Camptothecin prodrug as well as drug-loading liposome and application thereof

Abstract

The invention provides a camptothecin prodrug. The camptothecin prodrug provided by the invention has higher drug retention in the liposome and better stability, and the prodrug liposome can maintain long-time plasma concentration in plasma. In-vitro characterization of the camptothecin prodrug liposome shows that the camptothecin prodrug liposome has higher encapsulation efficiency, drug loading capacity and stability, and can achieve a slow release effect in plasma; the experimental result on the cellular level shows that the compound has higher cellular uptake rate; in-vivo experiments prove that the compound has a better tumor accumulation effect and antitumor activity, can tolerate a higher dosage, and has higher biological safety.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, relates to drug-loaded liposomes, and specifically relates to a camptothecin prodrug and an active drug-loaded liposome and application thereof. Background Art

[0002] Camptothecin (CPT) is a cytotoxic quinoline alkaloid that inhibits DNA replication by inhibiting DNA topoisomerase I, thereby inducing apoptosis of tumor cells. At present, camptothecin anti-tumor drugs include irinotecan, topotecan, hydroxycamptothecin, rubitecan and 9-aminocamptothecin. These camptothecin derivatives are mainly used in the form of injections. Although they have strong anti-cancer activity, they also have the problem of poor cell selectivity. CPT is prone to side effects such as bone marrow suppression, gastrointestinal toxicity and hemorrhagic cystitis, which greatly limits the clinical application of CPT drugs.

[0003] Liposomes are vesicle particles formed by phospholipids and cholesterol in water. Liposome preparations of many chemotherapy drugs have been successfully used in the treatment of tumors. Liposomes can change the distribution of chemotherapy drugs in normal tissues and tumor tissues, improve the pharmacokinetic behavior of drugs, and prolong the blood circulation time of drugs. Liposome preparations in clinical practice include amphotericin B liposomes, doxorubicin liposomes, and cytarabine liposomes, etc. Therefore, liposomes are considered to be the most successful nanocarriers for clinical treatment of tumors.

[0004] Liposomes are a feasible strategy to improve the therapeutic index of CPT. Stable encapsulation of CPT in liposomes is the basis for preparing CPT liposome drugs. However, as a hydrophobic molecule, CPT is mainly encapsulated in phospholipid membranes, which has the problems of fast drug release and low encapsulation rate. Most cationic drugs can be encapsulated by ammonium sulfate gradient active loading method. This method uses the ammonium sulfate gradient inside and outside the liposome to drive the cationic drug into the liposome and form a complex precipitation with sulfate ions, so that it is retained in the liposome, and then the encapsulation of the cationic drug is completed. However, CPT does not have an ionizable group, so it cannot be directly encapsulated by the ammonium sulfate gradient method. In order to solve the problem of efficient encapsulation of CPT liposomes by using the ammonium sulfate gradient loading method, the positively charged groups on CPT can be modified to enable it to be encapsulated in the liposome. However, although this CPT cationic prodrug can be encapsulated in the liposome, since the CPT cationic prodrug cannot form a stable complex precipitation with counterions such as ammonium sulfate, it leads to rapid release of the drug in the liposome, and the advantage of liposomes in improving the anti-tumor therapeutic index of CPT cannot be brought into play.

[0005] In summary, conventional CPT cationic prodrugs cannot be stably encapsulated in liposomes and have the disadvantage of fast drug release. Therefore, developing a CPT prodrug that can be stably encapsulated in liposomes to overcome the problem of easy leakage of conventional cationic drugs will hopefully promote the development and application of CPT liposome drugs. Summary of the invention

[0006] In order to solve the problems in the prior art, the present invention provides a camptothecin prodrug.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] According to the first aspect of the present invention, the camptothecin prodrug of the present invention has a structure shown in the following formula I or formula II:

[0009]

[0010] in

[0011] n is an integer of 1-9; R1 is a C1-C6 alkyl group; R2 is a C1-C6 alkyl group; R3 is H or a hydroxyl group; R4 is H or a C1-C6 alkyl group.

[0012] The C1-C6 alkyl group of the present invention is a C1-C6 straight-chain alkyl group or a branched-chain alkyl group. The straight-chain alkyl group is selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl; the branched-chain alkyl group is selected from isopropyl, sec-butyl, isobutyl, tert-butyl or neopentyl.

[0013] Preferably, the camptothecin prodrug of the present invention has the following structural formula:

[0014]

[0015] More preferably, the camptothecin prodrug of the present invention has the following structural formula:

[0016]

[0017] According to the second aspect of the present invention, the present invention provides a pharmaceutical composition of the camptothecin prodrug. The pharmaceutical composition of the camptothecin prodrug comprises the camptothecin prodrug and a pharmaceutically acceptable excipient. Further, the pharmaceutical composition is a drug-loaded liposome of the camptothecin prodrug.

[0018] The drug-loaded liposomes of the camptothecin prodrugs of the present invention include the camptothecin prodrugs, phospholipids, cholesterol, PEGylated phospholipids, and ammonium sulfate. The phospholipids are natural, semi-synthetic, and fully synthetic phospholipids such as egg yolk phosphatidylcholine (EPC), soybean phospholipids, sphingomyelin, distearoyl phosphatidylcholine (DSPC), hydrogenated soybean phospholipids (HSPC), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl phosphatidylcholine (DMPC), preferably hydrogenated soybean phospholipids (HSPC). The PEGylated phospholipids are couplings of PEG and DSPE connected by amide bonds, such as DSPE-PEG 2000 .

[0019] According to the third aspect of the present invention, the present invention provides the use of the above-mentioned drug-loaded liposomes of camptothecin prodrugs in the preparation of drugs for preventing or treating tumors.

[0020] Beneficial effects:

[0021] The present invention provides a camptothecin prodrug. The camptothecin prodrug of the present invention has higher drug retention and better stability in liposomes, and the prodrug liposomes of the present invention can maintain a long-term blood drug concentration in plasma. In vitro characterization of the camptothecin prodrug liposomes of the present invention shows that it has a higher encapsulation rate, drug loading and stability, and can achieve a sustained release effect in plasma; experimental results at the cellular level show that it has a higher cell uptake rate; in vivo experiments have shown that it has better tumor accumulation effect and anti-tumor activity, can tolerate higher doses, and has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is compound 2 1 H-NMR spectrum;

[0023] Figure 2 is compound 4 1 H-NMR spectrum;

[0024] Figure 3 is compound 5 1 H-NMR spectrum;

[0025] Figure 4 Compound 7 (Azo-CPT (1)) 1 H-NMR spectrum;

[0026] Figure 5 It is the change of encapsulation efficiency of Azo-CPT(1)lip at different drug loadings;

[0027] Figure 6 is the particle size and PDI graph of Azo-CPT(1)lip before and after drug loading;

[0028] Figure 7 is the transmission electron microscopy image of Azo-CPT(1)lip;

[0029] Figure 8 is the drug release profile of Azo-CPT(1)lip and Azo-CPT(1) in plasma;

[0030] Fig. 9 is a graph showing the stability changes of Azo-CPT(1)lip and Azo-CPT(1) prodrug in PBS;

[0031] Fig.10 The uptake of CPT, Azo-CPT(1)lip and Azo-CPT(1) in 4T1 cells: uptake at 2 h (A), uptake at different time points within 8 h (B);

[0032] Fig.11 It is the pharmacokinetic study of CPT and Azo-CPT(1)lip: blood drug concentration-time curve;

[0033] Fig.12 Azo-CPT(1)lip biosafety evaluation: mouse body weight changes (A), HE sections of various tissues (B);

[0034] Fig.13 This is a biodistribution study of Azo-CPT(1)lip: in vivo imaging of mice;

[0035] Fig.14 This is a study diagram of the tissue distribution of CPT and Azo-CPT(1)lip;

[0036] Fig.15 This is an in vivo efficacy study of Azo-CPT(1)lip: changes in tumor volume (A), changes in mouse body weight (B), in vitro tumor size (C), and HE sections of various tissues (D). DETAILED DESCRIPTION

[0037] In the following examples, the present invention is only exemplarily described, but those skilled in the art can make various modifications to the present invention without departing from the spirit and scope of the present invention after reading this patent application. Unless otherwise specified, the parts described in the present invention are all parts by weight, and the percentages are all weight percentages.

[0038] Related terms:

[0039] Encapsulation efficiency (EE) refers to the ratio of the drug encapsulated in the liposome to the total amount of drug. Calculation formula: EE = W (amount of drug in the liposome) / W (total amount of drug) × 100%

[0040] Drug loading capacity (LC) refers to the ratio of drug contained in liposomes to the total weight of liposomes. Calculation formula: LC = W (drug amount in liposomes) / W (total liposome weight) × 100%

[0041] Example 1 Synthesis of Azo-CPT (1) (Compound 7):

[0042]

[0043] (1) Synthesis steps of compound 2:

[0044] Accurately weigh 1g of methyl 4-aminobenzoate, i.e., compound 1 (1eq, 6.6mmol) in a 100mL clean reaction bottle, evacuate for 5min, add 20mL of anhydrous DCM to dissolve it, and fill with nitrogen. Accurately weigh 8g of Oxone (2eq, 13.2mmol) in a 50mL Eppendorf tube, add 40mL of ultrapure water to completely dissolve it into a colorless and transparent state, stir, and add the Oxone solution into the reaction bottle with a 20mL syringe, and stir and react at room temperature for 5h. After TLC confirmed that the reaction was completed, the reaction solution was poured into a separatory funnel for separation, the organic phase was collected, and the aqueous phase was extracted with DCM, which was repeated 3 times. The organic phases were combined and washed with appropriate amounts of saturated citric acid, saturated sodium bicarbonate, pure water, and saturated sodium chloride, respectively. The washed organic phase was dehydrated with an appropriate amount of anhydrous sodium sulfate, filtered, and the filtrate was dried by a rotary evaporator, and an appropriate amount of DCM was added for redissolution. The mixture was recrystallized in a -20°C refrigerator for 2 h to precipitate a light yellow solid. The filter cake was filtered to obtain compound 2 (M+H=166.0459), and the product was identified by nuclear magnetic resonance spectroscopy ( Figure 1 ) to confirm that the synthesis is successful.

[0045] (2) Synthesis steps of compound 4:

[0046] Accurately weigh 500 mg of compound 2 (1 eq, 3.03 mmol) in a 100 mL clean reaction bottle, add an appropriate amount of glacial acetic acid (HAc) and ultrasonically dissolve, add 455 μL of 4-((dimethylamino)methyl)aniline, i.e., compound 3 (1 eq, 3.03 mmol) under stirring, and react overnight at room temperature. After the reaction is completed, pour the reaction solution into a separatory funnel, add an appropriate amount of pure water, and then add an appropriate amount of DCM for extraction, repeat 3 to 4 times, combine the organic phases, wash the organic phases with pure water, saturated sodium bicarbonate, and pure water in turn, collect the organic phases, remove water with an appropriate amount of anhydrous sodium sulfate, filter, spin dry, re-dissolve with a small amount of DCM, and apply on a large silica gel plate for separation and purification (developing solvent is DCM: MeOH = 20: 1), to obtain compound 4 (M+H = 298.1511), and identify the product by nuclear magnetic resonance spectroscopy ( Figure 2) to confirm that the synthesis is successful.

[0047] (3) Synthesis steps of compound 5:

[0048] Accurately weigh 500 mg of compound 4 (1 eq, 1.68 mmol) in a 100 mL clean reaction bottle, evacuate for 5 min, add 20 mL of anhydrous DCM to dissolve, fill with nitrogen, add 4 mL of lithium aluminum hydride (LAH) (6 eq, 10.08 mmol) and 20 mL of tetrahydrofuran (THF) under stirring, and react at room temperature for 5 h. After the reaction is completed, the reaction solution is poured into a 500 mL beaker, and wet DCM is added dropwise. Bubbles are generated during the process. After the bubbles are slowed down, ultrapure water is added dropwise until no more bubbles are generated and Al(OH)3 precipitates are generated. Hydrochloric acid is added until the precipitate is completely dissolved. Saturated sodium bicarbonate is added to separate the solution until no more bubbles are generated. The organic phase is collected, and the aqueous phase is repeatedly extracted with DCM until the color of the aqueous phase becomes lighter. An appropriate amount of anhydrous sodium sulfate is added to the collected organic phase to remove water, filter, spin dry, add a small amount of DCM to re-dissolve, and apply it on a large silica gel plate for separation and purification (developing agent is DCM: MeOH = 10: 1) to obtain compound 5 (M+H = 270.1562), which is identified by nuclear magnetic resonance spectroscopy ( Figure 3 ) to confirm that the synthesis is successful.

[0049] (4) Synthesis steps of compound 7:

[0050] Accurately weigh 200 mg CPT (compound 6) (1.1 eq, 0.58 mmol) in a 100 mL clean reaction bottle, evacuate for 5 min, add 40 mL anhydrous DCM to dissolve it, and fill with nitrogen. Weigh 114 mg BTC (0.726 eq, 0.38 mmol) and 320 mg DMAP (4.95 eq, 2.62 mmol) in a 2 mL Eppendorf tube, add an appropriate amount of anhydrous DCM to dissolve, and add them to the reaction bottle with a 1 mL syringe. After reacting for 5 min, weigh 142 mg compound 5 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to orange-yellow and clear, and reacts at room temperature for 2 h. After the reaction was completed as determined by TLC, the reaction solution was poured into a separatory funnel, and a small amount of saturated citric acid and saturated sodium chloride were added for washing. The organic phase was collected, and an appropriate amount of anhydrous sodium sulfate was added for dehydration. The product was filtered and dried by spin drying. A small amount of DCM was added for re-dissolution, and the product was coated on a large silica gel plate for separation and purification (the developing solvent was first DCM:MeOH=20:1, and then DCM:MeOH=10:1, and the reaction was run twice in total) to obtain compound 7 (Azo-CPT(1), M+H=644.2464). The product was identified by nuclear magnetic resonance spectroscopy ( Figure 4 ) to confirm that the synthesis was successful.

[0051] Example 2 Synthesis of Azo-CPT (2) (Compound 14):

[0052]

[0053] (1) Synthesis steps of compound 9:

[0054] The synthesis method of intermediate 9 is as described in Example 1 (synthesis steps of compound 2).

[0055] (2) Synthesis steps of compound 11:

[0056] Accurately weigh 500 mg of compound 9 (1 eq, 3.03 mmol) in a 100 mL clean reaction bottle, add an appropriate amount of glacial acetic acid (HAc) and ultrasonically dissolve, add 540 μL of 4-(diethylaminomethyl)aniline compound 10 (1 eq, 3.03 mmol) under stirring, and react overnight at room temperature. After the reaction is completed, pour the reaction solution into a separatory funnel, add an appropriate amount of pure water, and then add an appropriate amount of DCM for extraction, repeat 3 to 4 times, combine the organic phases, wash the organic phases with pure water, saturated sodium bicarbonate, and pure water in turn, collect the organic phases, remove water with an appropriate amount of anhydrous sodium sulfate, filter, spin dry, re-dissolve with a small amount of DCM, and apply on a large silica gel plate for separation and purification (developing solvent is DCM: MeOH = 20: 1) to obtain compound 11 (M+H = 326.1824).

[0057] (3) Synthesis steps of compound 12:

[0058] 500 mg of compound 11 (1 eq, 1.54 mmol) was accurately weighed into a 100 mL clean reaction bottle, vacuumed for 5 min, 20 mL of anhydrous DCM was added to dissolve, and nitrogen was filled. Under stirring, 3.7 mL of lithium aluminum hydride (LAH) (6 eq, 9.24 mmol) was added, and then 20 mL of tetrahydrofuran (THF) was added, and the reaction was carried out at room temperature for 5 h. After the reaction is completed, the reaction solution is poured into a 500 mL beaker, and moistened DCM is added dropwise. Bubbles are generated during the process. After the bubbles are subsided, ultrapure water is added dropwise until no bubbles are generated and Al(OH)3 precipitates are generated. Hydrochloric acid is added until the precipitate is completely dissolved. Saturated sodium bicarbonate is added to separate the solution until no bubbles are generated. The organic phase is collected, and the aqueous phase is repeatedly extracted with DCM until the color of the aqueous phase becomes lighter. An appropriate amount of anhydrous sodium sulfate is added to the collected organic phase to remove water, filter, spin dry, add a small amount of DCM to re-dissolve, and coat on a large silica gel plate for separation and purification (developing solvent is DCM:MeOH=10:1) to obtain compound 12 (M+H=298.1875).

[0059] (4) Synthesis steps of compound 14:

[0060] Accurately weigh 200 mg CPT (compound 13) (1.1 eq, 0.58 mmol) in a 100 mL clean reaction bottle, evacuate for 5 min, add 40 mL anhydrous DCM to dissolve it, and fill with nitrogen. Weigh 114 mg BTC (0.726 eq, 0.38 mmol) and 320 mg DMAP (4.95 eq, 2.62 mmol) in a 2 mL Eppendorf tube, add an appropriate amount of anhydrous DCM to dissolve, and add them to the reaction bottle with a 1 mL syringe. After reacting for 5 min, weigh 155 mg compound 12 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to orange-yellow and clear, and reacts at room temperature for 2 h. After the reaction was completed as determined by TLC, the reaction solution was poured into a separatory funnel, and washed with a small amount of saturated citric acid and saturated sodium chloride. The organic phase was collected, and an appropriate amount of anhydrous sodium sulfate was added for dehydration. The phase was filtered and dried by spin drying. A small amount of DCM was added for re-dissolution, and the phase was coated on a large silica gel plate for separation and purification (first using DCM:MeOH=20:1 as the developing solvent, and then using DCM:MeOH=10:1, for a total of two runs) to obtain compound 14 (Azo-CPT(2), M+H=672.2777).

[0061] Example 3 Synthesis of Azo-CPT (3) (Compound 21):

[0062]

[0063] (1) Synthesis steps of compound 16:

[0064] The synthesis method of intermediate 16 is as described in Example 1 (synthesis steps of compound 2).

[0065] (2) Synthesis steps of compound 18:

[0066] Accurately weigh 500 mg of compound 16 (1 eq, 3.03 mmol) in a 100 mL clean reaction bottle, add an appropriate amount of glacial acetic acid (HAc) and ultrasonically dissolve, add 622 mg of 4-(4-methylpiperazine)aniline (compound 17) (1 eq, 3.03 mmol) under stirring, and react overnight at room temperature. After the reaction is completed, pour the reaction solution into a separatory funnel, add an appropriate amount of pure water, and then add an appropriate amount of DCM for extraction, repeat 3 to 4 times, combine the organic phases, wash the organic phases with pure water, saturated sodium bicarbonate, and pure water in turn, collect the organic phases, remove water with an appropriate amount of anhydrous sodium sulfate, filter, spin dry, and separate and purify through a silica gel column (developing solvent is 100 mL of pure DCM, DCM: MeOH = 5: 1) to obtain compound 18 (M + H = 353.1933).

[0067] (3) Synthesis steps of compound 19:

[0068] 500 mg of compound 18 (1 eq, 1.42 mmol) was accurately weighed into a 100 mL clean reaction bottle, vacuumed for 5 min, 20 mL of anhydrous DCM was added to dissolve, and nitrogen was filled. Under stirring, 3.5 mL of lithium aluminum hydride (LAH) (6 eq, 8.52 mmol) was added, and then 20 mL of tetrahydrofuran (THF) was added, and the reaction was carried out at room temperature for 5 h. After the reaction is completed, the reaction solution is poured into a 500 mL beaker, and moistened DCM is added dropwise. Bubbles are generated during the process. After the bubbles are subsided, ultrapure water is added dropwise until no bubbles are generated and Al(OH)3 precipitates are generated. Hydrochloric acid is added until the precipitate is completely dissolved, saturated sodium bicarbonate is added to separate the solution until no bubbles are generated, and the organic phase is collected. The aqueous phase is repeatedly extracted with DCM until the color of the aqueous phase becomes lighter. An appropriate amount of anhydrous sodium sulfate is added to the collected organic phase to remove water, filter, spin dry, add a small amount of DCM to re-dissolve, and coat on a large silica gel plate for separation and purification (developing solvent is DCM:MeOH=10:1) to obtain compound 19 (M+H=325.1984).

[0069] (4) Synthesis steps of compound 21:

[0070] Accurately weigh 200 mg CPT (compound 20) (1.1 eq, 0.58 mmol) in a 100 mL clean reaction bottle, evacuate for 5 min, add 40 mL anhydrous DCM to dissolve it, and fill with nitrogen. Weigh 114 mg BTC (0.726 eq, 0.38 mmol) and 320 mg DMAP (4.95 eq, 2.62 mmol) in a 2 mL Eppendorf tube, add an appropriate amount of anhydrous DCM to dissolve, and add them to the reaction bottle with a 1 mL syringe. After reacting for 5 min, weigh 170 mg compound 19 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to orange-yellow and clear, and reacts at room temperature for 2 h. After the reaction was completed as determined by TLC, the reaction solution was poured into a separatory funnel, and washed with a small amount of saturated citric acid and saturated sodium chloride. The organic phase was collected, and an appropriate amount of anhydrous sodium sulfate was added for dehydration. The phase was filtered and dried by spin drying. A small amount of DCM was added for re-dissolution, and the phase was coated on a large silica gel plate for separation and purification (first using DCM:MeOH=20:1 as the developing solvent, and then using DCM:MeOH=10:1, for a total of two runs) to obtain compound 21 (Azo-CPT(3), M+H=699.2886).

[0071] Example 4 Preparation of Azo-CPT (1) drug-loaded liposomes:

[0072] Preparation of blank ammonium sulfate liposomes:

[0073] Weigh 84mg HSPC, 40mg Chol, 27mg DSPE-PEG 2000, dissolved in 1 mL of anhydrous ethanol. Under vigorous stirring at 60°C, the above lipid ethanol solution was added to 5 mL of 300 mM ammonium sulfate solution to obtain a crude liposome solution. The liposome solution was removed of ethanol by a rotary evaporator at 45°C, and then extruded through an 80 nm polycarbonate nucleopore filter at room temperature. The obtained liposomes were transferred to a dialysis bag with a molecular weight cutoff of 3000 and dialyzed in a 150 mM NaCl solution at 37°C to remove the unencapsulated ammonium sulfate outside the liposomes and form a gradient difference in ammonium sulfate concentration inside and outside the liposomes. The dialysate was replaced every 3 hours, and replaced 4 times in total to obtain blank liposomes encapsulating ammonium sulfate.

[0074] Preparation of Azo-CPT(1) drug-loaded liposomes:

[0075] 2 mg of Azo-CPT (1) was weighed and dissolved in 100 μL of DMSO. Under vigorous stirring, it was first added to 1 mL of the blank liposomes at room temperature, and then the mixed liposome solution was stirred and incubated in a water bath at 60°C for 3 min. After the incubation, it can be observed that the liposome solution changed from turbid to clear, indicating that the drug loading was successful.

[0076] Example 5 Determination of encapsulation efficiency and drug loading of Azo-CPT (1) drug-loaded liposomes:

[0077] The encapsulation efficiency of Azo-CPT(1) drug-loaded liposomes was determined by fluorescence method. First, 5 μL of the prepared drug-loaded liposomes were taken, and the unloaded drug was removed without passing through a G50 chromatography column. After mixing with 1 mL of Na2S2O4 solution, the fluorescence intensity A1 was measured. Secondly, 100 μL of Triton was added to the mixed solution, and the fluorescence intensity A2 was measured after mixing. The encapsulation efficiency and drug loading of the drug-loaded liposomes were calculated by the following formula:

[0078]

[0079] Wherein, W1 is the amount of drug in the liposome, and W2 is the total amount of liposome. The measured encapsulation efficiency of drug-loaded liposomes is 95%, and the drug loading is 20% ( Figure 5 ).

[0080] Example 5 Determination of particle size, potential and appearance of Azo-CPT(1)lip drug-loaded liposomes:

[0081] The particle size and potential of Azo-CPT(1) drug-loaded liposomes were measured by Malvern laser particle size analyzer. The particle size and PDI before drug loading were 114.1nm and 0.085, respectively; after drug loading, the particle size and PDI were 131.4nm and 0.122( Figure 6The potential before drug loading was -17.1 mV; after drug loading, it was -15.7 mV. The appearance of Azo-CPT(1) drug-loaded liposomes was observed by transmission electron microscopy (TEM). 10 μL Azo-CPT(1) lip was dropped onto a 200-mesh carbon-coated copper grid and kept for 30 seconds. It was then stained with 0.2% phosphotungstic acid for 30 seconds and observed ( Figure 7 ), the results showed that obvious liposome vesicle structure could be observed.

[0082] Example 6 Drug release of Azo-CPT (1) drug-loaded liposomes:

[0083] Azo-CPT(1)lip (2 mg / mL) and Azo-CPT(1) were diluted to 10 μg / mL with rat plasma, and the mixture was incubated at 38°C. 10 μL of plasma samples were taken at the corresponding time points, 100 μL of 1% acetic acid methanol was added, vortexed for 1 min, ultrasonically extracted for 5 min, and centrifuged at 10000 r for 5 min. The supernatant was then analyzed by HPLC. Drug release was calculated by the following formula: A CPT / (A CPT +A Azo-CPT(1) )×100%, draw the drug release curve ( Figure 8 ). As shown in the figure, the drug release rate of Azo-CPT(1) reached more than 90% within 48 hours, while after being prepared into liposomes, the drug release rate was significantly slowed down, and only about 30% of the drug was released within 48 hours. This shows that Azo-CPT(1)lip drugs are not easy to leak, can maintain effective blood drug concentrations in the body for a long time, and are not easily eliminated by the body, which prolongs the drug action time.

[0084] Example 7 Study on the stability of Azo-CPT (1) drug-loaded liposomes:

[0085] Azo-CPT(1)lip (1 mg / mL) and Azo-CPT(1) were diluted to 10 μg / mL with PBS at pH 7.4, and the mixture was incubated at 38°C. 100 μL of the mixture was sampled at the corresponding time points, and 100 μL of 1% acetic acid methanol was added for HPLC analysis. The results are shown in Fig. 9 As shown in the figure, Azo-CPT(1) was degraded at 48h, and the drug was degraded by about 35% at 72h. However, Azo-CPT(1)lip existed as the prodrug Azo-CPT(1) within 72h, and no CPT peak was detected, indicating that compared with Azo-CPT(1), Azo-CPT(1)lip remained stable within 72h, and the prodrug in the liposome was not degraded. The above results show that the stability of Azo-CPT(1) was greatly increased after it was prepared into liposomes.

[0086] Example 8 Study on cellular uptake of Azo-CPT (1) drug-loaded liposomes:

[0087] 4T1 cells were plated at 3 × 10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate and incubated in a CO2 incubator at 37°C for 24 h. 1 μg / mL of free CPT, Azo-CPT(1) and Azo-CPT(1)lip were added to the cells and incubated at 37°C. After 2 h of incubation, the drug concentration in each well was determined by HPLC, and the drug concentrations in the wells of different groups were compared. In addition, the drug concentrations in the cells at different time points were determined by HPLC, and the drug concentration change curve was drawn. The results are shown in Figure 2. Fig.10 The results show that the uptake of Azo-CPT(1) by tumor cells is higher than that of CPT, whether incubated for 2 h (A) or at different time points (B).

[0088] Example 9 Pharmacokinetic study of Azo-CPT (1) drug-loaded liposomes:

[0089] 10 mg / kg of Azo-CPT(1)lip and CPT were injected into SD male rats through the tail vein. Blood samples were collected at given time intervals, 400 μL of 1% acetic acid and methanol were added, vortexed for 1 min, ultrasonically extracted for 5 min, and centrifuged at 10000 r for 5 min. The supernatant was analyzed by HPLC and the drug-time curve was drawn ( Fig.11 ). The results showed that the area under the drug-dose curve (AUC) of Azo-CPT(1)lip was much greater than that of CPT, indicating that Azo-CPT(1) was more completely absorbed by the body and had a better effect; and the blood concentration of Azo-CPT(1)lip decreased more slowly, indicating that it was cleared from the body more slowly and had a longer duration of action. In summary, in terms of pharmacokinetics, the therapeutic effect of Azo-CPT(1)lip was much greater than that of CPT.

[0090] Example 10 Biosafety Evaluation of Azo-CPT (1) Drug-Loaded Liposomes:

[0091] 4T1 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS at a concentration of 1×10 7 100 μL of cell suspension was inoculated subcutaneously on the right back of female BABL / c mice to establish a mouse breast cancer tumor model. 3At the time of the experiment, the tumor-bearing mice were divided into 4 groups according to the size of the tumor, with 3 mice in each group, and different doses of Azo-CPT(1)lip (10 mg / kg, 20 mg / kg, 40 mg / kg, 80 mg / kg) were injected through the tail vein. The drug was administered once every 3 days for a total of 4 times. The weight changes of the mice were measured every day, and the weight change curve was drawn to determine the toxicity of different doses of drug-loaded liposomes. On the fourth day after the last administration, all mice were killed, and the heart, liver, spleen, lung, kidney, and sternum were dissected for HE staining to analyze the pathological changes of each tissue. The mouse weight results are shown in the figure. Fig.12 The results showed that the weight of mice in the 80 mg / kg group dropped sharply after two doses, and the toxicity was the greatest. After the second dose, the weight of mice dropped from about 24 g to about 16 g. After comprehensive consideration, the third dose was not given. After three doses, the weight of mice in the 40 mg / kg group dropped from about 23 g to about 15 g. The toxicity was also very high. After comprehensive consideration, the fourth dose was not given. After four doses, the weight of mice in the 10 mg / kg and 20 mg / kg groups did not drop significantly, and the safety was excellent. HE results showed that significant pathological changes occurred in various tissues and organs in the 40 mg / kg and 80 mg / kg groups, especially in the sternum and liver; while there were no obvious pathological changes in the 10 mg / kg and 20 mg / kg groups. Therefore, after comprehensive consideration, the Azo-CPT(1)lip group was given a dose of 20 mg / kg in the subsequent in vivo efficacy experiments.

[0092] Example 11 Biodistribution study of Azo-CPT (1) drug-loaded liposomes:

[0093] 4T1 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS at a concentration of 1×10 7 100 μL of cell suspension was inoculated subcutaneously on the right back of female BABL / c mice to establish a mouse breast cancer tumor model. 3 At 0.5, 4, 8, 24, and 48 hours after administration, the mice were anesthetized with isoflurane and the fluorescence in the mice was imaged using the IVISLumina III imaging system. Fig.13 ). The results showed that in the Dir-Azo-CPT(1)lip group, fluorescence could be observed at the tumor site at 0.5h, and the fluorescence intensity at the tumor site gradually increased over time. After 48h, the tumor site still had strong fluorescence. In the Dir solution group, no fluorescence was observed at the tumor site within 48h, and only the tail had fluorescence. This indicates that after the drug-loaded liposomes enter the body, they accumulate rapidly, in large quantities, and for a long time at the tumor site, and are mainly distributed in the tumor.

[0094] Example 12 Study on the tissue distribution of Azo-CPT (1) drug-loaded liposomes:

[0095] 4T1 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS at a concentration of 1×10 7 100 μL of cell suspension was inoculated subcutaneously on the right back of female BABL / c mice to establish a mouse breast cancer tumor model. 3 At the same time, the mice were divided into 2 groups, 4 in each group, and Azo-CPT(1)lip (10 mg / kg) and free CPT (10 mg / kg) were injected through the tail vein. 24 hours after administration, the mice were killed, and 100 mg of each of the heart, liver, spleen, lung, kidney, small intestine, and tumor were dissected and placed in a 2mL Eppendorf tube. One large steel ball and two small steel balls were added and ground into a slurry in a tissue crusher. 100 μL of the above tissue suspension was placed in a 1.5mL Eppendorf tube, 400 μL of 1% acetic acid and methanol were added, vortexed for 1 min, ultrasonically extracted for 5 min, centrifuged at 10000r for 5 min, and the supernatant was used for HPLC to determine the drug concentration in the tissue, and a comparison of drug concentrations in different tissues was plotted ( Fig.14 The results showed that Azo-CPT(1)lip was mainly distributed in the tumor after entering the body, while only a small amount of free CPT was distributed in the tumor. Therefore, compared with free CPT, Azo-CPT(1)lip can selectively accumulate in the tumor site.

[0096] Example 13 Pharmacodynamics study of Azo-CPT (1) drug-loaded liposomes:

[0097] 4T1 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS at a concentration of 1×10 7 100 μL of cell suspension was inoculated subcutaneously on the right back of female BABL / c mice to establish a mouse breast cancer tumor model. 3 At the time of the experiment, the mice were divided into 3 groups according to the tumor volume, with 5 mice in each group (4 mice in the normal saline group). Normal saline, free CPT (10 mg / kg), and Azo-CPT (1) lip (20 mg / kg) were administered through the tail vein of the mice, once every 3 days, for a total of 4 times. After administration, the weight and tumor volume of the mice were measured and recorded every day. The tumor volume of the mice = (long diameter of the tumor × short diameter of the tumor) 2 ) / 2. On the 5th day after the last administration, all mice were killed, and the heart, liver, spleen, lung, kidney, and sternum were dissected and HE staining was performed to analyze the pathological changes of each tissue. Fig.15As shown in the results, compared with free CPT, Azo-CPT(1)lip showed significant antitumor activity, significantly inhibited tumor growth, and did not significantly cause weight loss in mice. HE section results showed that Azo-CPT(1)lip had no obvious pathological changes. In summary, the results indicate that Azo-CPT(1)lip can tolerate higher doses and has better antitumor efficacy in vivo.

Claims

1. A camptothecin prodrug having a structure as shown in Formula I or Formula II: in n is an integer of 1-9; R1 is a C1-C6 alkyl group; R2 is a C1-C6 alkyl group; R3 is H or a hydroxyl group; R4 is H or a C1-C6 alkyl group.

2. The camptothecin prodrug according to claim 1, characterized in that: The C1-C6 alkyl group is a C1-C6 straight-chain alkyl group or a branched-chain alkyl group; the straight-chain alkyl group is selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl; the branched-chain alkyl group is selected from isopropyl, sec-butyl, isobutyl, tert-butyl or neopentyl.

3. The camptothecin prodrug according to claim 1, characterized in that: It has the following structural formula:

4. The camptothecin prodrug according to claim 1, characterized in that: It has the following structural formula:

5. The pharmaceutical composition of camptothecin prodrug according to any one of claims 1 to 4, characterized in that: The invention comprises a camptothecin prodrug and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is a drug-loaded liposome of a camptothecin prodrug.

7. The drug-loaded liposome of camptothecin prodrug according to claim 6, characterized in that: Including camptothecin prodrug, phospholipids, cholesterol, PEGylated phospholipids, and ammonium sulfate.

8. The drug-loaded liposome according to any one of claims 1 to 4, characterized in that: The phospholipids are egg yolk phosphatidylcholine (EPC), soybean lecithin, sphingomyelin, distearoyl phosphatidylcholine (DSPC), hydrogenated soybean lecithin (HSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC) or hydrogenated soybean lecithin (HSPC).

9. The drug-loaded liposome according to any one of claims 1 to 4, characterized in that: The PEGylated phospholipid is a conjugate of PEG and DSPE connected via an amide bond.

10. Use of the drug-loaded liposome of the camptothecin prodrug according to any one of claims 6 to 9 in the preparation of drugs for preventing or treating tumors.

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