Crocetin bridged taxane dimer prodrug as well as preparation method and application thereof

Abstract

The invention discloses a crocetin bridged taxane dimer prodrug as well as a preparation method and application thereof, and belongs to the technical field of biological medicines, the dimer prodrug is formed by bridging one molecule of crocetin with two molecules of taxane compounds through an ester bond; the taxane compound has a C2 '-site hydroxyl group, and the hydroxyl group is covalently connected with a carboxyl group of crocetin through an ester bond. Specifically, crocetin serves as a molecular bridge, dicarboxyl of crocetin and C2'hydroxyl of two-molecule paclitaxel are subjected to esterification coupling, and the crocetin-bridged paclitaxel dimer prodrug is constructed, the lipophilicity of paclitaxel is enhanced through the prodrug, and meanwhile toxicity to normal tissue is reduced through the synergistic effect of crocetin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to development and application of a crocetin-bridged taxane dimer prodrug and a nano preparation thereof. The prodrug is bridged by an ester bond between a double carboxylic acid group of crocetin and a C2' hydroxyl group of a taxane drug, and realizes efficient delivery and low toxicity by combining with a hybrid liposome technology, and is suitable for oral or injection administration for anti-tumor treatment. BACKGROUND

[0002] Cancer is one of the diseases with the highest mortality rate worldwide, and chemotherapy, as the main treatment method, still faces challenges such as significant toxic side effects and low drug delivery efficiency in clinical practice. Taxane drugs (such as paclitaxel and docetaxel) have become first-line treatment drugs for various solid tumors such as breast cancer, ovarian cancer and lung cancer due to their broad-spectrum anti-tumor activity, but they can easily cause serious side effects such as bone marrow suppression, reproductive toxicity and neurotoxicity. In addition, the inherent extremely low water solubility of the drug molecules and the extremely low oral bioavailability caused by the first-pass effect limit the expansion of their administration routes. For example, paclitaxel injection needs to rely on polyoxyethylene castor oil (Cremophor EL) as a solubilizer, which can easily cause side effects such as allergic reactions and neurotoxicity; docetaxel lyophilized powder is prone to drug degradation during storage, and is prone to crystallization after reconstitution. Although existing technologies attempt to improve the above problems by using delivery systems such as liposomes (Liporaxel ® ® ), albumin nanoparticles (such as Abraxane ® ), they still face challenges such as low drug loading capacity and complex preparation process. Oral liposome preparations still face problems such as low oral bioavailability, gastrointestinal drug leakage and poor formulation stability.

[0003] In recent years, traditional Chinese medicine has attracted attention for its multi-target and low toxicity advantages in tumor treatment. Crocetin (CRT), as the main active ingredient of saffron, has been proven to have significant anti-tumor activity, and can exert its effects by inhibiting tumor cell proliferation, inducing apoptosis and inhibiting angiogenesis, while showing low toxicity to normal tissues. However, the anti-tumor effect of a single traditional Chinese medicine component is limited and cannot meet the clinical needs.

[0004] Based on the structural commonality (C2' hydroxyl activity) of taxane drugs, the present application innovatively proposes to convert the medicinal value of CRT from a single active ingredient to a functional bridge molecule, specifically couple two molecules of taxane drugs through the double carboxylic acid structure of CRT, and construct a dimer prodrug with high lipophilicity and high drug loading capacity. This design can be expanded to all taxane derivatives with C2' hydroxyl groups (such as docetaxel), and can solve the bottleneck of its clinical application by enhancing lipophilicity, reducing toxicity and improving targeting. SUMMARY

[0005] The present application aims at the long-standing technical problems of significant toxic side effects, poor water solubility and low oral bioavailability in the clinical application of taxane drugs, and proposes a synergistic solution based on the bridging of active molecules of traditional Chinese medicine. The present application uses crocetin acid (CRT) as a molecular bridge, and uses its double carboxyl group to esterify and couple with the C2' hydroxyl group of two molecules of taxane drugs to construct a taxane dimer prodrug. Taking paclitaxel as an example, a paclitaxel dimer prodrug bridged by crocetin acid (PCP) is designed and synthesized. The prodrug enhances the lipophilicity of paclitaxel, and the synergistic effect of CRT reduces the toxicity to normal tissues. Specifically, the preparation method of the paclitaxel dimer prodrug bridged by crocetin acid (PCP) covalently connects through esterification reaction of the double carboxyl group of CRT and the hydroxyl group of two PTX molecules to form a stable dimer structure.

[0006] The synthetic route is as follows:

[0007] In the aspect of formulation development, based on the above molecular characteristics, a hybrid liposome (PCP-LPL) suitable for oral administration is prepared by the thin film hydration method. The formulation uses natural soybean phospholipid PC-50 and poloxamer 188 (Pluronic F68) as mixed carriers, and is constructed by solvent evaporation-hydration method, which has the advantages of uniform particle size, high stability, high drug loading capacity, etc., can effectively avoid the premature release of the drug in the gastrointestinal tract, and can also improve the tumor targeting effect through the enhanced permeability and retention effect (EPR effect).

[0008] Among them, the molar ratio of natural soybean phospholipid PC-50 and poloxamer 188 is 1:0.5-1:2, the mass ratio of PCP and total excipients is 1:5-1:20, the drug loading capacity is 5%-15% (w / w), the average particle size is 100nm-150nm, the polydispersity index (PDI) is ≤0.2, and the Zeta potential is -20mV to -30mV.

[0009] In the aspect of formulation development, it also includes an injection, which is a lyophilized powder injection or a solution type injection, prepared by the following method: (a) dissolving the paclitaxel dimer prodrug bridged by crocetin acid in an injection-grade solvent selected from at least one of polyethylene glycol, ethanol, and propylene glycol; (b) adding a lyoprotectant (such as mannitol, sucrose) or a solubilizer (such as Tween 80), and adjusting the pH to 4.0-7.0; (c) after sterile filtration, dispensing, freeze-drying or nitrogen filling, the injection preparation is obtained.

[0010] In vitro experiments confirmed that PCP-LPL had an inhibitory effect on the proliferation of prostate cancer (RM-1) tumor cells comparable to that of free PTX, and had a significant inhibitory effect on breast cancer and other tumor cells, while the half inhibitory concentration (IC 50 The half inhibitory concentration (IC

[0011] It is worth emphasizing that the molecular design of the dimeric prodrug of the present application has the ability to adapt to universal dosage forms. For injection dosage forms, based on the mature technology of taxane injections (such as low-temperature vacuum drying of freeze-dried injections and solvent replacement of solution-type preparations), the skilled person in the art can directly dissolve the prodrug in injection-grade solvents (such as an ethanol-polyethylene glycol complex system) or perform freeze-drying treatment, without relying on Cremophor EL to achieve safe administration. Although such embodiments are not specifically developed in the present study, based on the enhanced lipophilicity of the prodrug and the common knowledge of existing taxane preparation technology, its feasibility has been fully theoretically supported. Therefore, the scope of protection of the present application covers all derivative structures with CRT-bridged taxane dimers as the core and their development and application in oral, injection, transdermal and other dosage forms.

[0012] The beneficial effects of the present application are: The present application provides an efficient and safe solution for the clinical transformation of paclitaxel through molecular design and formulation technology, which has important scientific value and application prospects.

[0013] The scope of protection of the present application covers all structural variants with CRT-bridged taxane dimers as the core and their delivery system applications, and the technology extension is not limited to the verified oral dosage form, but provides a universal solution for the dosage form innovation of taxane drugs.

[0014] The prodrug of the present application is suitable for treating solid tumors sensitive to taxane drugs, including but not limited to prostate cancer, breast cancer, ovarian cancer, and lung cancer. In tumor-bearing mouse models, the tumor volume inhibition rate of the prodrug after oral administration is ≥50%, and the blood routine index, major organ and reproductive system histopathology detection results have no significant difference from the blank control group. The dosage of the oral preparation of the prodrug is 25 mg / kg (calculated as paclitaxel), once a day, for 7 consecutive days.

[0015] The prodrug described in this invention avoids oral intestinal toxicity, does not cause bone marrow suppression, and does not cause significant damage to major organs; it can also alleviate testicular toxicity caused by paclitaxel, reduce testicular tissue structural damage, improve the problem of reduced mature sperm count, and is beneficial to the recovery of spleen function and morphology. It is an oral preparation with high development value. Attached Figure Description

[0016] Figure 1 This is a full-wavelength ultraviolet scan of the PTX, CRT, and PCP of the present invention; Figure 2 The infrared spectra of PTX, CRT, and PCP of the present invention; Figure 3 This is the proton NMR spectrum of the PCP of the present invention; Figure 4 The nuclear magnetic resonance carbon spectrum of the PCP of this invention; Figure 5 This is a high-resolution mass spectrum of the PCP of the present invention; Figure 6 This is a particle size distribution diagram of the nano-formulation of the present invention; Figure 7 This is a transmission electron microscope (TEM) image of the PCP phospholipid / Pluronic F68 hybrid liposome (PCP-LPL) of the present invention; Figure 8 The following images illustrate the in vivo antitumor effect of the nano-formulation of this invention in a mouse model of prostate cancer: A: Body weight change graph, B: Tumor volume change graph, C: Tumor weight statistics, D: Actual tumor sample. ** p <0.01, *** p <0.001; Figure 9 The results of routine blood tests in a mouse model of prostate cancer bearing the nano-formulation of the present invention are shown. * p <0.05, ** p <0.01, *** p <0.001; Figure 10 The above are the H&E staining results of the nano-formulation of the present invention in the gastrointestinal tissue of a prostate cancer-bearing mouse model; Figure 11 The above are the H&E staining results of the nano-formulation of the present invention in the major organs of a mouse model of prostate cancer. Figure 12 The image shows the H&E staining results of the nano-formulation of the present invention on the reproductive organs (testes) of a mouse model of prostate cancer. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the described embodiments are only used to exemplify the core innovations of the present invention and are not intended to limit the scope of protection. Based on the technical teachings of the present invention, those skilled in the art can make reasonable parameter adjustments and process adaptations without creative effort, and such implementations all fall within the protection scope of the present invention.

[0018] Example 1

[0019] Synthesis and characterization of crocin-bridged paclitaxel dimer prodrug (PCP): Under nitrogen protection, paclitaxel (PTX, 0.35 mmol), trans-crocin (CRT, 0.16 mmol), EDCl (0.77 mmol), and DMAP (0.05 mmol) were dissolved in 15 mL of anhydrous dichloromethane. The mixture was stirred at 30 °C for 1 hour, then EDCl (0.37 mmol) and DMAP (0.05 mmol) were added, and the reaction continued for another 24 hours. The reaction solution was extracted three times each with 0.1 mmol / L dilute hydrochloric acid and deionized water. After removing the solvent by rotary evaporation, the solution was purified by silica gel column chromatography (eluent: dichloromethane / methanol gradient) combined with recrystallization from methanol, finally yielding an orange powder product PCP (molecular formula C...). 144 H 122 N2O 30 ).

[0020] By ultraviolet spectroscopy analysis ( Figure 1 PCP exhibits the characteristic absorption peak of PTX at 229 nm, while the carbonyl characteristic peak of CRT red-shifts from 424 nm to 440 nm, confirming the successful formation of the ester bond.

[0021] Infrared spectroscopy analysis ( Figure 2 The vibrational absorption peak of the C=O group at CRT is 1663.65 cm⁻¹. -1 In comparison, the vibrational absorption peak of the carbonyl C=O group in PCP blue-shifted to 1720 cm⁻¹. -1 This demonstrates the formation of new ester bonds. Simultaneously, PCP also exhibits characteristic infrared peaks of PTX, such as the aromatic cyclization CH vibration absorption peak at 1358.11 cm⁻¹. -1 1238.56cm -1 1097.41cm -1 The absorption peak of the C=O vibration of aromatic ketones is at 1620 cm⁻¹. -1 This demonstrates the successful synthesis of the prodrug PCP.

[0022] Analysis by nuclear magnetic resonance hydrogen spectroscopy ( Figure 3 The results are as follows: 1H-NMR(400MHz,Chloroform-d): δ8.23-8.00(m, 4H), 7.91-7.82(m, 4H), 7.09-7.68(m, 26H), 6.86-6.76(m, 5H),6.62-6.53(m, 5H), 5.93(dd, J=8.91, 4.03Hz, 2H), 5.67(d, J=7.1Hz, 2H), 5.53(d,J=4Hz, 2H, PTX-C2'), 5.02(d, J=5.8Hz, 2H), 4.44(m, 5H), 4.36-4.16(m, 5H),4.05(d, J=12.0Hz, 2H), 2.58–2.25 (m, 7H), 2.10 (s, 18H), 2.04–1.97 (m, 3H), 1.93 (s, 6H), 1.86 (s, 6H), 1.37 (s, 3H), 1.35 (s, 6H), 1.33 (s, 9H). The protons and chemical shifts of the target compounds all correspond one-to-one. In PCP… 1 In the 1H-NMR spectrum, the characteristic peak of C2'-OH of PTX (3.61 ppm) disappeared, while other peaks were well preserved. Due to the formation of the ester bond, the proton shift of C2'-CH changed from 4.8 ppm to 5.5 ppm, indicating that PTX and CRT were successfully linked by an ester bond. (C1NMR spectrum) Figure 4 This also proves the successful synthesis of PCP.

[0023] Analysis by high-resolution mass spectrometry (MALDI-TOF) Figure 5 The molecular ion peak of the product is [M+Na]. + =2022.29, consistent with the theoretical molecular weight.

[0024] High-performance liquid chromatography (HPLC) analysis showed that the product purity was ≥98%, meeting the requirements for subsequent experiments.

[0025] Example 2

[0026] Preparation and characterization of PCP oral hybrid liposomes (PCP-LPL): Based on a PCP to total excipient mass ratio of 1:12 and a soybean lecithin PC-50 to poloxamer 188 molar ratio of 1:1.2, accurately weighed PCP (10 mg), soybean lecithin PC-50 (8.5 mg), and poloxamer 188 (111 mg) were dissolved in 10 mL of anhydrous ethanol. After rotary evaporation to form a film, 2 mL of deionized water was added, and the mixture was hydrated at 50 °C for 30 minutes. Following ultrasonic dispersion (150 W, 30 minutes) and filtration through a 0.22 μm filter membrane, a PCP-LPL hybrid liposome solution was obtained. The PCP-LPL solution was orange-yellow with a certain degree of opalescence. A significant Tyndall effect was observed under laser irradiation, indicating that the drug formed a nanostructure in the solution.

[0027] Dynamic light scattering results show that the average particle size of PCP-LPL ( Figure 6 The wavelength range was (132.90±5.07) nm, the PDI was 0.19±0.04, and the Zeta potential was -24.23±0.21 mV. (Transmission electron microscopy) Figure 7 The results showed that PCP-LPL particles were spherical, intact, uniform in size, and well dispersed. The drug loading determined by ultrafiltration centrifugation was (8.33±0.5)% (w / w).

[0028] It is worth noting that although this embodiment only describes in detail the preparation and evaluation of oral hybrid liposomes (PCP-LPL), those skilled in the art can refer to the conventional processes of taxane drug injections in the prior art (such as solvent selection and the addition of lyophilization protectants) to prepare the prodrug into an injectable formulation.

[0029] Example 3

[0030] In vitro antitumor activity and safety evaluation: The CCK-8 assay was used to detect the inhibitory effect of PCP-LPL on the proliferation of RM-1 (mouse prostate cancer cells), 4T1 (mouse breast cancer cells), U251 (human glioma cells), and WPMY-1 (normal human prostate cells). The results showed that after 48 hours of treatment, PCP-LPL significantly reduced the IC50 of RM-1 cells. 50 The value is (1.83±0.1) nM, which is higher than the IC of free PTX. 50 (0.12±0.1) nM, but still maintained a strong tumor-suppressive effect. Furthermore, after 48 hours of PCP-LPL treatment, the IC50 of 4T1 cells was significantly reduced. 50 The IC50 of U251 cells was (346.6 ± 2.1) nM. 50 The concentration was (92.17±1.3) nM, indicating that PCP-LPL has antitumor effects on various cancer cells. After 48 hours of treatment, the IC50 of PCP-LPL on WPMY-1 cells in normal prostate cells was (92.17±1.3) nM. 50The concentration was (309±0.1) nM, which is 15 times that of free PTX (20.66±0.1) nM, confirming that it significantly reduced the killing effect on normal cells and showed the characteristics of high efficiency and low toxicity in in vitro cell experiments.

[0031] Example 4

[0032] In vivo antitumor efficacy and toxicity assessment: In a C57BL / 6 mouse model of prostate cancer xenografts, tumor-bearing mice were randomly divided into four groups: saline group, PTX injection group (10 mg / kg), PTX oral solution group (25 mg / kg), and PCP-LPL oral group (equivalent to PTX 25 mg / kg). Normal mice without tumors served as a blank control group. The injection group received the medication three times a week, while the oral group received the medication daily by gavage for 7 days. During the treatment period, compared with PTX, the PCP-LPL group showed no significant decrease in body weight. Figure 8 A) There were no significant differences in tumor volume, tumor inhibition rate, and ex vivo tumor weight between the PCP-LPL group and the PTX group. Figure 8 B-8D). The most common adverse reaction to paclitaxel injection is bone marrow suppression, as indicated by blood routine results ( Figure 9 This indicates that, compared with PTX, PCP-LPL did not show a decrease in neutrophil count or bone marrow suppression. H&E section results of major organs and gastrointestinal tissues showed that ( Figure 10 , Figure 11 Compared to PTX, PCP-LPL showed no significant damage to any tissues or organs, demonstrating higher safety. Furthermore, since all chemotherapy drugs have potential reproductive system toxicity, we further performed H&E sectioning on mouse testes (…). Figure 12 The results showed that the PTX group suffered more severe damage to testicular tissue structure and a significant reduction in the number of mature sperm in the field of view. Compared with PTX, PCP-LPL alleviated the toxicity of PTX to testicular tissue and mature sperm. In conclusion, PCP-LPL is an oral formulation with high development value.

[0033] Example 5

[0034] Synthesis of crocin-bridged docetaxel dimer prodrug (DCP): Under nitrogen protection, docetaxel (0.35 mmol), trans-crocin (0.16 mmol), EDCl (0.77 mmol), and DMAP (0.05 mmol) were dissolved in 15 mL of anhydrous dichloromethane. The mixture was stirred at 30 °C for 1 hour, and then EDCl (0.37 mmol) and DMAP (0.05 mmol) were added to continue the reaction for another 24 hours. The reaction solution was extracted three times each with 0.1 mmol / L dilute hydrochloric acid and deionized water. After removing the solvent by rotary evaporation, the product was purified by silica gel column chromatography (eluent: dichloromethane / methanol gradient) combined with recrystallization from methanol to obtain a powdered product.

[0035] Example 6

[0036] Preparation of DCP oral hybrid liposomes (DCP-LPL): Based on a DCP to total excipient mass ratio of 1:12 and a soybean lecithin PC-50 to poloxamer 188 molar ratio of 1:1.2, accurately weigh 10 mg of DCP, 8.5 mg of soybean lecithin PC-50, and 111 mg of poloxamer 188 and dissolve them in 10 mL of anhydrous ethanol. After rotary evaporation to form a film, add 2 mL of deionized water and hydrate at 50 °C for 30 minutes. After ultrasonic dispersion (150 W, 30 minutes) and filtration through a 0.22 μm filter membrane, obtain the DCP-LPL hybrid liposome solution.

[0037] Example 7

[0038] Synthesis of crocin-bridged carbamate dimer prodrug (CCP): Under nitrogen protection, cabazitaxel (0.35 mmol), trans-crocin (0.16 mmol), EDCl (0.77 mmol), and DMAP (0.05 mmol) were dissolved in 15 mL of anhydrous dichloromethane. The mixture was stirred at 30 °C for 1 hour, and then EDCl (0.37 mmol) and DMAP (0.05 mmol) were added to continue the reaction for another 24 hours. The reaction solution was extracted three times each with 0.1 mmol / L dilute hydrochloric acid and deionized water. After removing the solvent by rotary evaporation, the solution was purified by silica gel column chromatography (eluent: dichloromethane / methanol gradient) combined with recrystallization from methanol to obtain a powdered product.

[0039] Example 8

[0040] Preparation of CCP oral hybrid liposomes (CCP-LPL): With a CCP to total excipient mass ratio of 1:12 and a soybean lecithin PC-50 to poloxamer 188 molar ratio of 1:1.2, CCP (10 mg), soybean lecithin PC-50 (8.5 mg), and poloxamer 188 (111 mg) were accurately weighed and dissolved in 10 mL of anhydrous ethanol. After rotary evaporation to form a film, 2 mL of deionized water was added, and the mixture was hydrated at 50 °C for 30 minutes. After ultrasonic dispersion (150 W, 30 minutes) and filtration through a 0.22 μm filter membrane, a CCP-LPL hybrid liposome solution was obtained.

Claims

1. A crocin-bridged taxane dimer prodrug, characterized in that, The dimer prodrug is formed by bridging two taxane compounds with one molecule of crocin via an ester bond; the taxane compounds have a hydroxyl group at the C2' position, and the hydroxyl group is covalently linked to the carboxyl group of crocin via an ester bond.

2. The crocin-bridged taxane dimer prodrug according to claim 1, characterized in that, The taxane compound is paclitaxel.

3. The crocin-bridged taxane dimer prodrug according to claim 2, characterized in that, When the taxane compound is paclitaxel, the structural formula of the crocin-bridged paclitaxel dimer prodrug is as follows: 。 4. An antitumor drug formulation, characterized in that, The drug comprises a taxane dimer prodrug bridged with crocin as described in any one of claims 1-3 and a pharmaceutically acceptable carrier; the dosage form of the antitumor drug formulation is an oral formulation or an injection, wherein the oral formulation is in the form of a hybrid liposome, and the injection is a lyophilized powder for injection or a solution for injection.

5. The antitumor drug formulation according to claim 4, characterized in that, The oral formulation uses natural soybean lecithin PC-50 and poloxamer 188 as a mixed carrier in a molar ratio of 1:0.5-1:

2. The mass ratio of crocin-bridged taxane dimer prodrug to total excipients is 1:5-1:

20. The drug loading is 5%-15%, the average particle size is 100nm-150nm, the PDI is ≤0.2, and the zeta potential is -20mV to -30mV.

6. The antitumor drug formulation according to claim 4, characterized in that, The injection is prepared by the following method: (a) Dissolve the taxane dimer prodrug bridged with crocin in an injection-grade solvent, wherein the solvent is selected from at least one of polyethylene glycol, ethanol, and propylene glycol; (b) Add a lyophilization protectant or solubilizer and adjust the pH to 4.0-7.0; (c) After sterile filtration, the product is dispensed, freeze-dried or stored under nitrogen to obtain the injectable formulation.

7. The use of the crocin-bridged taxane dimer prodrug according to any one of claims 1-3 or the antitumor drug formulation according to any one of claims 4-6 in the preparation of an antitumor drug.

8. The application according to claim 7, characterized in that, The tumors include prostate cancer, breast cancer, ovarian cancer, and lung cancer.

9. The use of the crocin-bridged taxane dimer prodrug according to any one of claims 1-3 in the preparation of a drug that alleviates paclitaxel-induced testicular toxicity, reduces testicular tissue structural damage, improves the problem of reduced mature sperm count, and is beneficial to the recovery of spleen function and morphology.

10. The application according to claim 7 or 9, characterized in that, The routes of administration include oral administration, injection administration, local administration, respiratory administration, and cavity administration.

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