Nanometer preparation for targeting glioblastoma as well as preparation and application of nanometer preparation

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to a nano preparation for targeting glioblastoma, and preparation and application thereof, in particular to a nano preparation of chlorambucil-mycophenolic acid prodrugs for targeting glioblastoma, especially temozolomide drug-resistant glioblastoma, and preparation and application thereof. According to the invention, firstly, a chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof is constructed, and then on the basis, a prodrug co-assembled nano preparation capable of realizing synergistic delivery of three drugs, namely chlorambucil, mycophenolic acid and MHY1485, is constructed. Meanwhile, the prodrug co-assembled nano preparation is subjected to targeted modification, so that the prodrug co-assembled nano preparation is endowed with the capability of crossing a blood brain barrier. A targeting peptide modified nano-drug delivery system is constructed through a prodrug co-assembly technology, blood brain barrier crossing and accurate delivery of drugs to tumor sites are achieved, and the synergistic anti-tumor effect of a drug combination is remarkably improved.

Description

Technical Field:

[0001] The present invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to a nano - preparation targeting glioblastoma, its preparation and application. Specifically, it relates to a nano - preparation of chlorambucil - mycophenolic acid prodrug targeting glioblastoma, especially temozolomide - resistant glioblastoma, and its preparation and application. Background Art:

[0002] Tumor is a major disease seriously threatening human life and health. Among them, glioblastoma is recognized as one of the most difficult - to - cure malignant tumors due to its characteristics such as high treatment difficulty, high disability rate and high fatality rate. At present, the standard treatment plan for glioblastoma mainly adopts a comprehensive treatment mode of surgical resection combined with radiotherapy and chemotherapy. However, this tumor has the biological characteristic of invasive growth and lacks a clear boundary with normal brain tissue, resulting in difficult radical resection by simple surgery. At the same time, high - dose radiotherapy and chemotherapy often cause irreversible damage to the neurological function of patients, seriously affecting the quality of life of patients.

[0003] In terms of drug treatment, temozolomide, as the only current first - line chemotherapy drug, faces a serious drug - resistance problem in clinical application. Research shows that glioblastoma can effectively remove methylated damage regions through O6 - methylguanine - DNA methyltransferase (MGMT) and base excision repair mechanisms, thus generating drug resistance to temozolomide. Therefore, developing new treatment strategies for temozolomide - resistant glioblastoma has important clinical significance for improving the survival rate of patients and improving the prognosis.

[0004] Among existing chemotherapy drugs, chlorambucil, as another important alkylating agent, has a significant difference in its action mechanism from temozolomide. This drug forms irreversible cross - links between DNA strands through SN 2 nucleophilic reaction. The alkylation damage generated is not recognized and cleared by MGMT, but is repaired depending on the base excision repair mechanism.

[0005] Currently, chlorambucil is mainly clinically applied to the maintenance treatment of chronic lymphocytic leukemia, Hodgkin lymphoma and ovarian cancer.

[0006] It is worth noting that the DNA damage repair process induced by chlorambucil highly depends on the base excision repair mechanism, and this repair process requires guanosine as a key raw material. The biosynthesis and metabolism of guanine are precisely regulated by inosine monophosphate dehydrogenase 2 (IMPDH2) and mammalian target of rapamycin complex 1 (mTORC1) respectively, which provides new ideas for developing new combination treatment strategies.

[0007] MHY1485 is a small molecule mTOR agonist that activates this pathway by stabilizing the GTP-binding state of Rag GTPase, a process that requires guanine nucleotide metabolism to provide GTP molecules for support. The regulation of mTORC1 by MHY1485 affects the homeostasis of intracellular purine nucleotide pools, particularly by remodeling the nucleotide metabolic network through altering guanylate kinase activity.

[0008]

[0009] In the drug treatment of glioblastoma, the blood-brain barrier constitutes a major obstacle to drug delivery. This barrier is composed of brain microvascular endothelial cells, pericytes, basement membrane, and end-feet astrocytes, and has highly selective permeability characteristics. In addition, the non-specific distribution of chemotherapeutic drugs in the brain tissue may lead to severe neurotoxicity, which is also an important issue to be considered in the treatment of glioblastoma. Therefore, the development of a delivery system that can effectively penetrate the blood-brain barrier and achieve site-specific drug release at the lesion site has important clinical value for improving the treatment effect of glioblastoma.

[0010] There are no relevant reports on the combined use of chlorambucil, mycophenolic acid, and MHY1485 in the prior art, and even less on the combined use of chlorambucil and mycophenolic acid after being made into prodrugs and then combined with MHY1485. Summary of the Invention:

[0011] Aiming at the limitations of the prior art in the treatment of glioblastoma, especially temozolomide-resistant glioblastoma, the present invention innovatively proposes a multi-drug synergistic treatment strategy and constructs an intelligent drug delivery system with high blood-brain barrier penetration ability. The core of this system lies in: first, using chlorambucil to generate specific DNA damage that is not regulated by MGMT; second, through a dual regulation mechanism of "opening sources and reducing expenditures", that is, using the mTORC1 agonist MHY1485 to activate the ribosome biosynthesis pathway to accelerate guanosine consumption, and at the same time using the IMPDH2 inhibitor mycophenolic acid to selectively inhibit the de novo synthesis pathway of guanosine, thereby effectively depleting the intracellular guanosine level and finally blocking the base excision repair pathway.

[0012] Based on the above treatment strategy, the present invention adopts a prodrug co-assembly technology. First, a chlorambucil-mycophenolic acid prodrug or its pharmaceutically acceptable salt is constructed, and then on this basis, a prodrug co-assembly nanon preparation that can achieve the synergistic delivery of three drugs is constructed. At the same time, the prodrug co-assembly nanon preparation is targeted modified, endowing it with the ability to cross the blood-brain barrier. The present invention provides a new treatment idea and technical solution for targeting glioblastoma, especially temozolomide-resistant glioblastoma.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] The present invention provides a chlorambucil - mycophenolic acid prodrug shown in Formula I or a pharmaceutically acceptable salt thereof:

[0015]

[0016] Wherein, X is a tumor microenvironment - sensitive chemical bond.

[0017] In the above structural formula, the tumor microenvironment - sensitive chemical bond is a pH - sensitive bond, a redox environment - sensitive bond, an enzyme - sensitive bond, or a hypoxia - sensitive bond. The pH - sensitive bond includes at least one of a hydrazone bond, an imine bond, an acetal bond, and a β - amino ester bond. The redox environment - sensitive bond includes at least one of a monothioether bond, a disulfide bond, a trisulfide bond, a monoselenide bond, a diselenide bond, and a borate ester bond. The enzyme - sensitive bond includes at least one of a metalloproteinase - sensitive bond, an esterase - sensitive bond, a phosphatase - sensitive bond, a transglutaminase - sensitive bond, and a thioredoxin reductase - sensitive bond. The hypoxia - sensitive bond includes at least one of an azo bond and a nitroaromatic bond.

[0018] Further, X is a redox environment - sensitive bond.

[0019] Specifically, the structure of the chlorambucil - mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof is as follows:

[0020]

[0021] The present invention further provides a method for synthesizing the above chlorambucil - mycophenolic acid prodrug, which specifically includes the following steps:

[0022] Step 1: Chlorambucil reacts with oxalyl chloride to form an acyl chloride intermediate, and then condenses with dithiodiethanol. After extraction and preparative liquid - phase separation, a chlorambucil - dithiodiethanol intermediate is obtained.

[0023] Step 2: After the chlorambucil - dithiodiethanol intermediate reacts with p - nitrophenyl chloroformate, mycophenolic acid and triethylamine are added, and the reaction is carried out overnight. After extraction and preparative liquid - phase separation, a chlorambucil - mycophenolic acid prodrug is obtained.

[0024] The purity of the chlorambucil - mycophenolic acid prodrug prepared by the present invention is above 98%.

[0025] The chlorambucil - mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof of the present invention can self - assemble into a chlorambucil - mycophenolic acid prodrug self - assembled nano - preparation (CMNP).

[0026] The aforementioned chlorambucil - mycophenolic acid prodrug self - assembled nano - formulation comprises chlorambucil - mycophenolic acid prodrug, PEG modifier / active targeting modifier.

[0027] The mass ratio of the aforementioned chlorambucil - mycophenolic acid prodrug to PEG modifier / active targeting modifier is 1:(0.1 - 1).

[0028] The aforementioned PEG modifier is an amphiphilic polymer or targeting group such as DSPE - PEG, TPGS, PEG - PLGA or PEG - P, and the active targeting modifier is a substance capable of targeting specific tissues such as brain - targeting peptide, antibody conjugate, ligand conjugate, cell - penetrating peptide conjugate, receptor - targeting conjugate, sugar residue, hormone, etc., selected from DSPE - PEG - SHp, DSPE - PEG - Angiopep, DSPE - PEG - T7, DSPE - PEG - RVG29, DSPE - PEG - cRGD, DSPE - PEG - Lactoferrin, DSPE - PEG - NGR, DSPE - PEG - TAT, DSPE - PEG - iRGD, DSPE - PEG - Mannose, DSPE - PEG - OTC, DSPE - PEG - GE11, DSPE - PEG - CREKA, DSPE - PEG - TH, DSPE - PEG - R8, DSPE - PEG - APOE, preferably phospholipid - polyethylene glycol - apolipoprotein E (DSPE - PEG - APOE).

[0029] Furthermore, the present invention provides a preparation method of the aforementioned chlorambucil - mycophenolic acid prodrug nano - formulation, which comprises the following steps:

[0030] Dissolve an appropriate amount of PEG modifier / active targeting modifier and chlorambucil - mycophenolic acid prodrug in an organic solvent, stir and mix well, and then slowly drop the mixed solution into the aqueous phase. Under the action of self - assembly of prodrug molecules, the system spontaneously forms uniformly dispersed nanoparticles. Subsequently, use the reduced - pressure rotary evaporation method to completely remove the organic solvent, and finally obtain a pure PEG - modified / active - targeting - modified chlorambucil - mycophenolic acid prodrug self - assembled nano - formulation.

[0031] The chlorambucil - mycophenolic acid prodrug of the present invention or its pharmaceutically acceptable salt can also be co - assembled with other small - molecule compounds as the main drug components into a chlorambucil - mycophenolic acid prodrug and small - molecule compound co - assembled nano - formulation (tNP).

[0032] The aforementioned small - molecule compound is MHY1485.

[0033] The molar ratio of the chlorambucil - mycophenolic acid prodrug or its pharmaceutically acceptable salt to MHY1485 is 5:1 - 1:2, preferably 3:1 - 5:1, more preferably 3:1 - 4:1.

[0034] The co - assembled nano - formulation of the chlorambucil - mycophenolic acid prodrug and the small - molecule compound contains the chlorambucil - mycophenolic acid prodrug, MHY1485, and a PEG modifier / active targeting modifier.

[0035] The mass ratio of the total mass of the chlorambucil - mycophenolic acid prodrug and MHY1485 to the mass of the PEG modifier / active targeting modifier is 1:(0.1 - 1).

[0036] The PEG modifier is an amphiphilic polymer or targeting group such as DSPE - PEG, TPGS, PEG - PLGA, or PEG - P, and the active targeting modifier is a substance capable of targeting specific tissues such as a brain - targeting peptide, antibody conjugate, ligand conjugate, cell - penetrating peptide conjugate, receptor - targeting conjugate, sugar residue, hormone, etc., selected from DSPE - PEG - SHp, DSPE - PEG - Angiopep, DSPE - PEG - T7, DSPE - PEG - RVG29, DSPE - PEG - cRGD, DSPE - PEG - Lactoferrin, DSPE - PEG - NGR, DSPE - PEG - TAT, DSPE - PEG - iRGD, DSPE - PEG - Mannose, DSPE - PEG - OTC, DSPE - PEG - GE11, DSPE - PEG - CREKA, DSPE - PEG - TH, DSPE - PEG - R8, DSPE - PEG - APOE, preferably phospholipid - polyethylene glycol - apolipoprotein E (DSPE - PEG - APOE).

[0037] The present invention also provides a preparation method for the co - assembled nano - formulation of the chlorambucil - mycophenolic acid prodrug and MHY1485, comprising the following steps:

[0038] An appropriate amount of the PEG modifier / active targeting modifier, the chlorambucil - mycophenolic acid prodrug, and MHY485 are dissolved in an organic solvent. After thorough stirring and mixing, the mixed solution is slowly added dropwise to the aqueous phase. Under the action of the self - assembly of the prodrug molecules, the system spontaneously forms uniformly dispersed nanoparticles. Subsequently, the organic solvent is completely removed by the method of reduced - pressure rotary evaporation to finally obtain a pure co - assembled nano - formulation of PEG - modified / active - targeting - modified chlorambucil - mycophenolic acid prodrug and MHY1485.

[0039] The organic solvent is an organic solvent miscible with water such as ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, N,N - dimethylformamide, etc., preferably acetone.

[0040] The volume ratio of the organic solvent to water is (0.1 - 1):1, preferably 0.2 - 0.5:1.

[0041] Advantages of the present invention:

[0042] (1) The present invention proposes an "open source and throttling" strategy. By depleting the guanosine raw materials required for tumor cells to repair DNA damage, it forms a highly efficient synergistic effect with chlorambucil, effectively circumventing the resistance mechanism of temozolomide. This strategy not only enriches the design ideas of combination drug use for temozolomide-resistant glioblastoma, but also provides a new solution for clinical treatment.

[0043] (2) The present invention designs and synthesizes a chlorambucil - mycophenolic acid prodrug, and successfully prepares self-assembled nanoformulations with regular morphology and uniform particle size and their co-assembled nanoformulations with MHY1485. The prepared nanoformulations have a particle size less than 200 nm, and the optimal range is 50 - 150 nm. The polydispersity index (PDI) < 0.15. The preparation method is simple and easy to operate, with good repeatability and stability.

[0044] (3) The present invention constructs a nano-drug delivery system modified with a targeting peptide through prodrug co-assembly technology, achieving crossing the blood-brain barrier and precisely delivering drugs to the tumor site, significantly enhancing the synergistic anti-tumor effect of the drug combination, and is expected to break through the key bottlenecks of low drug delivery efficiency and poor treatment effect in brain diseases. In addition, the tumor redox-responsive disulfide bond introduced in the prodrug design enables the nanoformulations to specifically activate drugs in tumor cells with high expression of reduction, while remaining inert in normal tissues, greatly reducing the damage of off-target effects to the brain parenchyma and achieving the treatment goal of reducing toxicity and enhancing efficacy. Brief description of the drawings:

[0045] Figure 1 It is the mass spectrum of the chlorambucil - mycophenolic acid prodrug in Example 1 of the present invention.

[0046] Figure 2 It is the 1 HNMR spectrum of the chlorambucil - mycophenolic acid prodrug in Example 1 of the present invention.

[0047] Figure 3 It is the particle size distribution diagram of CMNP and tNP in Example 3 of the present invention.

[0048] Figure 4 It is the transmission electron micrograph of CMNP and tNP in Example 3 of the present invention.

[0049] Figure 5 It is the colloidal stability diagram of CMNP and tNP in Example 4 of the present invention.

[0050] Figure 6This is the graph showing the changes in particle size and PDI of CMNP and tNP in Example 4 of the present invention during long-term storage.

[0051] Figure 7 This is the graph showing the potential changes of CMNP and tNP in Example 4 of the present invention during long-term storage.

[0052] Figure 8 This is the in vitro drug release graph of tNP in Example 5 of the present invention.

[0053] Figure 9 This is the in vitro drug release graph of CMNP in Example 5 of the present invention.

[0054] Figure 10 This is the comet assay graph of each formulation in Example 7 of the present invention.

[0055] Figure 11 This is the quantitative graph of the comet assay tail moment of each formulation in Example 7 of the present invention.

[0056] n.s. indicates no significant difference, and P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0057] Figure 12 This is the quantitative graph of the DNA content in the tail of the comet assay of each formulation in Example 7 of the present invention.

[0058] n.s. indicates no significant difference, and P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0059] Figure 13 This is the graph of the intracellular GTP concentration of each formulation in Example 7 of the present invention.

[0060] n.s. indicates no significant difference, and P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0061] Figure 14 This is the quantitative detection graph of AP sites of each formulation in Example 7 of the present invention.

[0062] n.s. indicates no significant difference, and P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0063] Figure 15 This is the in vivo tumor fluorescence signal graph of each formulation in Example 8 of the present invention.

[0064] Figure 16 This is the quantitative graph of the in vivo tumor fluorescence signal of each formulation in Example 8 of the present invention.

[0065] Figure 17 This is the fluorescence signal graph of the main tissues and tumors in vitro of each formulation in Example 8 of the present invention.

[0066] Figure 18 Quantitative fluorescence signal graphs of main tissues and tumors in vitro for each formulation of Example 8 of the present invention.

[0067] Figure 19 Fluorescence signal graph of the brain in vitro for each formulation of Example 8 of the present invention.

[0068] n.s. No significant difference, P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0069] Figure 20 Bioluminescence graph of temozolomide-resistant glioblastoma in mice of Example 9 of the present invention.

[0070] Figure 21 Quantitative bioluminescence graph of temozolomide-resistant glioblastoma in mice of Example 9 of the present invention.

[0071] n.s. No significant difference, P < 0.05 is considered to have a significant difference (one-way ANOVA).

[0072] Figure 22 Graph of the change in body weight of mice in Example 9 of the present invention.

[0073] Figure 23 Graph of the survival rate of mice in Example 9 of the present invention.

[0074] Figure 24 Graph of the median survival time of mice in Example 9 of the present invention.

[0075] Figure 25 H&E section graph of the brain tissue of mice in Example 9 of the present invention. Detailed implementation method:

[0076] The present invention will be further described in detail below in conjunction with the embodiments.

[0077] Example 1: Synthesis of chlorambucil-mycophenolic acid prodrug

[0078] Chlorambucil (5 mmol) and a catalytic amount of DMF were dissolved in anhydrous dichloromethane, cooled to 0 - 5 °C in an ice bath, and oxalyl chloride (6 mmol) was rapidly added dropwise under stirring. After the addition was completed, the reaction was carried out at room temperature for 4 hours, and the solvent was evaporated to dryness to obtain the intermediate acyl chloride. Subsequently, dithiodiethanol (6 mmol) and triethylamine (10 mmol) were dissolved in anhydrous dichloromethane, cooled to 0 - 5 °C in an ice bath, and the acyl chloride intermediate dissolved in anhydrous dichloromethane was slowly added dropwise. After the addition was completed, the reaction was carried out overnight at room temperature. The organic phase was extracted with 10% citric acid solution, evaporated to dryness and redissolved in acetonitrile, and purified by preparative liquid chromatography, and then evaporated to dryness to obtain the chlorambucil-dithiodiethanol intermediate.

[0079] Dissolve chlorambucil-disulfide diethanol intermediate (3 mmol) and DIPEA (6 mmol) in anhydrous dichloromethane, cool to 0 - 5 °C in an ice bath, slowly add dropwise p-nitrophenyl chloroformate (4 mmol) dissolved in anhydrous dichloromethane. After the addition is complete, raise the temperature to room temperature and react for 2 hours. Rotate to dry the solvent, redissolve in anhydrous DMF, add mycophenolic acid (3 mmol), stir at room temperature until completely dissolved, quickly add triethylamine (6 mmol), react overnight at room temperature. Extract the organic phase with saturated sodium bicarbonate and 10% citric acid solution respectively, rotate to dry and then redissolve in acetonitrile, separate and purify by preparative liquid phase, rotate to dry to obtain chlorambucil-mycophenolic acid prodrug.

[0080] Mass spectrometry and proton nuclear magnetic resonance spectroscopy were used to determine the structure of the chlorambucil-mycophenolic acid prodrug in Example 1, and the results are as Figure 1 、 Figure 2 shown. The spectral analysis results are as follows:

[0081] 1 H NMR (400 MHz, Chloroform-d) δ 7.17–6.96 (m, 2H, d), 6.64 (d, J = 8.5 Hz, 2H, c), 5.30 (s, 1H, p), 5.23–5.03 (m, 2H, k), 4.54 (t, J = 6.8 Hz, 2H, j), 4.34 (t, J = 6.5 Hz, 2H, h), 3.91–3.40 (m, 13H, a, b, n, o), 3.07 (t, J = 6.8 Hz, 2H, e), 2.95 - 2.56 (t, J = 6.4 Hz, 4H, i), 2.47–2.14 (m, 9H, g, r, s), 1.91 (p, J = 7.5 Hz, 2H, f), 1.79 (d, J = 1.4 Hz, 3H, q). MS (ESI) m / z: 808.1765 [M+Na] + .

[0082] Example 2: Prescription screening of brain-targeted prodrug nanoparticles

[0083] Select DSPE-PEG-APOE as the surface modification material, and prepare brain-targeted prodrug nanoparticles by one-step nanoprecipitation method. The specific preparation process is as follows:

[0084] Preparation of self-assembled nanoparticles of chlorambucil-mycophenolic acid prodrug: Dissolve chlorambucil-mycophenolic acid prodrug and DSPE-PEG-APOE (mass ratio 20%, w / w) in 0.5 mL of acetone to form a homogeneous solution. Under vigorous stirring conditions, slowly add this solution dropwise to 5 mL of deionized water. After thorough mixing, remove acetone by rotary evaporation under reduced pressure to finally obtain self-assembled nanoparticles of chlorambucil-mycophenolic acid prodrug (CMNP).

[0085] Preparation of co - assembled nanomedicine of chlorambucil - mycophenolic acid prodrug and MHY1485: The chlorambucil - mycophenolic acid prodrug and MHY1485 were mixed at different molar ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5, and dissolved in 0.5 mL of acetone with DSPE - PEG - APOE (mass ratio 20%, w / w) to form a homogeneous solution. Under vigorous stirring, this solution was slowly added dropwise to 5 mL of deionized water. After thorough mixing, acetone was removed by rotary evaporation under reduced pressure, and finally, the co - assembled nanomedicine of chlorambucil - mycophenolic acid prodrug and MHY1485 was obtained.

[0086] Characterization of prodrug nanomedicine: After diluting the prodrug nanomedicine 10 - fold with deionized water, its particle size, polydispersity index (PDI), and surface potential were measured using a Malvern particle size analyzer.

[0087] Determination of encapsulation efficiency and drug loading of prodrug nanomedicine: Centrifugation was used to remove the unencapsulated drugs in the prodrug nanomedicine, and high - performance liquid chromatography was used to determine the encapsulation efficiency. A C18 reverse - phase chromatographic column (4.6×250 mm, 5 μm) was used, with the mobile phase being methanol and water. The detection wavelength for the chlorambucil - mycophenolic acid prodrug was 258 nm, and the detection wavelength for MHY1485 was 340 nm. The encapsulation efficiency and drug loading of the chlorambucil - mycophenolic acid prodrug and MHY1485 were calculated according to the following formulas:

[0088] Encapsulation efficiency = (drug content in the formulation / total amount of drug input) × 100%;

[0089] Drug loading = theoretical drug loading × encapsulation efficiency × 100%.

[0090] Synergy index of prodrug nanomedicine: First, the MTT method was used to evaluate the cytotoxicity of the co - assembled nanomedicines at various ratios. The specific operation procedure was as follows: U87 / TR cells (temozolomide - resistant U87 cells) were seeded into 96 - well plates (2×10 3 cells per well) and incubated in an incubator for 12 hours to ensure stable cell adhesion. Subsequently, the original culture medium was replaced with culture media containing different concentration gradients of chlorambucil, mycophenolic acid, MHY1485, CMNP, and the co - assembled nanomedicines at various ratios. Untreated cells served as the control group. After incubating the cells in a 37°C incubator for another 48 hours, 20 μL of MTT solution was added to each well and incubated for 4 hours to allow the formation of formazan crystals. Subsequently, the culture medium was discarded, DMSO was added to dissolve the formazan crystals, and the absorbance value was measured at a wavelength of 490 nm using a multifunctional microplate reader. The half - maximal inhibitory concentration (IC 50)。In addition, using chlorambucil, mycophenolic acid, and MHY1485 as controls, the Chou-Talalay method was used for calculation, and the formula is as follows: 50 As a control, using the Chou-Talalay method for calculation, the formula is as follows:

[0091]

[0092] Among them, D 1 , D 1 , D 3 are the actual molar concentrations of chlorambucil, mycophenolic acid, and MHY1485 in the prodrug nanoparticles, respectively; IC 50,1 , IC 50,2 , IC 50,3 are the IC 50 values of chlorambucil, mycophenolic acid, and MHY1485 alone, respectively. CI 50 < 1 indicates a synergistic effect, CI 50 = 1 is an additive effect, and CI 50 > 1 indicates an antagonistic effect.

[0093] Morphological observation of the prodrug nanoparticles: The prodrug nanoparticles were diluted 10-fold with pure water to the sample loading volume, and then a small amount of the diluted solution was dropped onto a copper grid for electron microscopy and allowed to air-dry naturally. Then, 0.25% phosphotungstic acid solution was dropped for staining in the dark and allowed to air-dry at room temperature. After sample preparation, the morphological characteristics of the nanoparticles were observed using a transmission electron microscope (TEM).

[0094] By investigating the effects of different molar ratios of chlorambucil - mycophenolic acid prodrug to MHY1485 on the particle size, encapsulation efficiency, and drug loading of the nanoparticles, the optimal assembly ratio was determined. The results are shown in Table 1. When the molar ratio of chlorambucil - mycophenolic acid prodrug to MHY1485 was 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, colloidal nanoparticles were successfully prepared. However, at ratios of 1:3, 1:4, and 1:5, the drugs aggregated and precipitated. The blood-brain barrier consists of a dense monolayer of cell membranes, tight junctions, and a matrix, strictly controlling the entry and exit of substances. Even after surface modification, nanoparticles with too large a particle size (>200 nm) still have poor permeability and are difficult to cross the blood-brain barrier. Therefore, the co-assembled nanoparticles at ratios of 2:1, 1:1, and 1:2 cannot be used as candidate formulations due to their particle sizes being greater than 200 nm. When the molar ratio of chlorambucil - mycophenolic acid prodrug to MHY1485 was 5:1 - 3:1, the particle sizes of the nanoparticles were all less than 150 nm, and the dispersion coefficients were all less than 0.15. Therefore, the preferred molar ratio of chlorambucil - mycophenolic acid prodrug to MHY1485 is 5:1 - 3:1.

[0095] Table 1. Characterization of co-assembled nanoparticles of chlorambucil - mycophenolic acid prodrug and MHY1485 at different molar ratios

[0096]

[0097] a) The different ratios are the molar ratios of chlorambucil - mycophenolic acid prodrug to MHY1485

[0098] The encapsulation efficiency and drug loading of the nano - formulation are two key indicators for evaluating the performance of the nano - formulation. The higher the encapsulation efficiency, the stronger the self - assembly ability of the drug system of the nano - formulation, the better the reproducibility between different batches, and the more conducive to industrial production and quality controllability. The higher the drug loading, the lower the proportion of non - drug components in the nano - formulation, and the more effectively it can avoid the potential toxic and side effects brought by non - drug components. As shown in Table 2, when the molar ratio of chlorambucil - mycophenolic acid prodrug to MHY1485 is 5:1 - 1:2, the encapsulation efficiency of chlorambucil - mycophenolic acid prodrug can reach more than 75%, the encapsulation efficiency of MHY1485 reaches more than 60%, and the total drug loading is above 50%. When the ratio of the two is 5:1 - 3:1, the encapsulation efficiency of chlorambucil - mycophenolic acid prodrug can reach more than 98%, the encapsulation efficiency of MHY1485 is about more than 90%, and the total drug loading reaches more than 60%. Especially in the co - assembled nano - formulation of chlorambucil - mycophenolic acid prodrug and MHY1485 with a ratio of 3:1, the encapsulation efficiencies of chlorambucil - mycophenolic acid prodrug and MHY1485 both reach more than 99%, and the total drug loading is as high as 65.43%.

[0099] Table 2. Encapsulation efficiency and drug loading of co - assembled nano - formulations of chlorambucil - mycophenolic acid prodrug and MHY1485 with different molar ratios

[0100]

[0101]

[0102] a) The different ratios are the molar ratios of chlorambucil - mycophenolic acid prodrug to MHY1485. b) The total drug loading is the sum of the drug loadings of chlorambucil, mycophenolic acid and MHY1485

[0103] The IC of co - assembled nano - formulations with each ratio 50 and CI 50 As shown in Table 3. The co - assembled nano - formulations constructed from chlorambucil - mycophenolic acid prodrug and MHY1485 at molar ratios of 4:1, 3:1 and 1:1 show significant synergistic effects (CI 50 <1), while the combinations of 5:1, 2:1 and 1:2 ratios show antagonistic effects (CI 50 >1). It is worth noting that the co - assembled nano - formulation constructed from chlorambucil - mycophenolic acid prodrug and MHY1485 at a molar ratio of 3:1 has the lowest IC 50(15.7μM) and the smallest CI 50 (0.39). Based on the results of particle size, encapsulation efficiency and drug loading, the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is preferably 3:1-4:1. Moreover, when the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is 3:1, the co-assembled nanoformulation has the best pharmaceutical properties and synergistic effect. Therefore, 3:1 is determined to be the optimal ratio for constructing the co-assembled nanoformulation of chlorambucil-mycophenolic acid prodrug and MHY1485.

[0104] Table 3. IC of nanoformulations co-assembled with chlorambucil-mycophenolic acid prodrug and MHY1485 at different molar ratios 50 With CI 50

[0105]

[0106]

[0107] a) Molar ratio of chlorambucil: mycophenolic acid: MHY1485 corrected by encapsulation efficiency b) IC 50 Unit: μM

[0108] The following studies were conducted using chlorambucil-mycophenolic acid prodrug self-assembled nanoformulation (CMNP) and chlorambucil-mycophenolic acid prodrug and MHY1485 co-assembled nanoformulation (tNP) constructed with a molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 of 3:1.

[0109] like Figure 3 and Figure 4 As shown, the particle size of CMNP and tNP is about 90nm, and has a uniform particle size distribution (PDI < 0.15). The surface potential of both nanoformulations is about -50mV, ensuring the biocompatibility of intravenous administration. In addition, transmission electron microscopy observation results show that both have a uniform spherical structure and have good morphology.

[0110] Example 3: Preparation of brain-targeted prodrug nanoformulations

[0111] (1) Preparation of Chlorambucil-Mycophenolic Acid Prodrug Self-Assembled Nanoformulation (CMNP):

[0112] Chlorambucil-mycophenolic acid prodrug and DSPE-PEG-APOE (mass ratio 20%, w / w) were dissolved in 0.5 mL of acetone to form a uniform solution. The solution was slowly added dropwise to 5 mL of deionized water under vigorous stirring, and after thorough mixing, the acetone was removed by reduced pressure rotary evaporation to obtain the product.

[0113] (2) Preparation of the co-assembled nanoformulation (tNP) of chlorambucil - mycophenolic acid prodrug and MHY1485:

[0114] Mix the chlorambucil - mycophenolic acid prodrug and MHY1485 in a molar ratio of 3:1, and dissolve them with DSPE - PEG - APOE (mass ratio 20%, w / w) in 0.5 mL of acetone to form a homogeneous solution. Under vigorous stirring conditions, slowly add this solution dropwise to 5 mL of deionized water. After thorough mixing, remove the acetone by rotary evaporation under reduced pressure to obtain the product.

[0115] Example 4: Colloidal stability and long - term storage stability of the brain - targeting prodrug nanoformulation

[0116] Colloidal stability: Dilute the CMNP and tNP prepared in Example 3 in a ratio of 1:10 with PBS solution containing 10% fetal bovine serum (FBS), and place them in a shaker at 37 °C for incubation. Samples are taken at the 0, 2, 4, 8, 12, 24, and 48 hours of incubation, and a Malvern particle size analyzer is used to measure their particle size and polydispersity index (PDI).

[0117] Long - term storage stability: Store the CMNP and tNP prepared in Example 3 in a 4 °C refrigerator. Samples are taken on the 1st, 7th, 15th, and 30th days respectively. After diluting the prodrug nanoformulation 10 - fold with deionized water, a Malvern particle size analyzer is used to measure their particle size, polydispersity index (PDI), and surface potential.

[0118] Use PBS containing 10% FBS to simulate the in - vivo physiological environment. The results are as Figure 5 shown. Within 12 hours of incubation, both CMNP and tNP can remain stable without obvious particle size changes. However, after 12 hours, the particle size and PDI of CMNP increase rapidly, indicating the disintegration of its nanostructure. In contrast, tNP can remain stable within 48 hours. This result shows that tNP may have better in - vivo stability.

[0119] The results of long - term storage are as Figure 6 and Figure 7 shown. During the 30 - day storage period, there are no significant changes in the particle size and surface potential of CMNP and tNP, and the PDI also remains below 0.2, indicating that the prodrug nanoformulations can maintain their physical properties during long - term storage and are suitable for subsequent applications.

[0120] Example 5: In vitro drug release of the brain - targeting prodrug nanoformulation

[0121] The PBS solution containing 20% ethanol was used as the release medium, and different concentrations of dithiothreitol (DTT) were added thereto, with the final concentrations set at 1 and 10 mM respectively, and the blank release medium was used as the control. The CMNP and tNP prepared in Example 3 were respectively loaded into dialysis bags. After sealing, the dialysis bags were placed in the corresponding release medium and incubated with shaking at 37 °C. To evaluate the drug release process, samples were taken at different time points (1, 2, 4, 8, and 12 hours) after incubation, and the samples at each time point were repeated three times. The collected samples were quantitatively analyzed by high performance liquid chromatography (HPLC), and chlorambucil, mycophenolic acid, and MHY1485 were detected at wavelengths of 258 nm, 250 nm, and 340 nm respectively.

[0122] As Figure 8 and Figure 9 shown, tNP and CMNP exhibited excellent reduction-responsive drug release characteristics, and both showed similar trends in the release behaviors of chlorambucil and mycophenolic acid. In the absence of DTT, the amount of drug released from the prodrug nanoparticles within 12 hours was less than 10%. While at a DTT concentration of 1 mM, almost all drugs could be completely released within 12 hours. When the DTT concentration was increased to 10 mM, chlorambucil and MHY1485 were completely released from tNP or CMNP within 4 hours, and mycophenolic acid was completely released within 8 hours. It should be particularly noted that MHY1485 itself was not prodrug-modified and thus did not possess reduction-responsive characteristics. However, when it was co-assembled with the chlorambucil-mycophenolic acid prodrug bridged by a disulfide bond, the reduction-responsive effect of the chlorambucil-mycophenolic acid prodrug led to the disassembly of the tNP nanostructure, thereby endowing MHY1485 with synchronous reduction-responsive ability. This characteristic enables tNP to achieve intelligent responsive release of triple drugs, thus providing a unique advantage for reducing toxic side effects and improving the therapeutic effect.

[0123] Example 6: Evaluation of the cytotoxicity and selectivity of the brain-targeted prodrug nanoparticles

[0124] The MTT method was used to evaluate the cytotoxicity of the prodrug nanoparticles. The specific operation procedure was as follows: Human glioblastoma U87 cells, U87 / TR cells (temozolomide-resistant U87 cells), and mouse hippocampal neurons HT22 cells were seeded into 96-well plates (2×10 3cells), and incubate them in an incubator for 12 hours to ensure that the cells can adhere stably. Subsequently, replace the original culture medium with a culture medium containing temozolomide solution, chlorambucil, mycophenolic acid and MHY1485 in different concentration gradients (Mixed sol, molar ratio: chlorambucil: mycophenolic acid: MHY1485 = 3:3:1, the same as the ratio of the three drugs in tNP), CMNP or tNP. Untreated cells were used as the control group. After incubating the cells in each group in a 37 °C incubator for 48 hours, add 20 μL of MTT solution to each well and continue to incubate for 4 hours to allow the formation of formazan crystals. Subsequently, discard the culture medium, add DMSO to dissolve the formazan crystals, measure the absorbance value using a multifunctional microplate reader at a wavelength of 490 nm, and calculate the half-maximal inhibitory concentration (IC 50 ) according to the measured data using graphpad software. Calculate the selection index (SI) according to the obtained IC 50 , and the formula is as follows: Selection index = IC 50 of the formulation in tumor cells / IC 50 of the formulation in HT22 cells. The results are shown in Table 4.

[0125] Table 4. IC 50 and selection index of each formulation

[0126]

[0127] The results showed that in two glioblastoma cells (U87 and U87 / TR), since the prodrug nanoplatforms need to exert their activity after drug release, their IC 50 values were higher than those of Mixed sol. As a first-line treatment drug for glioblastoma, the cytotoxicity of temozolomide was significantly weaker than that of other combination drug groups. Especially in drug-resistant U87 / TR cells, compared with wild-type U87, the IC 50 of temozolomide increased by 8.6 times, while the IC 50 of other combination formulations increased in drug-resistant cells, but the increase did not exceed 2 times. Among them, the IC 50 of the two prodrug nanoplatforms only increased by 1.2 times. It is worth noting that among the prodrug nanoplatforms, tNP showed stronger cytotoxicity due to the synergistic effect of the triple drugs. These results indicate that the combination drug strategy designed in the present invention can target glioblastoma cells and further effectively overcome the drug resistance of glioblastoma to temozolomide.

[0128] In addition, mouse hippocampal neuron HT22 cells were used instead of normal brain tissue to evaluate the off-target toxicity of each preparation. The experimental results showed that Mixed sol had the strongest toxicity in HT22 cells, while the prodrug nanoplatforms with strong anti-tumor activity in glioma cells had significantly reduced toxicity in HT22 cells. To further analyze the selectivity of the drugs, the selectivity index was calculated, which is used to measure the relative toxicity of drugs to tumor cells and normal cells. The larger the value, the stronger the toxicity of the drug to tumor cells and the lower the toxicity to normal cells. The results showed that the selectivity index of temozolomide was even lower than 1, indicating that it had high toxic side effects on normal brain tissue. In contrast, tNP had the highest selectivity index, which may be attributed to its responsive drug release mechanism in the tumor microenvironment and the lower proportion of chlorambucil (a highly toxic drug) at the same molar concentration. This property not only endows tNP with stronger anti-glioblastoma activity, but also reduces its non-specific toxicity to brain tissue and is not affected by temozolomide resistance, demonstrating good therapeutic advantages.

[0129] Example 7: Anti-tumor mechanism of brain-targeted prodrug nanoplatforms

[0130] Comet assay (single cell gel electrophoresis): U87 / TR cells were seeded in 6-well plates at a density of 2×10 5 cells per well and incubated in an incubator for 12 hours to allow them to adhere. The medium was discarded and replaced with medium containing temozolomide (1000 μM), Mixed sol, CMNP, and tNP (total drug amount 10 μM), and a blank cell control was set, and incubation was continued for 24 hours. The cells were collected and washed, mixed with low melting point agarose, and then spread on pre-treated slides. After solidification, the slides were placed in lysis solution and incubated at 4 °C in the dark to lyse the cell membranes and release DNA, and then transferred to alkaline solution to unwind the DNA and expose the damaged sites, and then electrophoresed in alkaline electrophoresis buffer to form a comet-like tail of the damaged DNA. After electrophoresis, the slides were washed with neutralization buffer, stained with EB, and images were recorded under a fluorescence microscope, and the tail DNA content was analyzed using Image J software.

[0131] Intracellular GTP concentration detection: U87 / TR and U87 cells were seeded in 6-well plates at a density of 2×10 5Inoculate into a 6-well plate and incubate in an incubator for 12 hours to promote cell adhesion. Discard the culture medium in the U87 / TR cell wells and replace it with a culture medium containing temozolomide (1000 μM), Mixed sol, CMNP, and tNP (total drug dose 10 μM). Set blank U87 / TR and U87 cells as controls and continue to incubate for 24 hours. Collect cell samples, lyse them with cell lysate, and centrifuge to obtain the supernatant. According to the operation of the GTP concentration detection kit, add a known concentration of GTP standard and the cell lysate supernatant to an enzyme-linked immunosorbent assay (ELISA) plate coated with specific antibodies and incubate. After washing the plate, add the enzyme-labeled secondary antibody and incubate and wash again. Add the substrate solution to develop color in the dark. When the color development is appropriate, add the stop solution. Finally, measure the absorbance at 450 nm with an ELISA reader and obtain the intracellular GTP concentration by comparing with the standard curve.

[0132] Quantitative detection of AP sites: Seed U87 / TR cells at a density of 2×10 5 cells per well in a 6-well plate and incubate in an incubator for 12 hours to allow cell adhesion. Discard the culture medium and replace it with a culture medium containing temozolomide (1000 μM), Mixed sol, CMNP, and tNP (total drug dose 10 μM). Set blank cell controls and continue to incubate for 24 hours. At the end of incubation, collect the cells. According to the operation of the AP site detection kit, first equilibrate the materials and reagents to room temperature, purify genomic DNA and dilute it to 100 μg / mL, perform the ARP reaction, and dilute the ARP-derived DNA sample to 1 μg / mL after processing the reaction product. Add the standard dilution and the sample to a DNA high-binding plate, sequentially add the DNA binding solution, streptavidin-enzyme conjugate, and substrate solution, incubate and wash after each addition, add the stop solution, and measure the absorbance at 450 nm with a microplate reader. Calculate the intracellular AP site content by comparing with the standard curve.

[0133] The comet assay (single cell gel electrophoresis) is a highly sensitive method for detecting DNA damage. Its principle is that damaged DNA fragments migrate through electrophoresis, thus forming a typical "comet-like" structure. Usually, the tail length, tail moment, and tail DNA content of the comet can quantitatively reflect the degree of DNA damage. The higher the value, the more severe the damage. As Figure 10 、 Figure 11 and Figure 12As shown, only a very small amount of tailing was observed in temozolomide-treated U87 / TR cells, indicating a weak DNA damage effect on drug-resistant cells. The comet tails in the Mixed sol group were the longest, with the highest tail moment and tail DNA content, indicating the most significant induction of DNA damage, followed by tNP. This trend was consistent with the results of the cytotoxicity experiment, further demonstrating that it is difficult to effectively induce DNA damage by using temozolomide alone in temozolomide-resistant U87 / TR cells, while combination drug regimens, especially Mixed sol and tNP, can still significantly disrupt the DNA integrity of drug-resistant glioblastoma cells.

[0134] The temozolomide resistance mechanism in glioblastoma usually involves the upregulation of guanine nucleotide metabolism to enhance DNA repair ability and cope with chemotherapy-induced DNA damage. As Figure 13 shown, compared with wild-type U87 cells, the intracellular GTP concentration in U87 / TR cells was significantly increased, indicating that the guanine nucleotide metabolic pathway was more active in drug-resistant cells. Under different drug treatments, temozolomide even further increased the GTP level, while Mixed sol and tNP, based on the "open source and cut off the flow" guanine nucleotide regulation strategy, that is, MHY1485 activates the mTOR pathway to promote guanine nucleotide consumption (open source), and at the same time IMPDH2 inhibits de novo guanine nucleotide synthesis (cut off the flow), ultimately resulting in a significant decrease in intracellular GTP concentration and causing guanine nucleotide depletion. In contrast, although CMNP also decreased the GTP level, it only inhibited guanine nucleotide synthesis through a single mechanism of IMPDH2, and its effect was far less than the "open source and cut off the flow" strategy of Mixed sol and tNP.

[0135] AP sites (apurinic sites) refer to the abasic sites formed after the loss of purine bases in the DNA strand due to factors such as hydrolysis, oxidation, or chemical modification. In tumor cells, when chlorambucil induces DNA strand crosslinking, the cell needs to excise the damaged bases and activate the base excision repair pathway for DNA repair, and guanine nucleotide is an essential raw material in this repair process. When intracellular guanine nucleotide is depleted, DNA repair is blocked, and the unrepaired AP sites accumulate, thus exacerbating DNA damage. As Figure 14 shown, the change trend of the number of AP sites was consistent with the DNA damage trend and was negatively correlated with the intracellular GTP concentration. This result indicates that the combination drug regimen based on the "open source and cut off the flow" strategy significantly reduces the intracellular guanine nucleotide level, exacerbates the DNA damage induced by chlorambucil, and effectively inhibits the viability of temozolomide-resistant glioblastoma cells, suggesting its therapeutic advantage in overcoming drug-resistant glioblastoma.

[0136] Example 8: Biodistribution of Brain-Targeted Prodrug Nanopreparations

[0137] Preparation of DiR-labeled CMNP and tNP: DiR-labeled CMNP and tNP were prepared using the same method as in Example 3, with the only difference being the additional addition of 10% DiR (mass ratio, w / w) as a fluorescent marker during the preparation process to facilitate fluorescence tracer analysis.

[0138] Preparation of DiR-labeled non-brain-targeted tNP (tNP / nT): The preparation method of tNP / nT was consistent with the tNP preparation method described in Example 3. The difference was that DSPE-PEG-APOE was replaced with an equal mass of DSPE-PEG during the preparation process, and 10% DiR (mass ratio, w / w) was additionally added as a fluorescent marker to achieve fluorescence tracing. Finally, the prepared nanoparticles were named tNP / nT.

[0139] Construction of temozolomide-resistant glioblastoma model: An orthotopic glioblastoma model was constructed using immunodeficient Balb / c nude mice. After anesthesia with isoflurane, the nude mice were fixed on a stereotaxic apparatus. After drilling a hole in the skull, digested U87 / TR-luc cells (5×10 5 cells, diluted with PBS) were slowly injected into the right caudate nucleus region. After the injection was completed, the wound was sealed with tissue glue. Ten days after tumor inoculation, potassium fluorescein (150 mg / kg) was injected intraperitoneally, and the bioluminescence signal of the tumor was recorded and analyzed using a live imaging system. Subsequently, the successfully modeled nude mice were randomly grouped for subsequent experiments.

[0140] Biodistribution experiment: DiR solution, DiR-labeled CMNP, tNP, and tNP / nT were administered to the modeled mice by tail vein injection. According to the DiR equivalent calculation, the administration dose was 2 mg / kg. At 4, 8, 12, and 24 hours after administration, the mice were anesthetized, and DiR fluorescence images of the tumor site were collected using a live imaging system, and quantitative analysis was performed simultaneously. At the 24-hour endpoint, the mice were sacrificed, and the main organs (heart, liver, spleen, lung, and kidney) and brain tissue were isolated, and the DiR fluorescence signal of each tissue was imaged and quantitatively analyzed using a live imaging instrument to evaluate the tissue distribution of the drug.

[0141] The experimental results are as Figure 15 and Figure 16 shown. Only extremely low fluorescence signals were detected in the brain tissue for the DiR solution and non-brain-targeted modified DiR-labeled tNP / nT, while obvious fluorescence accumulation was shown in the brain for both DiR-labeled CMNP and tNP. Over time, the fluorescence intensity of the two brain-targeted prodrug nanoparticles in the brain gradually increased and reached a peak at 12 hours. This phenomenon indicates that both CMNP and tNP can successfully cross the blood-brain barrier and accumulate in the brain tumor region, verifying their good brain-targeted delivery ability and emphasizing the importance of brain-targeted modification.

[0142] At 24 hours, the fluorescence distribution in major organs and brain tissue was as follows Figure 17 and Figure 18 , and the results were consistent with those observed by in vivo fluorescence imaging. There were only extremely low fluorescence signals of DiR solution and DiR-labeled tNP / nT without brain targeting modification in the brain, and they were mainly distributed in the lungs and liver. In contrast, both DiR-labeled CMNP and tNP successfully crossed the blood-brain barrier and were significantly enriched in the brain tumor region. As shown in Figure 19 , compared with DiR solution, the fluorescence intensity of DiR-labeled tNP / nT was only increased by 1.5 times, while DiR-labeled CMNP and tNP were increased by 6.1 times and 6.5 times respectively, further demonstrating their effective accumulation in the brain. There was no significant difference in the accumulation effect of the two prodrug nanoplatforms in brain tumors, which might be related to their same targeting modification and similar surface physicochemical properties, resulting in similar in vivo distribution characteristics. In summary, the brain-targeted prodrug nanoplatform constructed in this study can achieve precise targeted delivery to glioblastoma, laying an important foundation for its further anti-tumor effect.

[0143] Example 9: Investigation of anti-tumor effect of brain-targeted prodrug nanoplatform in vivo

[0144] An orthotopic model of glioblastoma was established using immunodeficient Balb / c nude mice. After anesthesia with isoflurane, the nude mice were fixed on a stereotaxic apparatus. After drilling a hole in the skull, digested U87 / TR-luc cells (5×10 5 cells, diluted with PBS) were slowly injected into the right caudate nucleus region. After the injection, the wound was sealed with tissue glue. Ten days after tumor inoculation, potassium fluorescein (150 mg / kg) was injected intraperitoneally, and the bioluminescence signal of the tumor was recorded and analyzed using an in vivo imaging system. Subsequently, the successfully modeled nude mice were randomly grouped for subsequent experiments.

[0145] In vivo anti-tumor experiment: The in situ model of nude mice with glioblastoma was used to evaluate the anti-tumor efficacy of different drugs. Temozolomide was administered by intraperitoneal injection at a dose of 60 mg / kg (309 μM / kg); Mixed sol, CMNP, and tNP were all administered by intravenous injection, and the total drug dose was set at 21.87 μM / kg. The specific doses are shown in Table 5, with PBS as the control. Administration started on day 0, once every 3 days for a total of 5 times. On days 0, 4, 8, 12, and 16, potassium fluorescein (150 mg / kg) was injected intraperitoneally, and the bioluminescence signal of the tumor was recorded and analyzed using a living imaging system to monitor tumor growth. When the mouse body weight decreased to 20% of the initial body weight, the experiment was terminated and euthanasia was performed. On day 16, mouse brain tissues were collected for H&E staining, and the remaining mice continued to undergo survival experiments. The body weight changes and survival rate were monitored every 2 days until the experiment ended.

[0146] Table 5. Specific administration doses of each prescription

[0147]

[0148]

[0149] a) (μ) represents μM / kg; (m) represents mg / kg

[0150] The tumor growth conditions are as Figure 20 , Figure 21 and Figure 22 shown, and the mouse survival conditions are as Figure 23 and Figure 24 shown. In the PBS control group, the tumor grew rapidly, the mouse body weight continued to decrease, and the median survival period was only 18 days. In the temozolomide-resistant glioblastoma model, the first-line clinical drug temozolomide could hardly inhibit tumor growth, and the mice started to die on day 14, failing to extend the median survival period. In addition, although Mixed sol showed good anti-tumor activity in cell experiments, it also failed to effectively inhibit tumor growth in the animal model. Its anti-tumor effect was similar to that of temozolomide, and the mice also started to die on day 14, and the median survival period was even shortened to 16 days, lower than that of the PBS control group. This result indicates that Mixed sol in the free drug form cannot cross the blood-brain barrier and is difficult to reach the brain tumor area to play a role, thus emphasizing the key role of drug delivery strategies in the treatment of gliomas.

[0151] In contrast, due to their brain-targeting ability, CMNP and tNP demonstrated more excellent anti-tumor effects in the drug-resistant glioblastoma model. Among them, tNP showed a stronger inhibitory effect, not only significantly slowing down tumor growth but also effectively delaying the weight loss of mice and extending the median survival period to 30 days. Since the brain-targeted delivery ability of CMNP and tNP was verified to be comparable in Example 8, the stronger anti-tumor activity of tNP was attributed to the synergistic effect of the triple drugs based on the "open source and reduce expenditure" strategy. As Figure 25 , the H&E staining results of mouse brain tissue further verified that tNP significantly inhibited the growth of drug-resistant glioblastoma, followed by CMNP, while the tumor sizes of the temozolomide and Mixed sol treatment groups were close to those of the PBS control group, which was consistent with the pharmacodynamic results.

[0152] In summary, the brain-targeted prodrug nanoparticle tNP constructed in the present invention enhances the sensitivity of cells to the alkylating agent chlorambucil by exacerbating the depletion of guanosine monophosphate in tumor cells through the "open source and reduce expenditure" strategy. At the same time, the brain-targeting modification enables tNP to efficiently cross the blood-brain barrier and achieve targeted delivery to the brain tumor region, ultimately significantly inhibiting the growth of temozolomide-resistant glioblastoma. This study provides an innovative treatment strategy for overcoming the drug resistance of glioblastoma and offers new theoretical basis and potential application value for the precision treatment of temozolomide-resistant patients in clinical practice.

Claims

1. Chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof as shown in formula I: in, X is a redox environment sensitive bond.

2. The chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof according to claim 1:

3. A pharmaceutical composition, comprising the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and MHY1485, wherein the molar ratio of the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof to MHY1485 is 5:1-1:2, preferably 5:1-3:

1.

4. The nanoformulation of chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof according to claim 1 or 2, comprising chlorambucil-mycophenolic acid prodrug and a PEG modifier / active targeting modifier.

5. A co-assembled nanoformulation of chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof and MHY1485, comprising the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof of claim 1 or 2, MHY1485 and a PEG modifier / active targeting modifier.

6. The co-assembled nanoformulation of chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof and MHY1485 according to claim 5, characterized in that: The molar ratio of chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof to MHY1485 is 5:1-1:2, preferably 5:1-3:

1.

7. The nanoformulation according to any one of claims 4 to 6, characterized in that: The PEG modifier is an amphiphilic polymer of DSPE-PEG, TPGS, PEG-PLGA or PEG-P, and the active targeting modifier is a brain targeting peptide, an antibody conjugate, a ligand conjugate, a cell penetrating peptide conjugate, a receptor targeting conjugate, a sugar residue, a hormone that can target a specific tissue, preferably DSPE-PEG-SHp, DSPE-PEG-Angiopep, DSPE-PEG-T7, DSPE-PEG-RVG29, DSPE-PEG-cRGD, DSPE-PEG-Lactoferrin, DSPE-PEG-NGR, DSPE-PEG-TAT, DSPE-PEG-iRGD, DSPE-PEG-Mannose, DSPE-PEG-OTC, DSPE-PEG-GE11, DSPE-PEG-CREKA, DSPE-PEG-TH, DSPE-PEG-R8, and DSPE-PEG-APOE.

8. The nanoformulation according to any one of claims 4 to 6, characterized in that: The mass ratio of the chlorambucil-mycophenolic acid prodrug to the PEG modifier / active targeting modifier is 1:0.1-1; the mass ratio of the total mass of the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof and MHY1485 to the PEG modifier / active targeting modifier is 1:0.1-1.

9. Use of the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3, or the nanoformulation according to any one of claims 4 to 8 in the preparation of a drug for treating glioblastoma.

10. Use of the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3, or the nanoformulation according to any one of claims 4 to 8 in the preparation of a drug for treating temozolomide-resistant glioblastoma.

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