conjugated lipid polymer

[0110] An N-acetylhistidine conjugated lipopolymer was prepared according to the procedure described in Example 1.

[0111] More specifically, DSPE-PEG 5k -NH225mg, N-acetylhistidine (NAcHis) 4.93mg, DCC 5.2mg, N-hydroxysuccinimide (NHS) 2.9mg, 4-dimethylaminopyridine (DMAP) 3.1mg were dissolved in 0.4mL anhydrous DMSO. The mixture was stirred at room temperature for 48 hours. Subsequently, 2.5mL DMSO was added to the mixture, and the by-product dicyclohexylcarbodiurea was filtered repeatedly. The filtrate was then dialyzed with DMSO (molecular weight cut-off of 3500) for 3 days to remove residual NAcHis, NHS and DMAP. The final product NAcHis-PEG was obtained by freeze drying. 5k -DSPE.

[0112] Example 9: Preparation and characterization of N-acetylhistidine-conjugated CBZ-mPM

[0113] The PEG-containing 5k -DSPE and NAcHis-PEG 5k -CBZ-mPM, DHB001 from DSPE.

[0114] More specifically, mPEG 5k -b-PCL 2k 20mg, NAc His-PEG 5k -DSPE 2mg, PEG 5k -DSPE1mg, Cabazitaxel 4mg were dissolved in 1mL 60°C chloroform / methanol (9:1v / v), and the solvent was removed by rotary evaporation to obtain a dry film or dry cake. Then water was added to dissolve the dry film or dry cake at room temperature, and DHB001 was spontaneously formed during the dissolution process. Finally, the unencapsulated Cabazitaxel was removed by filtration using a 0.22μm PVDF membrane to obtain a pharmaceutical composition.

[0115] Table 6 below shows the characterization data of DHB001. It was characterized by five parameters including particle size, polydispersity index (PDI), cabazitaxel (CBZ) concentration, drug loading (DL) and encapsulation efficiency (EE). The particle size was analyzed by laser particle size analyzer (Beckman Delsa TMThe particle size and polydispersity index were determined by HPLC. The amount of cabazitaxel encapsulated in DHB001 was determined by HPLC. The encapsulation efficiency and drug loading were calculated using the formula in Example 2.

[0116] In Table 6, "C" represents NAcHis-PEG 5k -DSPE, "particle size", "PDI", "CBZ", "DL" and "EE" are as defined above. For the definition of "A" and "B", please see Example 2.

[0117] Table 6. Characterization of N-acetylhistidine conjugated CBZ-mPM

[0118]

[0119] As shown in Table 6, DHB001, like other CBZ-mPMs of the present invention, unexpectedly exhibited a higher encapsulation efficiency and a narrower particle size distribution.

[0120] Other embodiments

[0121] All features disclosed in this specification may be combined in any form. Each feature disclosed in this specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly provided, each feature disclosed is only an example of a series of equivalent or similar features.

[0122] In addition, from the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and can make various changes and modifications to the present invention without departing from the essence and scope of the present invention to adapt it to various uses and conditions. Therefore, other embodiments also fall within the claims of this application.

Claims

1. A pharmaceutical composition comprising mixed polymer micelles having a particle size of 1 to 1000 nm and a drug encapsulated in the micelles; The mixed polymer micelles are composed of an amphiphilic block copolymer and a first lipid polymer conjugated to a ligand; wherein the amphiphilic block copolymer is mPEG-PCL; The first lipid polymer is 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-methoxypolyethylene glycol (PEG-DSPE); The ligand is folic acid; The drug in question is cabazitaxel; The first lipid polymer accounts for 4-25% of the weight of the mixed polymer micelle.

2. A pharmaceutical composition comprising mixed polymer micelles having a particle size of 1 to 1000 nm and a drug encapsulated in the micelles; wherein the mixed polymer micelles are composed of an amphiphilic block copolymer and a first lipid polymer conjugated to a ligand; and a second lipid polymer not conjugated to a ligand; wherein the amphiphilic block copolymer is mPEG-PCL; The first lipid polymer conjugated with the ligand is folic acid-PEG-DSPE; the second lipid polymer is PEG-DSPE; The drug is cabazitaxel; The first lipid polymer and the second lipid polymer together account for 4-25% of the weight of the mixed polymer micelle.

3. A method for preparing a pharmaceutical composition comprising mixed polymer micelles and a drug, wherein the particle size of the mixed polymer micelles is 1 to 1000 nm, and the drug is encapsulated in the micelles; the method comprises: preparing starting materials, the starting materials comprising an amphiphilic block copolymer, a lipid polymer and a drug; mixing the starting materials with a solvent; removing the solvent to obtain a dry film or a dry cake; adding water to dissolve the dry film or dry cake to form a solution containing mixed polymer micelles; Filtering the solution, the filtrate is the pharmaceutical composition; wherein the amphiphilic block copolymer is mPEG-PCL; The lipid polymer is PEG-DSPE; The drug is cabazitaxel; The lipid polymer accounts for 4-25% of the weight of the mixed polymer micelle.

4. The method of claim 3, wherein the lipid polymer is not conjugated to a ligand.

5. A pharmaceutical composition prepared according to the method of claim 3.

6. The pharmaceutical composition of claim 5, wherein the lipid polymer is not conjugated to a ligand.

7. Use of the pharmaceutical composition according to claim 1 in preparing a drug for treating cancer.

8. Use of the pharmaceutical composition according to claim 5 in preparing a drug for treating cancer.

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