Tumor microenvironment response pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention provides a tumor microenvironment response pharmaceutical composition as well as a preparation method and application thereof. The tumor microenvironment response pharmaceutical composition comprises a pH-sensitive amphiphilic prodrug and a stabilizer, wherein the pH-sensitive amphiphilic prodrug is formed by condensing a small molecular chemical drug and an auxiliary molecule; the stabilizer comprises lipid and cholesterol, the lipid is non-ionizable neutral lipid, and the phase change temperature range is-20 to 80 DEG C. The drug release is accurate, the targeting property is strong, the stability of the drug in vivo is high, systematic toxic and side effects are reduced, the in-vivo circulation time of the drug is prolonged, and the drug loading efficiency (gt; 70%) and treatment effect. The medicine is suitable for treating various cancers such as breast cancer, lung cancer and prostatic cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a tumor microenvironment responsive drug composition and a preparation method and use thereof. Background Art

[0002] With the development of modern medicine, tumor treatment has become a key area of ​​global research. However, due to the complexity of the tumor microenvironment, the effectiveness of traditional drugs in tumor treatment is often limited. The tumor microenvironment usually exhibits characteristics such as acidic pH, hypoxia, and immunosuppression, which have adverse effects on the release, delivery, and ultimate efficacy of drugs. For example, many chemotherapy drugs cannot be effectively concentrated in tumor tissue when distributed throughout the body, resulting in increased systemic toxic side effects and limited therapeutic effects. In addition, the acidic environment in the tumor microenvironment may cause drug instability or inactivation, further reducing the efficiency of treatment.

[0003] To meet these challenges, the application of nanotechnology in tumor treatment has received widespread attention in recent years. Nanomedicines, due to their unique size and surface properties, can accumulate more effectively in tumor tissues through enhanced permeation and retention effects (EPR effects). In addition, nanocarriers can achieve targeting through surface modification, reduce the distribution of drugs in normal tissues, and thus reduce toxic side effects. Nanotechnology not only improves the bioavailability of drugs, but also controls the release rate of drugs to achieve more precise therapeutic effects. However, nanomedicines still face some challenges in the complex tumor microenvironment, especially in how to achieve precise drug release and improve therapeutic effects. Summary of the invention

[0004] In response to these problems, the present invention proposes an amphiphilic, microenvironment-sensitive prodrug. By combining drug molecules with auxiliary molecules with opposite hydrophilicity and hydrophobicity, a prodrug structure that can respond to the characteristics of the tumor microenvironment can be designed. Under the acidic conditions of tumor tissue, the prodrug can quickly dissociate and release active drug molecules, thereby achieving precise drug release.

[0005] Combined with previous research, the inventors realized that in an acidic microenvironment, the imine nitrogen of C=N is easily attacked by protons, resulting in protonation. This step makes the nitrogen of C=N carry a positive charge, increasing the polarity, causing the carbon atom to become more electrophilic. As the electrophilicity of the carbon atom increases, the water molecule can attack the carbon atom as a nucleophilic reagent, and finally the oxygen atom of the water molecule combines with the carbon atom to form an intermediate. The transition state intermediate undergoes further rearrangement, resulting in the rupture of the C=N bond, forming an amine intermediate (R 2 C-NH 2+) and an aldehyde or ketone intermediate. The intermediate forms an aldehyde or ketone after the reaction and releases a hydrazine compound. Finally, by removing the proton, the hydrazone compound is hydrolyzed to form an aldehyde or ketone and a free hydrazine compound.

[0006] In the present invention, pH-sensitive amphiphilic prodrugs and stabilizers are selected to synthesize acidic microenvironment-sensitive nanomedicines. In a microenvironment with a pH of 6 to 7, the drug is released by more than 70%, preferably more than 80%, and more preferably more than 90% within 24 hours.

[0007] In the present invention, in order to synthesize pH-sensitive amphiphilic prodrugs, auxiliary molecules with opposite hydrophilicity and hydrophobicity are selected to provide self-assembly characteristics for small molecule drugs. Just like the composition principle of cell membranes, self-assembly is the basis for building complex structures in biological systems and is also a core concept for developing new materials in nanotechnology and materials science. In aqueous solution, amphiphilic molecules spontaneously tend to minimize the free energy of the system. To achieve this, the molecules will spontaneously arrange so that the hydrophobic part is away from water, while the hydrophilic part is in contact with water. Ultimately, the amphiphilic molecules are able to form a variety of ordered structures, such as micelles and double-layer membrane structures, through their self-assembly ability.

[0008] In the present invention, cholesterol and lipids are selected as stabilizers of the drug in order to improve the in vivo stability of the drug. Cholesterol and lipids increase particle stability mainly by adjusting the structure and physical properties of the membrane and enhancing the rigidity and integrity of the membrane. In addition, the addition of cholesterol and lipids can also reduce aggregation and chemical degradation between particles.

[0009] In the present invention, the rigidity of the nanoparticle film of the tumor microenvironment responsive drug composition is 1000kPa-10MPa. Preferably, the rigidity of the nanoparticle film of the tumor microenvironment responsive drug composition is 1000kPa-10MPa, and in a microenvironment with a pH of 6 to 7, the drug is released more than 90% within 24 hours; or, the rigidity of the nanoparticle film of the tumor microenvironment responsive drug composition is 1300kPa-10MPa, and in a microenvironment with a pH of 6 to 7, the drug is released more than 85% within 24 hours; or, the rigidity of the nanoparticle film of the tumor microenvironment responsive drug composition is 3000 kPa-10MPa, and in a microenvironment with a pH of 6 to 7, the drug is released more than 70% within 24 hours.

[0010] In a specific embodiment, the ratio of the tumor microenvironment responsive drug composition released within 24 hours under a microenvironment of pH 6.5: the ratio of the drug composition released within 24 hours under a microenvironment of pH 7.4 is >8, preferably >8.5. Further preferably, the rigidity of the nanoparticle membrane of the tumor microenvironment responsive drug composition is 1000 kPa-10 MPa, and the ratio of the drug composition released within 24 hours under a microenvironment of pH 6.5: the ratio of the drug composition released within 24 hours under a microenvironment of pH 7.4 is >8, preferably >8.5; or, the rigidity of the nanoparticle membrane of the tumor microenvironment responsive drug composition is 3000 kPa-10 MPa, and the ratio of the drug composition released within 24 hours under a microenvironment of pH 6.5: the ratio of the drug composition released within 24 hours under a microenvironment of pH 7.4 is >8.5.

[0011] In the present invention, the preferred molar ratio of the stabilizer is 7-9:1-3, and the preferred molar ratio of the lipid and cholesterol components is 7:2. The inventors found that at this ratio, the tumor microenvironment response drug will obtain the highest drug loading efficiency (>70%) and better in vivo circulation time (>48h).

[0012] Preferably, the lipid is a non-ionizable neutral lipid with a phase transition temperature below 80°C; preferably, the phase transition temperature is between -20 and 75°C; further preferably, the lipid is one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0013] In a specific embodiment, the lipid is a combination of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and one or two of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Preferably, in the lipid, the molar ratio of DSPC: (DSPE+DPPE) is (2-5): 1. In this embodiment, the stability of the tumor microenvironment responsive pharmaceutical composition can be further improved.

[0014] In the present invention, there is no specific limitation on the type of drug, and commonly used anticancer drugs containing amino groups can be selected as needed, such as drugs including but not limited to one or more of melphalan, methotrexate, imatinib, and doxorubicin.

[0015] In the present invention, there is no specific limitation on the type of auxiliary molecule, and the auxiliary molecule only needs to be a small molecule having a benzene ring structure and at least one aldehyde group. The auxiliary molecule is a small molecule with opposite hydrophilicity and hydrophobicity to the small molecule drug.

[0016] Preferably, the hydrophobic auxiliary molecule has a fluorine group, and is selected from one or more of 3,4,5-trifluorobenzaldehyde, 2,3,4,5,6-pentafluorobenzaldehyde, 3,5-difluorobenzaldehyde, and 2,3,5,6-tetrafluorobenzaldehyde.

[0017] Preferably, the hydrophilic auxiliary molecule has a hydroxyl or carboxyl group, and is selected from one or more of 3-hydroxybenzaldehyde, 2,3,4-trihydroxybenzaldehyde, 2,4,6-trihydroxybenzaldehyde, 2,4,5-trihydroxybenzaldehyde, 3-carboxybenzaldehyde, 2-carboxybenzaldehyde, 3-formylbenzoic acid, and p-formylbenzoic acid.

[0018] A second aspect of the present invention provides a method for preparing a tumor microenvironment responsive drug, the method comprising the following steps: 1) uniformly dispersing the small molecule drug and the auxiliary molecule in an anhydrous polar solvent to obtain a solution A; preferably, the mass ratio of the small molecule drug to the auxiliary molecule is (1-4): (4-1), more preferably (2-3): (2-3); preferably, in the solution A, the concentration of the small molecule drug is 5-20 mg / ml, more preferably 10-15 mg / ml; 2) adding a trace amount of catalyst to solution A, such as one or more of hydrochloric acid, organic acid, and zinc salt; preferably, the amount of the catalyst added is 0.5-2% of the volume of solution A, more preferably 1%; 3) mechanically stirring for 18-30 hours, preferably 24 hours, in the dark, and controlling the reaction temperature to 20-40° C., preferably 30° C., to obtain product B with impurities; 4) purifying the obtained product B by column chromatography to obtain a pH-sensitive amphiphilic prodrug C with a purity greater than 99.9%; 5) Dispersing the prodrug C and the stabilizer uniformly in a glycerol hydration solution with a volume ratio of 3-15%, preferably a glycerol hydration solution with a volume ratio of 10%, according to a molar ratio of (5-10):1, to obtain a suspension D; 6) subjecting the suspension D to ultrasonic cavitation at an ultrasonic power of 5-200 kW for a time of 1-50 min; 7) Centrifugal purification.

[0019] Furthermore, in step 1, the anhydrous polar solvent is preferably an organic solvent with higher polarity such as anhydrous ethanol and anhydrous methanol, which provides a reaction environment for small molecule drugs and auxiliary molecules.

[0020] Furthermore, in step 2, in order to reduce the introduction of inorganic impurities, an organic acid is preferably used as a catalyst for the reaction, such as one or more of anhydrous acetic acid, anhydrous formic acid and p-toluenesulfonic acid (PTSA).

[0021] Furthermore, in step 4, column chromatography is used to purify the product B. First, a solvent system is selected by thin layer chromatography to determine the polarity of the eluent; preferably, dichloromethane and methanol are selected as eluents in a ratio of (1-5): (1-5), preferably 3:1.

[0022] Further, in step 5, in order to improve the in vivo stability of the drug, preferably, the prodrug C and the stabilizer are uniformly dispersed in a 10% by volume glycerol hydrate according to a molar ratio of 8:1 to obtain a suspension D; in step 5, the concentration of the prodrug C in the suspension D is preferably 0.1-5 mg / ml, preferably 1-2 mg / ml.

[0023] Furthermore, in step 6, in order to improve the synthesis efficiency of the drug, preferably, the power of ultrasonic cavitation is 5-100 kW and the time is 1-15 min.

[0024] The present invention adopts a unique design of combining pH-sensitive amphiphilic prodrug and stabilizer, so that the drug shows significant advantages in the tumor microenvironment. The following are the beneficial effects of the present invention.

[0025] 1. Precise drug release: Since the C=N bond in the prodrug structure is sensitive to the acidic environment, when the drug enters the acidic microenvironment of the tumor, the C=N is rapidly hydrolyzed to release the active drug molecules, thereby achieving precise drug release. This mechanism greatly improves the concentration and therapeutic effect of the drug at the tumor site.

[0026] 2. Efficient tumor targeting: By combining the amphiphilic prodrug with the stabilizer, the present invention can form stable nanoparticles, which can effectively aggregate in tumor tissues through the EPR effect, reduce the distribution of the drug in normal tissues, and significantly reduce systemic toxic side effects. The tumor microenvironment responsive drug of the present invention can be used to prepare drugs for the treatment of breast cancer, lung cancer, prostate cancer, colon cancer, and liver cancer. When administering the drug, the administration cycle is one dose every 5 days to one dose every 60 days.

[0027] 3. Enhanced drug stability: The stabilizers in the present invention include cholesterol and lipids, which enhance the rigidity (1000kPa-10MPa) and integrity of the membrane by regulating the structure and physical properties of the nanoparticle membrane, thereby improving the stability and bioavailability of the drug in the body. In particular, in the tumor microenvironment, the drug's in vivo circulation time is extended, further improving the therapeutic effect.

[0028] 4. High drug loading efficiency: By rationally designing the ratio of stabilizer to prodrug, the present invention achieves a high drug loading efficiency (>70%), which means that more active drugs can be released at the same dosage, further enhancing the therapeutic effect.

[0029] 5. Reduce drug degradation and aggregation: Cholesterol and lipids in the stabilizer effectively reduce the aggregation between nanoparticles and the chemical degradation of the drug, which not only extends the shelf life of the drug but also ensures the effectiveness of the drug after administration.

[0030] In summary, the tumor microenvironment responsive drug composition and preparation method thereof provided by the present invention not only solve the problems of poor stability, low targeting and large toxic side effects of drugs in the prior art in the tumor microenvironment, but also significantly improve the effect of tumor treatment through precise release and efficient targeting, and have important clinical application value and broad market prospects. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below by way of examples. The examples described in the present invention are only a part of the examples of the present invention, rather than all of the examples. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the materials used in the following examples were all commercially available analytically pure.

[0033] Preparation of pH-sensitive amphiphilic prodrugs.

[0034] Preparation Example 1.

[0035] Dissolve 50 mg of methotrexate in 5 ml of anhydrous ethanol to obtain a methotrexate ethanol solution.

[0036] Another 30 mg of 3,4,5-trifluorobenzaldehyde was dissolved in 5 ml of anhydrous ethanol to obtain a 3,4,5-trifluorobenzaldehyde ethanol solution.

[0037] The methotrexate ethanol solution and the 3,4,5-trifluorobenzaldehyde ethanol solution were mixed, and 0.1 ml of anhydrous acetic acid was added as a catalyst.

[0038] Under light-proof conditions, the solution was mechanically stirred for 24 hours, and the reaction temperature was controlled at 30° C. to obtain a reaction product.

[0039] The product was purified by column chromatography using dichloromethane and methanol (3:1) as eluent.

[0040] Finally, a pH-sensitive amphiphilic prodrug with a purity greater than 99.9% was obtained and was recorded as C1.

[0041] Preparation Example 2.

[0042] 100 mg of imatinib was dissolved in 5 ml of anhydrous ethanol to obtain an imatinib ethanol solution.

[0043] Another 30 mg of 2,3,4-trihydroxybenzaldehyde was dissolved in 5 ml of anhydrous ethanol to obtain a 2,3,4-trihydroxybenzaldehyde ethanol solution.

[0044] The imatinib ethanol solution and the 2,3,4-trihydroxybenzaldehyde ethanol solution were mixed, and 0.1 ml of anhydrous acetic acid was added as a catalyst.

[0045] Under light-proof conditions, the solution was mechanically stirred for 24 hours, and the reaction temperature was controlled at 30° C. to obtain a reaction product.

[0046] The product was purified by column chromatography using dichloromethane and methanol (3:1) as eluent.

[0047] Finally, a pH-sensitive amphiphilic prodrug with a purity greater than 99.9% was obtained, which was recorded as C2.

[0048] Preparation Example 3.

[0049] This preparation example was carried out according to the method of Preparation Example 1, except that the auxiliary molecule was 2,3,5,6-tetrafluorobenzaldehyde and the catalyst was p-toluenesulfonic acid. The obtained pH-sensitive amphiphilic prodrug was denoted as C3.

[0050] Preparation Example 4.

[0051] This preparation example was carried out according to the method of Preparation Example 2, except that the auxiliary molecule was 3-formylbenzoic acid and the catalyst was hydrochloric acid. The obtained pH-sensitive amphiphilic prodrug was denoted as C4.

[0052] Preparation of tumor microenvironment responsive pharmaceutical compositions.

[0053] Example 1.

[0054] Prodrug C1 was mixed with a stabilizer (composed of DPPE lipid and cholesterol in a molar ratio of 7:2) at a molar ratio of 8:1.

[0055] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0056] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0057] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0058] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0059] Finally, the tumor microenvironment responsive drug composition G1 was obtained.

[0060] Example 2.

[0061] Prodrug C1 was mixed with a stabilizer (consisting of DSPE lipid and cholesterol in a molar ratio of 8:3) at a molar ratio of 8:1.

[0062] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0063] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0064] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0065] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0066] Finally, the tumor microenvironment responsive drug composition G2 was obtained.

[0067] Example 3.

[0068] Prodrug C2 was mixed with a stabilizer (composed of DPPE lipid and cholesterol in a molar ratio of 7:2) at a molar ratio of 8:1.

[0069] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0070] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0071] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0072] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0073] Finally, the tumor microenvironment responsive drug composition G3 was obtained.

[0074] Example 4.

[0075] Prodrug C3 was mixed with a stabilizer (composed of DSPC, DPPE lipid, and cholesterol in a molar ratio of 5:2:2) at a molar ratio of 10:1.

[0076] The mixture was evenly dispersed in 3% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 2 mg / ml.

[0077] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0078] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0079] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0080] Finally, the tumor microenvironment responsive drug composition G4 was obtained.

[0081] Example 5.

[0082] Prodrug C4 was mixed with a stabilizer (composed of DSPC, DSPE, DPPE lipid, and cholesterol in a molar ratio of 6:1:1:2) at a molar ratio of 5:1.

[0083] The mixture was evenly dispersed in 15% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0084] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0085] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0086] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0087] Finally, the tumor microenvironment responsive drug composition G5 was obtained.

[0088] Example 6.

[0089] Prodrug C1 was mixed with a stabilizer (composed of DSPC, DSPE, DPPE lipid, and cholesterol in a molar ratio of 5:1:1:2) at a molar ratio of 8:1.

[0090] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0091] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0092] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0093] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0094] Finally, the tumor microenvironment responsive drug composition G6 was obtained.

[0095] Example 7.

[0096] Prodrug C2 was mixed with a stabilizer (composed of DSPC, DPPE lipid, and cholesterol in a molar ratio of 7:2:2) at a molar ratio of 8:1.

[0097] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0098] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0099] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0100] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0101] Finally, the tumor microenvironment responsive drug composition G7 was obtained.

[0102] Comparative example 1.

[0103] The prodrug C1 was uniformly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, and the concentration of the prodrug in the suspension was 1 mg / ml.

[0104] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0105] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0106] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0107] Finally, the tumor microenvironment responsive drug composition H1 was obtained.

[0108] Comparative example 2.

[0109] Prodrug C1 was mixed with a stabilizer (composed of DPPE lipid and cholesterol in a molar ratio of 1:1) at a molar ratio of 8:1.

[0110] The mixture was evenly dispersed in 10% (volume ratio) glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0111] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0112] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0113] The composition was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0114] Finally, the tumor microenvironment responsive drug composition H2 was obtained.

[0115] Comparative example 3.

[0116] Prodrug C1 was mixed with a stabilizer (composed of DPPE lipid and cholesterol in a molar ratio of 7:2) at a molar ratio of 15:1.

[0117] The mixture was evenly dispersed in 10% glycerol hydrate to obtain a suspension, in which the concentration of the prodrug was 1 mg / ml.

[0118] The suspension was subjected to ultrasonic cavitation treatment, with the ultrasonic power set to 50 kW and the treatment time being 10 minutes.

[0119] After cavitation treatment, a micron-scale or nano-scale pharmaceutical composition is obtained.

[0120] The combination was placed in a centrifuge and centrifuged at 10,000 rpm for 30 minutes to remove unreacted prodrug and other impurities.

[0121] Finally, the tumor microenvironment responsive drug composition H3 was obtained.

[0122] Testing of drug release behavior.

[0123] The pharmaceutical compositions prepared in the examples and comparative examples were placed in buffer solutions with a pH of 7.4 (simulating a normal physiological environment) and a pH of 6.5 (simulating a tumor microenvironment), respectively.

[0124] At 37°C, samples were taken regularly to detect drug release.

[0125] The test results are recorded in Table 1.

[0126] Table 1

[0127] As can be seen from Table 1, the drug release rate of the pharmaceutical compositions G1-G7 of the present invention is relatively slow in a buffer solution of pH 7.4, with only about 10% released within 24 hours. In a buffer solution of pH 6.5, the drug release rate increases significantly, with more than 30% released within 6 hours and more than 70% released within 24 hours.

[0128] In comparison, comparative drug H1 does not contain the stabilizer of the present invention, so its rigidity is lower; comparative drug H2 does not use the specific ratio of lipid and cholesterol of the present invention, and although its rigidity is higher, its release ratio within 24 hours is lower in a buffer solution with a pH of 6.5; comparative drug H3 does not use the ratio of the stabilizer and prodrug of the present invention, so its rigidity is lower.

[0129] The experimental results of the present invention verify that the pharmaceutical composition of the present invention can rapidly release drugs in an acidic tumor microenvironment while maintaining high rigidity, thereby improving the therapeutic effect.

Claims

1. A tumor microenvironment responsive pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a pH-sensitive amphiphilic prodrug and a stabilizer; wherein the pH-sensitive amphiphilic prodrug is formed by condensation of a small molecule chemical drug and an auxiliary molecule; the stabilizer comprises lipid and cholesterol, wherein the lipid is a non-ionizable neutral lipid with a phase transition temperature range of -20-80°C.

2. The tumor microenvironment responsive pharmaceutical composition according to claim 1, characterized in that: The pH-sensitive amphiphilic prodrug has a hydrophilic end and a hydrophobic end, and the hydrophilic end and the hydrophobic end are provided by small molecule drugs and auxiliary molecules, respectively.

3. The tumor microenvironment responsive pharmaceutical composition according to claim 1, characterized in that: The stabilizer is composed of lipid and cholesterol in a molar ratio of (7-9):(1-3); preferably, the molar ratio of lipid and cholesterol is 7:

2.

4. The tumor microenvironment responsive pharmaceutical composition according to claim 2, characterized in that: The small molecule chemical drug has one or more amino groups in its structure; preferably, the small molecule chemical drug is one or more of melphalan, methotrexate, imatinib, and doxorubicin.

5. The tumor microenvironment responsive pharmaceutical composition according to claim 2, characterized in that: The auxiliary molecule has a benzene ring structure and at least one aldehyde group, and its hydrophilicity is opposite to that of the small molecule drug, and its molecular weight is less than 500; Preferably, the hydrophobic auxiliary molecule has a fluorine group, and is selected from one or more of 3,4,5-trifluorobenzaldehyde, 2,3,4,5,6-pentafluorobenzaldehyde, 3,5-difluorobenzaldehyde, and 2,3,5,6-tetrafluorobenzaldehyde; Preferably, the hydrophilic auxiliary molecule has a hydroxyl or carboxyl group, and is selected from one or more of 3-hydroxybenzaldehyde, 2,3,4-trihydroxybenzaldehyde, 2,4,6-trihydroxybenzaldehyde, 2,4,5-trihydroxybenzaldehyde, 3-carboxybenzaldehyde, 2-carboxybenzaldehyde, 3-formylbenzoic acid, and p-formylbenzoic acid.

6. A method for preparing a tumor microenvironment responsive drug, characterized in that: The tumor microenvironment responsive drug is prepared by dehydration condensation of a small molecule chemical drug and an auxiliary molecule through a Schiff base reaction; the tumor microenvironment responsive drug contains the tumor microenvironment responsive drug composition according to any one of claims 1-5.

7. The method for preparing a tumor microenvironment responsive drug according to claim 6, characterized in that: The method comprises the following steps: 1) The small molecule drug and the auxiliary molecule are uniformly dispersed in an anhydrous polar solvent to obtain solution A; 2) Adding a trace amount of catalyst to solution A, such as one or more of hydrochloric acid, organic acid, and zinc salt; 3) Mechanically stir for 18-30 hours in the dark, and control the reaction temperature to 20-40°C to obtain product B with impurities; 4) Purifying the obtained product B to obtain a pH-sensitive amphiphilic prodrug C with a purity greater than 99.9%; 5) Dispersing the prodrug C and the stabilizer uniformly in a glycerol hydrate solution having a volume ratio of 3-15% at a molar ratio of (5-10):1 to obtain a suspension D; 6) subjecting the suspension D to ultrasonic cavitation, with an ultrasonic power of 5-200 kW and a time of 1-50 min; 7) Centrifugal purification.

8. The preparation method according to claim 7, characterized in that: Preferably, the molar ratio of prodrug C to stabilizer is (8-10):1; the power of ultrasonic cavitation is 5-100 kW, and the time is 1-15 min.

9. Use of the tumor microenvironment responsive drug composition according to any one of claims 1 to 5, or the tumor microenvironment responsive drug prepared according to the preparation method according to any one of claims 6 to 8 in the preparation of drugs for treating breast cancer, lung cancer, prostate cancer, colon cancer, and liver cancer.

10. The use according to claim 9, characterized in that The dosing cycle of the tumor microenvironment responsive drug is from once every 5 days to once every 60 days.

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