Camptothecin prodrugs and their drug-loaded liposomes and applications

Abstract

This invention provides a camptothecin prodrug. The camptothecin prodrug of this invention exhibits higher drug retention and better stability in liposomes, and the prodrug liposomes can maintain a prolonged plasma drug concentration. In vitro characterization of the camptothecin prodrug liposomes of this invention shows that they have higher encapsulation efficiency, drug loading, and stability, and can achieve a sustained-release effect in plasma; cellular level experiments show that they have higher cellular uptake rate; in vivo experiments demonstrate that they have better tumor accumulation effect and antitumor activity, can tolerate higher doses, and have higher biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations and relates to drug-loaded liposomes, specifically to a camptothecin prodrug, its active drug-loaded liposome, and its applications. Background Technology

[0002] Camptothecin (CPT) is a cytotoxic quinoline alkaloid that inhibits DNA replication by suppressing DNA topoisomerase I, thereby inducing apoptosis in tumor cells. Currently, camptothecin-based antitumor drugs include irinotecan, topotecan, hydroxycamptothecin, rubitecan, and 9-aminocamptothecin. These camptothecin derivatives are mainly used clinically in injectable form. While they possess strong anticancer activity, they also suffer from poor cell selectivity. CPT is prone to causing toxic side effects such as myelosuppression, gastrointestinal toxicity, and hemorrhagic cystitis, significantly limiting the clinical application of CPT drugs.

[0003] Liposomes are vesicles formed from phospholipids and cholesterol in water. Liposome formulations of many chemotherapy drugs have been successfully used in cancer treatment. Liposomes can alter the distribution of chemotherapy drugs in normal and tumor tissues, improve drug pharmacokinetic behavior, and prolong drug circulation time. Clinically used liposomal formulations include amphotericin B liposomes, doxorubicin liposomes, and cytarabine liposomes, among others. Therefore, liposomes are considered the most successful nanocarriers for clinical cancer treatment.

[0004] Liposomes are a feasible strategy for improving the therapeutic index of cytokine phospholipids (CPT). Stable encapsulation of CPT within liposomes is fundamental to the preparation of CPT liposomal drugs. However, as a hydrophobic molecule, CPT is primarily encapsulated within phospholipid membranes, leading to rapid drug release and low encapsulation efficiency. Most cationic drugs can be encapsulated using the ammonium sulfate gradient active drug delivery method. This method utilizes the ammonium sulfate gradient inside and outside the liposome to drive the cationic drug into the liposome and form a complex precipitate with sulfate ions, thus retaining it within the liposome and completing the encapsulation. However, CPT lacks ionizable groups, therefore it cannot be directly encapsulated using the ammonium sulfate gradient method. To address the issue of efficient CPT liposome encapsulation using the ammonium sulfate gradient method, positively charged groups can be modified onto CPT to enable encapsulation within liposomes. However, while this CPT cationic prodrug can be encapsulated within liposomes, the inability of the CPT cationic prodrug to form a stable complex precipitate with counterions such as ammonium sulfate leads to rapid drug release within the liposome, failing to leverage the advantages of liposomes in improving the antitumor therapeutic index of CPT.

[0005] In summary, conventional CPT cationic prodrugs cannot be stably encapsulated within liposomes, resulting in rapid drug release. Therefore, developing a CPT prodrug that can be stably encapsulated within liposomes to overcome the leakage problem of conventional cationic drugs is expected to promote the development and application of CPT liposomal drugs. Summary of the Invention

[0006] To address the problems in the prior art, the present invention provides a camptothecin-based prodrug.

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

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

[0009]

[0010] in

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

[0012] The C1-C6 alkyl groups described in this invention are straight-chain alkyl groups or branched alkyl groups of C1-C6. The straight-chain alkyl groups are selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, or n-octyl; the branched alkyl groups are 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 a second aspect of the present invention, the present invention provides a pharmaceutical composition of the above-mentioned camptothecin prodrug. The pharmaceutical composition of the above-mentioned camptothecin prodrug comprises a camptothecin prodrug and pharmaceutically acceptable excipients. Further, the above-mentioned pharmaceutical composition is a drug-loaded liposome of a camptothecin prodrug.

[0018] The drug-loaded liposome of the camptothecin prodrug of this invention comprises the above-mentioned camptothecin prodrug, phospholipids, cholesterol, PEGylated phospholipids, and ammonium sulfate. The phospholipids are natural, semi-synthetic, or fully synthetic phospholipids such as egg yolk lecithin (EPC), soybean lecithin, sphingomyelin, distearate phosphatidylcholine (DSPC), hydrogenated soybean lecithin (HSPC), dipalmitoyl phosphatidylcholine (DPPC), and myristoyl phosphatidylcholine (DMPC), preferably hydrogenated soybean lecithin (HSPC). The PEGylated phospholipids are couplings of PEG and DSPE linked by amide bonds, such as DSPE-PEG. 2000 .

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

[0020] Beneficial effects:

[0021] This invention provides a camptothecin prodrug. The camptothecin prodrug of this invention exhibits higher drug retention and better stability in liposomes, and the prodrug liposomes can maintain a prolonged plasma drug concentration. In vitro characterization of the camptothecin prodrug liposomes of this invention shows that they have higher encapsulation efficiency, drug loading, and stability, and can achieve a sustained-release effect in plasma; cellular level experiments show that they have higher cellular uptake rate; in vivo experiments demonstrate that they have better tumor accumulation effect and antitumor activity, can tolerate higher doses, and have higher biocompatibility. Attached Figure Description

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

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

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

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

[0026] Figure 5 This is a graph showing the encapsulation efficiency of Azo-CPT(1)lip under different drug loading levels;

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

[0028] Figure 7 This is a transmission electron microscope image of Azo-CPT(1)lip;

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

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

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

[0032] Figure 11 Pharmacokinetic studies of CPT and Azo-CPT(1)lip: blood concentration-time curves;

[0033] Figure 12 Biosafety evaluation of Azo-CPT(1)lip: Mouse weight change (A), HE slices of various tissues (B);

[0034] Figure 13 Biological distribution study of Azo-CPT(1)lip: in vivo mouse imaging;

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

[0036] Figure 15 This is an in vivo efficacy study of Azo-CPT(1)lip: tumor volume changes (A), mouse weight changes (B), tumor size in vitro (C), HE slices of various tissues (D). Detailed Implementation

[0037] In the following embodiments, the invention has only been described as an example; however, those skilled in the art can make various modifications to the invention after reading this patent application without departing from its spirit and scope. Unless otherwise specified, all parts and percentages mentioned in this invention are by weight.

[0038] Related terms:

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

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

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

[0042]

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

[0044] Accurately weigh 1 g of methyl 4-aminobenzoate, i.e., compound 1 (1 eq, 6.6 mmol), into a clean 100 mL reaction flask, evacuate for 5 min, add 20 mL of anhydrous DCM to dissolve it, and purge with nitrogen. Accurately weigh 8 g of Oxone (2 eq, 13.2 mmol) into a 50 mL Eppendorf tube, add 40 mL of ultrapure water to completely dissolve it until it becomes colorless and transparent, and add the Oxone solution to the reaction flask using a 20 mL syringe while stirring. Stir the reaction at room temperature for 5 h. After the reaction was confirmed by TLC, the reaction solution was poured into a separatory funnel and separated. The organic phase was collected, and the aqueous phase was extracted with DCM. This process was repeated three times. The organic phases were combined and washed successively with appropriate amounts of saturated citric acid, saturated sodium bicarbonate, pure water, and saturated sodium chloride. The washed organic phase was dehydrated with an appropriate amount of anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness using a rotary evaporator. An appropriate amount of DCM was added to redissolve the filtrate, and the mixture was recrystallized at -20°C for 2 hours. A pale yellow solid precipitated. The filter cake was collected to obtain compound 2 (M+H=166.0459). The product was identified by nuclear magnetic resonance spectroscopy. Figure 1 The synthesis was confirmed to be successful.

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

[0046] Accurately weigh 500 mg of compound 2 (1 eq, 3.03 mmol) into a clean 100 mL reaction flask, add an appropriate amount of glacial acetic acid (HAc) and sonicate to dissolve. While stirring, add 455 μL of 4-((dimethylamino)methyl)aniline, i.e., compound 3 (1 eq, 3.03 mmol), and react overnight at room temperature. After the reaction is complete, 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 3-4 times, combine the organic phases, and wash the organic phase successively with pure water, saturated sodium bicarbonate, and pure water. Collect the organic phase, remove water with an appropriate amount of anhydrous sodium sulfate, filter, evaporate to dryness, redissolve with a small amount of DCM, and spread onto a large silica gel plate for separation and purification (developing solvent: DCM:MeOH = 20:1), to obtain compound 4 (M+H = 298.1511). The product is identified by nuclear magnetic resonance spectroscopy. Figure 2The synthesis was confirmed to be successful.

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

[0048] Accurately weigh 500 mg of compound 4 (1 eq, 1.68 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 20 mL of anhydrous DCM to dissolve, and purge with nitrogen. While stirring, add 4 mL of lithium aluminum hydride (LAH) (6 eq, 10.08 mmol), then add 20 mL of tetrahydrofuran (THF), and react at room temperature for 5 h. After the reaction was complete, the reaction solution was poured into a 500 mL beaker, and moistened DCM was added dropwise. Bubbles were generated during the process. After the bubbling subsided, ultrapure water was added dropwise until no more bubbles were generated and Al(OH)3 precipitate was formed. Hydrochloric acid was added until the precipitate was completely dissolved. Saturated sodium bicarbonate was added to separate the solution into layers until no more bubbles were generated. The organic phase was collected, and the aqueous phase was repeatedly extracted with DCM until the color of the aqueous phase became lighter. The collected organic phase was dehydrated with an appropriate amount of anhydrous sodium sulfate, filtered, evaporated to dryness, and reconstituted with a small amount of DCM. The solution was then coated onto a large silica gel plate for separation and purification (developing solvent: DCM:MeOH = 10:1) to obtain compound 5 (M+H = 270.1562). The product was identified by nuclear magnetic resonance spectroscopy. Figure 3 The synthesis was confirmed to be successful.

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

[0050] Accurately weigh 200 mg CPT (compound 6) (1.1 eq, 0.58 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 40 mL of anhydrous DCM to dissolve it, and purge with nitrogen. Weigh 114 mg BTC (0.726 eq, 0.38 mmol) and 320 mg DMAP (4.95 eq, 2.62 mmol) into 2 mL Eppendorf tubes, dissolve them in an appropriate amount of anhydrous DCM, and add them to the reaction flasks using a 1 mL syringe. After reacting for 5 min, weigh 142 mg of compound 5 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to clear orange-yellow. React at room temperature for 2 h. After the reaction was confirmed by TLC, the reaction solution was poured into a separatory funnel, washed with a small amount of saturated citric acid and saturated sodium chloride, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added to remove water, filtered, evaporated to dryness, and redissolved with a small amount of DCM. The solution was then spread onto a large silica gel plate for separation and purification (the developing solvent was first DCM:MeOH = 20:1, then DCM:MeOH = 10:1, for a total of two runs), yielding compound 7 (Azo-CPT(1), M+H = 644.2464). The product was identified by nuclear magnetic resonance spectroscopy. Figure 4 The synthesis was confirmed to be successful.

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

[0052]

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

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

[0055] (2) The steps for synthesizing compound 11:

[0056] Accurately weigh 500 mg of compound 9 (1 eq, 3.03 mmol) into a clean 100 mL reaction flask, add an appropriate amount of glacial acetic acid (HAc) and sonicate to dissolve. While stirring, add 540 μL of 4-(diethylaminomethyl)aniline compound 10 (1 eq, 3.03 mmol) and react overnight at room temperature. After the reaction is complete, 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 3-4 times, combine the organic phases, and wash the organic phase successively with pure water, saturated sodium bicarbonate, and pure water. Collect the organic phase, remove water with an appropriate amount of anhydrous sodium sulfate, filter, evaporate to dryness, redissolve with a small amount of DCM, and spread on a large silica gel plate for separation and purification (developing solvent: DCM:MeOH = 20:1) to obtain compound 11 (M+H = 326.1824).

[0057] (3) The steps for synthesizing compound 12:

[0058] Accurately weigh 500 mg of compound 11 (1 eq, 1.54 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 20 mL of anhydrous DCM to dissolve, and purge with nitrogen. While stirring, add 3.7 mL of lithium aluminum hydride (LAH) (6 eq, 9.24 mmol), then add 20 mL of tetrahydrofuran (THF), and react at room temperature for 5 h. After the reaction was complete, the reaction solution was poured into a 500 mL beaker, and moistened DCM was added dropwise. Bubbles were generated during the process. After the bubbling subsided, ultrapure water was added dropwise until no more bubbles were generated and Al(OH)3 precipitate was formed. Hydrochloric acid was added until the precipitate was completely dissolved. Saturated sodium bicarbonate was added to separate the solution into layers until no more bubbles were generated. The organic phase was collected, and the aqueous phase was repeatedly extracted with DCM until the color of the aqueous phase became lighter. The collected organic phase was dehydrated by adding an appropriate amount of anhydrous sodium sulfate, filtered, evaporated to dryness, and reconstituted with a small amount of DCM. The solution was then coated onto a large silica gel plate for separation and purification (developing solvent: DCM:MeOH = 10:1) to obtain compound 12 (M+H = 298.1875).

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

[0060] Accurately weigh 200 mg of CPT (compound 13) (1.1 eq, 0.58 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 40 mL of anhydrous DCM to dissolve it, and purge with nitrogen. Weigh 114 mg of BTC (0.726 eq, 0.38 mmol) and 320 mg of DMAP (4.95 eq, 2.62 mmol) into 2 mL Eppendorf tubes, dissolve them in an appropriate amount of anhydrous DCM, and add them to the reaction flasks using a 1 mL syringe. After reacting for 5 min, weigh 155 mg of compound 12 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to clear orange-yellow. React at room temperature for 2 h. After the reaction was confirmed by TLC, the reaction solution was poured into a separatory funnel, a small amount of saturated citric acid and saturated sodium chloride were added for washing, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added to remove water, filtered, evaporated to dryness, a small amount of DCM was added to redissolve, and it was spread on a large silica gel plate for separation and purification (the developing solvent was first DCM:MeOH = 20:1, then 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) Synthetic steps of compound 16:

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

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

[0066] Accurately weigh 500 mg of compound 16 (1 eq, 3.03 mmol) into a clean 100 mL reaction flask, add an appropriate amount of glacial acetic acid (HAc) and sonicate to dissolve. While stirring, add 622 mg of 4-(4-methylpiperazine)aniline (compound 17) (1 eq, 3.03 mmol) and react overnight at room temperature. After the reaction is complete, 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 3-4 times, combine the organic phases, and wash the organic phase successively with pure water, saturated sodium bicarbonate, and pure water. Collect the organic phase, remove water with an appropriate amount of anhydrous sodium sulfate, filter, evaporate to dryness, and purify by silica gel column chromatography (eluent: 100 mL pure DCM, DCM:MeOH = 5:1) to obtain compound 18 (M+H = 353.1933).

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

[0068] Accurately weigh 500 mg of compound 18 (1 eq, 1.42 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 20 mL of anhydrous DCM to dissolve, and purge with nitrogen. While stirring, add 3.5 mL of lithium aluminum hydride (LAH) (6 eq, 8.52 mmol), then add 20 mL of tetrahydrofuran (THF), and react at room temperature for 5 h. After the reaction was complete, the reaction solution was poured into a 500 mL beaker, and moistened DCM was added dropwise. Bubbles were generated during the process. After the bubbling subsided, ultrapure water was added dropwise until no more bubbles were generated and Al(OH)3 precipitate was formed. Hydrochloric acid was added until the precipitate was completely dissolved. Saturated sodium bicarbonate was added to separate the solution into layers until no more bubbles were generated. The organic phase was collected, and the aqueous phase was repeatedly extracted with DCM until the color of the aqueous phase became lighter. The collected organic phase was dehydrated by adding an appropriate amount of anhydrous sodium sulfate, filtered, evaporated to dryness, and reconstituted with a small amount of DCM. The solution was then coated onto a large silica gel plate for separation and purification (developing solvent: DCM:MeOH = 10:1) to obtain compound 19 (M+H = 325.1984).

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

[0070] Accurately weigh 200 mg of CPT (compound 20) (1.1 eq, 0.58 mmol) into a clean 100 mL reaction flask, evacuate for 5 min, add 40 mL of anhydrous DCM to dissolve it, and purge with nitrogen. Weigh 114 mg of BTC (0.726 eq, 0.38 mmol) and 320 mg of DMAP (4.95 eq, 2.62 mmol) into 2 mL Eppendorf tubes, dissolve them in an appropriate amount of anhydrous DCM, and add them to the reaction flasks using a 1 mL syringe. After reacting for 5 min, weigh 170 mg of compound 19 (1 eq, 0.52 mmol) and add it. The reaction solution gradually changes from milky white to clear orange-yellow. React at room temperature for 2 h. After the reaction was confirmed by TLC, the reaction solution was poured into a separatory funnel, a small amount of saturated citric acid and saturated sodium chloride were added for washing, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added to remove water, filtered, evaporated to dryness, a small amount of DCM was added to redissolve, and it was spread on a large silica gel plate for separation and purification (the developing solvent was first DCM:MeOH = 20:1, then 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 2000The liposome was dissolved in 1 mL of anhydrous ethanol. Under vigorous stirring at 60°C, the ethanol solution was added to 5 mL of 300 mM ammonium sulfate solution to obtain a crude liposome solution. The ethanol was removed from the liposome solution by rotary evaporation at 45°C, and then extruded through an 80 nm polycarbonate core-porous membrane at room temperature. The resulting liposomes were transferred to a dialysis bag with a molecular weight cutoff of 3000 and dialyzed at 37°C in 150 mM NaCl solution to remove unencapsulated ammonium sulfate and create an ammonium sulfate concentration gradient between the inside and outside of the liposomes. The dialysate was changed every 3 hours for a total of 4 times to obtain blank liposomes encapsulated with ammonium sulfate.

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

[0075] Weigh 2 mg of Azo-CPT(1) and dissolve it in 100 μL of DMSO. Under vigorous stirring, first add it to 1 mL of the above blank liposomes at room temperature, and then incubate the mixed liposome solution in a water bath at 60 °C for 3 min with stirring. After incubation, the liposome solution can be observed to change 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 was taken, without removing unloaded drug through a G50 chromatography column, and mixed with 1 mL of Na2S2O4 solution before measuring the fluorescence intensity A1. Next, 100 μL of Triton was added to the above mixture, and after mixing, the fluorescence intensity A2 was measured. The encapsulation efficiency and drug loading of the drug-loaded liposomes were calculated using the following formula:

[0078]

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

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

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

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

[0083] Azo-CPT(1)lip (2 mg / mL) and Azo-CPT(1) were diluted with rat plasma to a concentration of 10 μg / mL, and the mixture was incubated at 38 °C. At each corresponding time point, 10 μL of plasma sample was taken, 100 μL of 1% acetic acid methanol was added, the mixture was vortexed for 1 min, sonicated for 5 min, centrifuged at 10000 rpm for 5 min, and the supernatant was collected for HPLC analysis. Drug release was calculated using the following formula: A CPT / (A CPT +A Azo-CPT(1) )×100%, plot the drug release curve ( Figure 8 As shown in the figure, Azo-CPT(1) achieved a drug release rate of over 90% within 48 hours. However, after being prepared as liposomes, the drug release rate slowed down significantly, with only about 30% of the drug released within 48 hours. This indicates that Azo-CPT(1) lip drugs are not easily leaked and can maintain an effective blood drug concentration in the body for a long time. They are not easily cleared by the body, thus prolonging the duration of drug action.

[0084] Example 7: Stability study 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. At each corresponding time point, 100 μL of the mixture was taken and 100 μL of 1% acetic acid-methanol was added for HPLC analysis. The results are as follows: Figure 9 As shown, Azo-CPT(1) degraded at 48 h, and approximately 35% of the drug degraded at 72 h. However, Azo-CPT(1)lip remained in the form of the prodrug Azo-CPT(1) within 72 h, with no CPT peak detected. This indicates that Azo-CPT(1)lip remained stable within 72 h compared to Azo-CPT(1), and the prodrug in the liposomes did not degrade. These results demonstrate that preparing Azo-CPT(1) into liposomes significantly increased its stability.

[0086] Example 8: Cellular uptake study of Azo-CPT(1) drug-loaded liposomes:

[0087] 4T1 cells were stored at 3 × 10⁴ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 12-well plates and incubated at 37°C in a CO2 incubator for 24 h. 1 μg / mL of free CPT, Azo-CPT(1), and Azo-CPT(1)lip were added to the cells and co-incubated at 37°C. After 2 h of incubation, the drug concentration in each well was determined by HPLC, and a comparison of drug concentrations in different groups was plotted. Additionally, the drug concentration in cells at different time points was determined by HPLC, and a drug concentration change curve was plotted. Results are as follows: Figure 10 The results showed that, regardless of whether the incubation time was 2 hours (A) or different incubation time points (B), the uptake of Azo-CPT (1) by tumor cells was higher than that of CPT.

[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 male SD rats via the tail vein. Blood samples were collected at given time intervals, and 400 μL of 1% acetic acid methanol was added. The mixture was vortexed for 1 min, sonicated for 5 min, and centrifuged at 10000 r for 5 min. The supernatant was then analyzed by HPLC, and a drug-time curve was plotted. Figure 11 The results showed that the area under the drug-time curve (AUC) of Azo-CPT(1)lip was much larger than that of CPT, indicating that Azo-CPT(1) was absorbed more completely by the body and had a better effect. Furthermore, the rate of decrease in blood concentration of Azo-CPT(1)lip was slower, 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 is far superior to that of CPT.

[0090] Example 10 Biosafety evaluation of Azo-CPT(1) drug-loaded liposomes:

[0091] 4T1 cells in logarithmic growth phase were digested with trypsin, centrifuged, resuspended in PBS, and a concentration of 1×10⁻⁶ cells was collected. 7 100 μL of cell suspension was subcutaneously injected into the right posterior back of female BABL / c mice to establish a mouse mammary tumor model. The tumor was allowed to grow to 100 mm. 3Mice bearing tumors were divided into four groups of three mice each, based on tumor size. Each group was injected with different doses of Azo-CPT(1)lip (10 mg / kg, 20 mg / kg, 40 mg / kg, and 80 mg / kg) via tail vein injection. The administration was repeated every three days for a total of four doses. Daily weight changes were measured, and weight change curves were plotted to assess the toxicity of different doses of the drug-loaded liposomes. Four days after the last administration, all mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and sternum were dissected and subjected to HE staining to analyze the pathological changes in each tissue. Mouse weight results are shown below. Figure 12 The results showed that the 80 mg / kg group exhibited the most significant toxicity, with mouse weight decreasing linearly after two doses, dropping from approximately 24 g to approximately 16 g after the second dose. Therefore, a third dose was not administered. Similarly, the 40 mg / kg group showed a significant toxicity, with mouse weight decreasing from approximately 23 g to approximately 15 g after three doses, and a fourth dose was not administered. The 10 mg / kg and 20 mg / kg groups showed no significant weight loss after four doses, indicating excellent safety. HE results showed significant pathological changes in various tissues and organs in the 40 mg / kg and 80 mg / kg groups, particularly in the sternum and liver; while no significant pathological changes were observed in the 10 mg / kg and 20 mg / kg groups. Therefore, considering all factors, a dose of 20 mg / kg was chosen for the Azo-CPT(1)lip group in subsequent in vivo efficacy experiments.

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

[0093] 4T1 cells in logarithmic growth phase were digested with trypsin, centrifuged, resuspended in PBS, and a concentration of 1×10⁻⁶ cells was collected. 7 100 μL of cell suspension was subcutaneously injected into the right posterior back of female BABL / c mice to establish a mouse mammary tumor model. The tumor was allowed to grow to 200 mm. 3 Mice were divided into two groups, one mouse in each group, and injected via tail vein with Dir-Azo-CPT(1)lip (20 mg / kg, 0.2% Dir) and Dir solution, respectively. At 0.5, 4, 8, 24, and 48 hours after administration, mice were anesthetized with isoflurane, and in vivo fluorescence was captured using an IVISLumina III imaging system. Figure 13 The results showed that fluorescence was observed at the tumor site in the Dir-Azo-CPT(1)lip group at 0.5 h, and the fluorescence intensity at the tumor site gradually increased over time, with strong fluorescence still present at the tumor site after 48 h. In the Dir solution group, no fluorescence was observed at the tumor site within 48 h, except for fluorescence at the tail. 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, mainly distributed within the tumor.

[0094] Example 12: Tissue distribution study of Azo-CPT(1) drug-loaded liposomes:

[0095] 4T1 cells in logarithmic growth phase were digested with trypsin, centrifuged, resuspended in PBS, and a concentration of 1×10⁻⁶ cells was collected. 7 100 μL of cell suspension was subcutaneously injected into the right posterior back of female BABL / c mice to establish a mouse mammary tumor model. The tumor was allowed to grow to 200 mm. 3 Mice were divided into two groups of four each, and were injected with Azo-CPT (1)lip (10 mg / kg) and free CPT (10 mg / kg) via tail vein injection, respectively. 24 hours after administration, the mice were sacrificed, and 100 mg each of heart, liver, spleen, lung, kidney, small intestine, and tumor tissue were collected and placed in 2 mL Eppendorf tubes. One large steel ball and two small steel balls were added, and the mixture was ground into a paste using a tissue homogenizer. 100 μL of the tissue suspension was placed in a 1.5 mL Eppendorf tube, 400 μL of 1% acetic acid methanol was added, the mixture was vortexed for 1 min, ultrasonically extracted for 5 min, centrifuged at 10000 r for 5 min, and the supernatant was used for HPLC determination of the drug concentration in the tissue. A comparison of drug concentrations in different tissues was plotted. Figure 14 The results showed that Azo-CPT(1)lip was mainly distributed in the tumor after entering the body, while free CPT was only distributed in small amounts in the tumor. Therefore, compared with free CPT, Azo-CPT(1)lip can selectively accumulate in the tumor site.

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

[0097] 4T1 cells in logarithmic growth phase were digested with trypsin, centrifuged, resuspended in PBS, and a concentration of 1×10⁻⁶ cells was collected. 7 100 μL of cell suspension was subcutaneously injected into the right posterior back of female BABL / c mice to establish a mouse mammary tumor model. The tumor was allowed to grow to 100 mm. 3 Mice were divided into three groups of five mice each (four mice in the saline group) based on tumor size. Saline, free CPT (10 mg / kg), and Azo-CPT (1)lip (20 mg / kg) were administered via the tail vein every three days for a total of four administrations. After administration, mouse weight and tumor volume were measured and recorded daily. Tumor volume was calculated as (tumor long axis × tumor short axis). 2 ) / 2. Five days after the last administration, all mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and sternum were dissected for HE staining to analyze the pathological changes in each tissue. The results are as follows: Figure 15As shown, compared with free CPT, Azo-CPT(1)lip exhibited significant antitumor activity, significantly inhibiting tumor growth without significantly causing weight loss in mice. Furthermore, HE section analysis revealed no significant pathological changes in Azo-CPT(1)lip. These results indicate that Azo-CPT(1)lip can be tolerated at higher doses and possesses better in vivo antitumor efficacy.

Claims

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

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

3. The camptothecin prodrug as described in claim 1, characterized in that, It has the following structural formula: 。 4. The camptothecin prodrug as described in claim 1, characterized in that, It has the following structural formula: 。 5. The pharmaceutical composition of a camptothecin prodrug as described in any one of claims 1-4, characterized in that, This includes 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 pharmaceutical composition according to claim 6, characterized in that, The drug-loaded liposomes include camptothecin prodrug, phospholipids, cholesterol, PEGylated phospholipids, and ammonium sulfate.

8. The pharmaceutical composition according to claim 6, characterized in that, The phospholipids are egg yolk lecithin (EPC), soybean phospholipids, sphingomyelin, distearate phosphatidylcholine (DSPC), hydrogenated soybean phospholipids (HSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or hydrogenated soybean phospholipids (HSPC).

9. The pharmaceutical composition according to claim 6, characterized in that, The PEGylated phospholipid is a coupling of PEG and DSPE linked by amide bonds.

10. Use of the pharmaceutical composition according to any one of claims 6-9 in the preparation of a drug for the prevention or treatment of tumors.

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