Application of crocetin and cyclodextrin inclusion compound thereof in assisting in resisting tumors and relieving toxic and side effects of tumor treatment

Abstract

The invention discloses application of crocetin and a cyclodextrin inclusion compound thereof in assisting in resisting tumors and relieving toxic and side effects in tumor treatment, belongs to the technical field of biological medicines, and particularly relates to application of crocetin or pharmaceutically acceptable salt thereof or combined administration of the cyclodextrin inclusion compound thereof and an anti-tumor chemotherapeutic drug to relieving toxic and side effects in tumor treatment. According to the crocetin cyclodextrin inclusion compound, the water solubility, the stability and the bioavailability of crocetin are remarkably improved. When the clathrate compound is combined with paclitaxel for use, the anti-tumor effect can be enhanced, and nervous system injury, myelosuppression and multi-organ toxicity caused by paclitaxel can be remarkably relieved. Experimental results show that the drug combination scheme shows the dual advantages of synergistic interaction and toxicity reduction in solid tumor models such as prostatic cancer and the like, and has important clinical application prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of combined administration of crocetin or a pharmaceutically acceptable salt thereof or a cyclodextrin inclusion compound thereof and an antitumor chemotherapeutic drug in reducing toxicity and increasing efficacy. BACKGROUND

[0002] In recent years, the main means of tumor treatment include surgical resection, radiotherapy, chemotherapy and targeted therapy. Among them, chemotherapy, as a systemic treatment method, plays an irreplaceable role in inhibiting tumor metastasis and recurrence. However, the chemotherapy drugs represented by cisplatin, cyclophosphamide, doxorubicin and paclitaxel, while killing tumor cells, will cause extensive damage to normal tissues and organs, and further cause serious side effects such as bone marrow suppression, liver and kidney toxicity, cardiac toxicity and reproductive system toxicity.

[0003] The toxic damage of chemotherapy drugs to the reproductive system has been widely reported. Cisplatin can induce apoptosis of testicular spermatogenic cells, destroy the protective blood-testis barrier, and inhibit the activity of key antioxidant enzymes, ultimately leading to a decrease in sperm count, a decrease in sperm motility and testicular atrophy. Paclitaxel, as an important microtubule stabilizer, although it shows significant efficacy in breast cancer, ovarian cancer and the like, its reproductive toxicity cannot be ignored. Studies have confirmed that paclitaxel (PTX) can damage male fertility, which is manifested as interference with the process of sperm production, reduction in sperm count and decrease in sperm motility. In addition, the drug can also interfere with the normal meiosis of oocytes, induce apoptosis of ovarian granulosa cells, and reduce the level of estrogen in the body, ultimately leading to premature ovarian failure and a significant decrease in female fertility. In the face of these serious reproductive and other organ toxicities, clinical attempts have been made to apply protective agents such as amifostine and gonadotropin-releasing hormone analogues (GnRHa). However, the effect of these protective measures is often limited, and may be accompanied by additional risks such as hypotension and endocrine disorders.

[0004] The damage of chemotherapy drugs to the nervous system also cannot be ignored. Peripheral nervous system damage is a common adverse reaction during chemotherapy. Damage to the central nervous system or peripheral nervous system caused by toxic substances can be divided into types such as neuropathy, axonopathy, myelinopathy and nerve terminal degeneration.

[0005] Crocetin (CRT), as a natural carotenoid compound, is mainly derived from saffron (Crocus sativus L.) and gardenia and the like, and has been confirmed by a large number of studies to have various pharmacological activities such as anti-tumor, anti-oxidation, anti-inflammatory and anti-apoptosis. Crocus sativus L. ) and gardenia and the like, and has been confirmed by a large number of studies to have various pharmacological activities such as anti-tumor, anti-oxidation, anti-inflammatory and anti-apoptosis.

[0006] However, crocin has extremely poor solubility in water, being difficult to dissolve in water and most organic solvents. Furthermore, crocin is sensitive to light, heat, enzymes, and oxygen. Cyclodextrin molecules have hydroxyl groups distributed on their outer surface, while their internal cavity structure is nonpolar. Therefore, they can accommodate poorly water-soluble guest drugs to form inclusion complexes, thereby improving the water solubility, stability, cell membrane permeability, relative bioavailability, reducing drug toxicity, and masking unpleasant odors of the guest drug molecules. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a combined drug delivery regimen that enhances efficacy and reduces toxicity, thereby improving the anti-tumor effects of radiotherapy and chemotherapy while reducing their toxic side effects. In this invention, the combined use of crocin and anti-tumor chemotherapy drugs can alleviate side effects caused by chemotherapy drugs, such as bone marrow suppression, neurotoxicity, organ toxicity, and reproductive toxicity, demonstrating promising application prospects in adjuvant chemotherapy treatment.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the use of crocin or a pharmaceutically acceptable salt thereof or a cyclodextrin inclusion complex thereof in the preparation of a medicament for use in combination with an antitumor chemotherapy drug.

[0009] The combined administration method described in this invention is used to: (a) enhance the antitumor efficacy of antitumor chemotherapeutic drugs; and / or (b) reduce the toxic side effects caused by antitumor chemotherapeutic drugs. Enhancing antitumor efficacy is manifested in improving the survival time of subjects and / or increasing the tumor inhibition rate; reducing toxic side effects is manifested in alleviating one or more of the following toxicities: bone marrow suppression, neurotoxicity, intestinal toxicity, or other organ toxicity.

[0010] Furthermore, the reduction of toxic side effects also includes promoting the functional and morphological recovery of the spleen damaged by tumors or chemotherapy.

[0011] Furthermore, the cyclodextrin inclusion complex of crocin is selected from any one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and α-cyclodextrin.

[0012] The antitumor chemotherapy drug is selected from platinum drugs, taxane drugs, topoisomerase inhibitors, antimetabolites, alkylating agents, or plant alkaloids; preferably one or more of cisplatin, oxaliplatin, carboplatin, paclitaxel, docetaxel, irinotecan, topotecan, 5-fluorouracil, gemcitabine, cyclophosphamide, and doxorubicin.

[0013] The combination therapy is targeted at tumors such as prostate cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, or ovarian cancer.

[0014] Furthermore, the dosage ratio of crocin or its pharmaceutically acceptable salt or its cyclodextrin inclusion complex to the antitumor chemotherapeutic drug is 0.1:1 to 20:1 (w / w). The antitumor chemotherapeutic drug is preferably paclitaxel; the preferred dosage ratio is 0.5:1 to 20:1 (w / w).

[0015] The pharmaceutical formulations of this invention include powders, tablets, lozenges, granules, capsules, suspensions, and injections, as well as their corresponding sustained-release or targeted nano-formulations, or are formulated as inclusion complexes, solid dispersions, liposomes, microspheres, nanoparticles, or emulsions for intracavitary administration, external use, oral administration, or injection.

[0016] Furthermore, combination regimens of crocin or its pharmaceutically acceptable salts or cyclodextrin inclusion complexes with antitumor chemotherapy drugs include: Crocin or its pharmaceutically acceptable salts or its cyclodextrin inclusion complexes are prepared into oral formulations; Preparation of antitumor chemotherapy drug formulations; The two preparations mentioned above can be used in combination.

[0017] Furthermore, the cyclodextrin inclusion complex of crocin can significantly improve the water solubility, stability, and bioavailability of crocin. In this inclusion complex, the molar ratio of crocin to cyclodextrin is 1:1 to 1:5, preferably 1:3.

[0018] The combined drug delivery regimen of this invention is used in the treatment of prostate cancer: By combining intraperitoneal injection of antitumor chemotherapy drugs (such as paclitaxel) with oral administration of crocin or its pharmaceutically acceptable salts or cyclodextrin inclusion complexes, tumor growth can be inhibited while reducing the toxicity of antitumor chemotherapy drugs.

[0019] The above combined administration regimens are applicable to: a) solid tumor chemotherapy regimens: including but not limited to prostate cancer, breast cancer, and ovarian cancer; b) hematologic malignancy chemotherapy regimens: including but not limited to leukemia and lymphoma; c) adjuvant chemotherapy and palliative chemotherapy scenarios.

[0020] The uses of the above-mentioned combined administration regimens include: a) inhibiting cancer cell proliferation; b) promoting cancer cell apoptosis; c) inhibiting cancer cell metastasis; d) inhibiting cancer cell spread; e) inhibiting tumor growth; f) inhibiting tumor metastasis; and g) preventing and / or treating tumors.

[0021] The above combined administration regimen can increase the tumor inhibition rate of paclitaxel against prostate cancer RM-1 cells to 66.01% (49.18% in the paclitaxel monotherapy group), without bone marrow suppression, significantly improve paclitaxel-induced neurological damage, and reduce paclitaxel toxicity to major organs.

[0022] The application of crocin or its pharmaceutically acceptable salts or cyclodextrin inclusion complexes in the preparation of drugs that enhance the efficacy of antitumor chemotherapy drugs and reduce their toxicity. This toxicity reduction and efficacy enhancement includes improving bone marrow suppression induced by antitumor chemotherapy drugs, reducing the toxicity of antitumor chemotherapy drugs to normal tissues and organs, and mitigating damage to the nervous system; the nervous system refers to both the central and peripheral nervous systems.

[0023] The symptoms of neurological damage include, but are not limited to: a) numbness and pain in the extremities; b) glove-and-stocking sensation; c) paresthesia such as coldness or burning pain in the skin; d) tingling sensation; e) weakness in holding objects; f) walking on cotton; g) sharp and stabbing pain.

[0024] Our team discovered through systematic research that a simple combination of crocin or its pharmaceutically acceptable salts or cyclodextrin inclusion complexes with antitumor chemotherapeutic drugs such as paclitaxel can produce a breakthrough synergistic effect. In animal models, the combination therapy group showed a significant increase in neutrophil count and a reduction in pathological damage to normal tissues compared to the paclitaxel monotherapy group, while maintaining an equivalent tumor suppression effect. This discovery breaks through the technical limitations of traditional combination therapy, and its core innovations are: (1) it does not change the existing clinical administration mode of chemotherapeutic drugs; (2) it does not require complex formulation processes or administration timing control; and (3) it achieves simultaneous optimization of toxicity control and efficacy maintenance.

[0025] The beneficial effects of this invention are: (1) Crocin can alleviate the toxic side effects of the antitumor drug paclitaxel, such as sudden weight loss, bone marrow suppression, neurotoxicity, and testicular damage, without affecting the antitumor efficacy of paclitaxel. (2) Toxicity control can be achieved through a simple combination therapy, without the need for complex formulation processes or strict dosing sequences. (3) It provides a solution that can be seamlessly integrated with existing chemotherapy systems. Paclitaxel is administered at the standard dose, and crocin is used simultaneously in the form of oral formulations, which facilitates clinical translation. (4) This invention not only provides a new use for crocin and a new scientific basis for the development of new antitumor adjuvant drugs, but also has great significance for the development and utilization of traditional Chinese medicine. It also provides a new treatment regimen for prostate cancer and has good application prospects in the prevention and treatment of prostate cancer. (5) This invention is the first to demonstrate that crocin can produce a synergistic effect when used in combination with conventional chemotherapy drugs, significantly reducing their toxic damage to normal organs while maintaining the antitumor activity of chemotherapy drugs. This technical solution can be widely applied to the clinical treatment of solid tumors such as prostate cancer, gastric cancer, and liver cancer, providing an innovative solution to the problem of dose-limiting toxicity of chemotherapy drugs. (6) This invention uses CRT cyclodextrin inclusion complex, which can improve the solubility of CRT and thus improve bioavailability, and better play the role of reducing toxicity and enhancing efficacy when used in combination with chemotherapy drugs. (7) The CRT cyclodextrin inclusion complex of this invention has broad potential for combined application. In addition to being used in combination with antitumor drugs, it may also be used in combination with drugs for the treatment of other types of diseases such as cardiovascular and cerebrovascular diseases and Alzheimer's disease. Attached Figure Description

[0026] Figure 1 This is a liquid chromatogram of a CRT standard.

[0027] Figure 2 Fourier transform infrared (FTIR) characterization results for the CRT / CD series; Group A is the CRT / α-CD series, Group B is the CRT / HP-β-CD series, and Group C is the CRT / γ-CD series. 1: Wavenumbers in the range of 4000-500 cm⁻¹ -1 Overall diagram within the range, 2: wavenumber in the range of 1800-1400 cm⁻¹ -1 A local view within the specified area.

[0028] Figure 3 The results are for dissolution in a pH 6.8 dissolution medium.

[0029] Figure 4 The study showed the changes in body weight in RM-1 tumor-bearing mice treated with CRT combined with PTX for prostate cancer.

[0030] Figure 5The following are the results of CRT combined with PTX treatment on RM-1 prostate cancer-bearing mice: A. In vitro tumor image of tumor-bearing mice after treatment; B. Changes in tumor volume of tumor-bearing mice during treatment; C. Tumor weight of tumor-bearing mice after treatment; D. Tumor inhibition rate of mice 7 days after drug administration.

[0031] Figure 6 Tumor tissue sections from RM-1 prostate cancer-bearing mice after treatment; A. H&E staining of mouse tumor tissue; B. Tunel immunohistochemical staining of mouse tumor tissue; C. Apoptosis index of mouse tumor tissue.

[0032] Figure 7 The complete blood count results of RM-1 prostate cancer-bearing mice after treatment are as follows: A. White blood cell count and its subsets; B. Red blood cell count; C. Platelet count; D. Mean platelet volume.

[0033] Figure 8 The images show histopathological sections of RM-1 prostate cancer-bearing mice after treatment. In the PTX group, the arrows indicate that PTX alone causes some degree of organ damage; however, when PTX is used in combination with CRT, this organ damage is significantly improved. In the MOD group, the arrows indicate that the spleen of tumor-bearing mice exhibits pathological changes with blurred boundaries between the red and white pulp; PTX alone failed to improve this pathological condition, but after combined administration of PTX and CRT, the morphology and structure of the spleen in the mice were restored, showing a pattern essentially consistent with the non-tumor-bearing CON group.

[0034] Figure 9 Survival curves of RM-1 tumor-bearing mice treated with CRT combined with PTX for prostate cancer.

[0035] Figures 4-9 In the study, CON represented normal mice without tumors, MOD represented saline, PTX represented 10 mg / kg paclitaxel, CP-L represented 10 mg / kg paclitaxel + 20 mg / kg crocin-γ-cyclodextrin inclusion complex, CP-M represented 10 mg / kg paclitaxel + 100 mg / kg crocin-γ-cyclodextrin inclusion complex, and CP-H represented 10 mg / kg paclitaxel + 200 mg / kg crocin-γ-cyclodextrin inclusion complex.

[0036] Figure 10 Figure 1 shows the results of the HT22 cell line experiment; A. Effect of different concentrations of PTX on relative cell viability; B. Effect of different concentrations of crocin on relative cell viability; C. Effect of different concentrations of crocin synergistic with PTX on relative cell viability; D. Cell density and morphology of each experimental group under a microscope; E. Representative results of cell apoptosis; F. Statistical results of cell apoptosis rate.

[0037] Figure 11Figure 1 shows the results of the RSC96 cell line experiment; A. Effect of different concentrations of PTX on relative cell viability; B. Effect of different concentrations of crocin on relative cell viability; C. Effect of different concentrations of crocin synergistic with PTX on relative cell viability; D. Cell density and morphology of each experimental group under a microscope; E. Representative results of cell apoptosis; F. Statistical results of cell apoptosis rate.

[0038] Figure 12 The figures show the results of the hot plate test and the new object recognition test in animal experiments. A and B show the results of the hot plate test performed on mice after the modeling process was completed. C shows the results of the new object recognition test performed on mice after the modeling process was completed. 2W represents the duration of the experiment, which is 2 weeks, corresponding to the end of the modeling phase. 4W represents the duration of the experiment, which is 4 weeks, corresponding to the end of the treatment phase.

[0039] Figure 13 Figure A shows the results of a water maze experiment in animal behavioral science; Figure B shows the changes in escape latency time for different groups from day 2 to day 6 of the experiment; Figure C shows the duration of mouse movement during the training and testing phases for each group; Figure D shows the number of times mice in each group crossed the original platform location; Figure E shows the escape latency results for each group.

[0040] Figure 14 Figure A shows the results of detecting key factors in serum after animal experiments; Figure B shows the results of detecting NO in the serum of mice in each group using a kit; Figure B shows the results of detecting NfL in the serum of mice in each group using a kit.

[0041] Figures 5-14 In the mean ± SD, n = 3, * P <0.05;** P <0.01; *** P <0.001.

[0042] Figure 15 Image showing the results of HE staining of nervous system tissues (hippocampus and spinal cord) after sampling for animal experiments.

[0043] Figure 16 Image showing the results of LFB staining of nervous system tissues (hippocampus and spinal cord) after samples were collected for animal experiments.

[0044] Figure 17 Image showing the results of HE staining of nervous system tissue (sciatic nerve) after obtaining samples for animal experiments.

[0045] Figure 18 Image showing the results of transmission electron microscopy examination of nervous system tissue (sciatic nerve) after samples were collected for animal experiments. Detailed Implementation

[0046] Unless otherwise specified, the following examples were conducted under standard conditions or conditions recommended by the manufacturer. Reagents and instruments used, unless otherwise specified, are commercially available products. Cyclodextrin purity > 98%, crocin purity > 98%.

[0047] Example 1

[0048] Preparation of CRT-α cyclodextrin inclusion complex (CRT / α-CDIC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an α-CD aqueous solution and stirred until mixed. The molar ratio of CRT to α-CD was 1:3. The mixture was sonicated for 3 hours, and the pH of the system was adjusted to 4.5–7 with 0.1 M hydrochloric acid. The mixture was then filtered through a 0.22 µM microporous membrane. The filtrate was freeze-dried to obtain the CRT / α-CD IC.

[0049] Example 2

[0050] Preparation of CRT-β-cyclodextrin inclusion complex (CRT / β-CDIC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous β-CD solution and stirred until mixed. The molar ratio of CRT to β-CD was 1:3. The mixture was sonicated for 3 hours, and the pH was adjusted to 4.5–7 with 0.1 M hydrochloric acid. The mixture was then filtered through a 0.22 µM microporous membrane. The filtrate was freeze-dried to obtain the CRT / β-CD IC.

[0051] Example 3

[0052] Preparation of CRT-HP-β cyclodextrin inclusion complex (CRT / HP-β-CD IC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous HP-β-CD solution and stirred until mixed. The molar ratio of CRT to HP-β-CD was 1:3. The mixture was sonicated for 3 hours, and the pH was adjusted to 4.5–7 with 0.1 M hydrochloric acid. The mixture was then filtered through a 0.22 µM microporous membrane. The filtrate was freeze-dried to obtain the CRT / HP-β-CD IC.

[0053] Example 4

[0054] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred until mixed. The molar ratio of CRT to γ-CD was 1:2. The mixture was sonicated for 3 hours, and the pH was adjusted to 4.5–7 with 0.1 M hydrochloric acid. The mixture was then filtered through a 0.22 µM microporous membrane. The filtrate was freeze-dried to obtain the CRT / γ-CD IC.

[0055] Example 5

[0056] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred. The molar ratio of CRT to γ-CD was 1:3. Subsequent experimental procedures and parameter settings were the same as in Example 4.

[0057] Example 6

[0058] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 50.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred. The molar ratio of CRT to γ-CD was 1:5. Subsequent experimental procedures and parameter settings were the same as in Example 4.

[0059] Example 7

[0060] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 500.0 mg of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred. The molar ratio of CRT to γ-CD was 1:3. Subsequent experimental procedures and parameter settings were the same as in Example 4.

[0061] Example 8

[0062] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 5.0 mg of cis-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred. The molar ratio of CRT to γ-CD was 1:3. Subsequent experimental procedures and parameter settings were the same as in Example 4.

[0063] Example 9

[0064] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 5.0 g of trans-CRT was dissolved in 0.1 M sodium hydroxide to obtain a CRT solution. The CRT solution was then added to an aqueous γ-CD solution and stirred. The molar ratio of CRT to γ-CD was 1:3. Subsequent experimental procedures and parameter settings were the same as in Example 4.

[0065] Example 10

[0066] Preparation of CRT-γ cyclodextrin inclusion complex (CRT / γ-CD IC): 5.0 g of trans-CRT was dissolved in anhydrous ethanol, and then the CRT solution was added dropwise to the γ-CD aqueous solution and stirred to perform inclusion. The molar ratio of CRT to γ-CD was 1:3. After filtration, the solution was freeze-dried or spray-dried to obtain CRT / γ-CD IC.

[0067] Example 11

[0068] Fourier transform infrared spectroscopy (FR-IR): 1. Experimental Methods Appropriate amounts of CRT, α-CD, HP-β-CD, γ-CD, CRT / α-CD PM (a physical mixture of CRT and α-CD, molar ratio 1:3), CRT / HP-β-CD PM (a physical mixture of CRT and HP-β-CD, molar ratio 1:3), CRT / γ-CD PM (a physical mixture of CRT and γ-CD, molar ratio 1:3), CRT / α-CD IC prepared in Example 1, CRT / HP-β-CD IC prepared in Example 3, and CRT / γ-CD IC prepared in Example 5 were respectively placed in an agate mortar, and potassium bromide powder (sample to potassium bromide mass ratio approximately 1:200) was added, mixed, ground, and pressed into tablets. Infrared spectroscopy analysis was then performed. Measurement conditions: KBr tablet method, scanning range: 4000-400 cm⁻¹ -1 , humidity 25%, room temperature.

[0069] 2. Experimental Results Figure 2 A represents the infrared characterization of the CRT / α-CD series. A-1 represents wavenumbers in the 4000-500 cm⁻¹ range. -1 The overall diagram within the range, A-2 shows the wavenumbers in the 1800-1500 cm⁻¹ range. -1 A partial view within the range. In Figure A-1, the CRT is at 1660.98 cm. -1 The characteristic peak at 1575.44 cm⁻¹ represents the C=O stretching vibration at the left and right ends of the CRT. -1 The characteristic peak at α-CD is the C=C stretching vibration in the conjugated long chain. The peak at α-CD is at 3390.24 cm⁻¹. -1 The characteristic peak at 2927.41 cm⁻¹ corresponds to the stretching vibration of OH.-1 The characteristic peak at 1639.22 cm⁻¹ corresponds to the -CH stretching vibration of -CH₂. -1 This corresponds to the stretching vibration of HOH. The overall infrared spectrum of CRT / α-CD PM is similar to that of α-CD, but differs at 1656.04 cm⁻¹. -1 1576.53cm -1 The characteristic peak of CRT is shown at 3394.11 cm⁻¹. -1 2927.45cm -1 The presence of a characteristic peak for α-CD at this location indicates that no new interaction force has formed between CRT and α-CD in the physical mixture; the characteristic peak is a simple combination of peaks from CRT and α-CD. In contrast, in the spectrum of the CRT / α-CD inclusion complex, CRT is at 1575.44 cm⁻¹. -1 The wavenumbers of the nearby characteristic peaks shift towards lower wavenumbers, and the wavenumber of the C=C stretching vibration shifts to 1543.76 cm⁻¹. -1 Compared to PM, the wavenumber of the stretching vibration of CRT shifts to 1631.85 cm⁻¹. -1 This indicates that CRT and α-CD may interact in IC. The characteristic peak shape of IC in Figure A-2 is broader than that of CRT and PM, and the peak shape variation is similar to that of CRT (1600 cm⁻¹). -1 The absence of ) may be due to the formation of interaction forces (such as hydrogen bonds) between CRT and α-CD, which provides preliminary evidence for CRT entering the α-CD cavity and forming inclusion complexes.

[0070] Figure 2 B represents the infrared characterization of the CRT / HP-β-CD series. B-1 represents the wavenumbers in the 4000-500 cm⁻¹ range. -1 The overall diagram within the range, B-2 represents wavenumbers in the 1800-1400 cm⁻¹ range. -1 A partial view within the range. In Figure B-1, the CRT is at 1660.41 cm. -1 The characteristic peak at 1575.55 cm⁻¹ corresponds to the C=O stretching vibration at the end of the CRT. -1 The characteristic peak at 3399.89 cm⁻¹ corresponds to the stretching vibration of C=C. HP-β-CD reaches this peak at 3399.89 cm⁻¹. -1 The characteristic peak at 2928.36 cm⁻¹ corresponds to the stretching vibration of OH. -1 The characteristic peak at 1640.81 cm⁻¹ corresponds to the -CH stretching vibration of -CH₂. -1 The characteristic peak at 1658.73 cm⁻¹ represents the stretching vibration of HOH. The overall infrared spectrum of the CRT / HP-β-CD PM reveals the structure of HP-β-CD glycosides, but the peak at 1658.73 cm⁻¹ is significant. -1 1575.11cm -1The characteristic peak of CRT is shown at 3399.65 cm⁻¹. -1 2927.42cm -1 The presence of a characteristic peak for HP-β-CD at [location missing] indicates that no new interaction force has formed between CRT and HP-β-CD in the physical mixture; the characteristic peak is a simple combination of the peaks from CRT and HP-β-CD. In contrast, in the spectrum of the inclusion compound, CRT is at 1575.55 cm⁻¹. -1 The wavenumbers of the nearby characteristic peaks shift towards lower wavenumbers, and the wavenumber of the C=C stretching vibration shifts to 1546.74 cm⁻¹. -1 Compared to physical mixtures, CRT showed a value of 1658.73 cm⁻¹. -1 The wavenumber of the stretching vibration at that location shifted to 1639.32 cm. -1 This indicates that CRT and CD may have interacted within the inclusion complex. The characteristic peak shape of the inclusion complex in Figure B-2 is broader than that of the CRT and physical mixture, for the same reason as the CRT / α-CD system. This provides preliminary evidence that CRT enters the HP-β-CD cavity and forms an inclusion complex.

[0071] Figure 2 C represents the infrared characterization of the CRT / γ-CD series. C-1 represents the wavenumbers in the 4000-500 cm⁻¹ range. -1 The overall diagram within the range, C-2 represents wavenumbers in the 1800-1400 cm⁻¹ range. -1 A partial view within the range. In Figure C-1, CRT is at 1660.41 cm. -1 The characteristic peak at this location corresponds to the characteristic peak of C=O at the end of the CRT, at 1577.48 cm⁻¹. -1 The characteristic peak at 3399.88 cm⁻¹ represents the stretching vibration of C=C in the conjugated long chain. -1 The characteristic peak at 2923.66 cm⁻¹ corresponds to the stretching vibration of OH. -1 The characteristic peak at 1157.08 cm⁻¹ is due to the -CH stretching vibration of -CH₂. -1 This corresponds to the sugar ring vibration of COC. The overall infrared structure of the physical mixture is similar to that of γ-CD, but at 1660.41 cm⁻¹. -1 1573.62cm -1 The characteristic peak of CRT was shown at 2923.66 cm⁻¹. -1 The presence of a characteristic γ-CD peak at this location indicates that no new interaction force has formed between CRT and γ-CD in the physical mixture; the characteristic peak is a simple combination of CRT and γ-CD peaks. In the spectrum of the inclusion compound, CRT is at 1577.48 cm⁻¹. -1 The characteristic peak at that point shifted towards lower wavenumbers, and the wavenumber of the C=C stretching vibration shifted to 1544.72 cm⁻¹. -1 Compared to physical mixtures, CRT showed a value of 1660.41 cm⁻¹.-1 The wavenumber of the stretching vibration at that location shifted to 1637.26 cm. -1 This indicates that CRT and CD may interact within the inclusion complex. In partial figure C-2, the characteristic peak shape of the inclusion complex is also broader than that of the CRT and physical mixture, for the same reason as the CRT / α-CD system. The broadened peak shape of the inclusion complex and the characteristic peak of CRT (1600 cm⁻¹) are related. -1 The absence of ) initially indicates the successful fabrication of CRT / γ-CD IC.

[0072] Hydrogen bonding causes the stretching vibrations of hydrogen-bonded groups to shift significantly towards lower wavenumbers, while also broadening the characteristic peaks. After CRT and CD form inclusion complexes, the characteristic peaks of CRT shift significantly towards lower wavenumbers and become broader in all three inclusion complexes. These changes are likely due to inclusion forces such as hydrogen bonds and van der Waals forces between CRT and CD, providing a basis for the successful formation of CRT / CD ICs.

[0073] Example 12

[0074] In vitro dissolution test: 1. Experimental Methods Weigh appropriate amounts of CRT, CRT / γ-CD PM (CRT to γ-CD molar ratio of 1:3), and CRT / γ-CD IC (prepared in Example 9), three portions of each. Dissolution conditions: The dissolution medium was pH 6.86 phosphate buffer (900 mL), the temperature was set to 37 ± 0.5 °C, and the rotation speed was 100 r / min. After the dissolution medium reached 37 °C, the samples were added. 5 mL samples were taken at 5, 10, 15, 30, 45, and 60 min, and the same volume of dissolution medium preheated to 37 °C was added each time. The solution was filtered through a 0.22 μm microporous membrane and appropriately diluted. The concentration of CRT in the solution was calculated using HPLC. The HPLC chromatogram of the CRT standard is shown below. Figure 1 As shown.

[0075] 2. Experimental Results Dissolution test results are as follows Figure 3As shown in the dissolution profiles, the cumulative dissolution rate of the active pharmaceutical ingredient (CRT) was 1.32% at 30 min and only 1.77% at 60 min. For CRT / γ-CD PM, the dissolution rate of CRT in CRT / γ-CD PM was slightly higher than that of the active pharmaceutical ingredient (API). At 60 min, the cumulative dissolution rate of CRT in CRT / γ-CD PM was 2.57%. However, the cumulative dissolution rate of CRT in CRT / γ-CD IC reached 98.57% at 5 min. CRT in CRT / γ-CD IC was almost completely dissolved at 5 min. These results indicate that CRT encapsulation with CD significantly improves the water solubility and dissolution rate of the drug CRT, increasing the release rate and extent of CRT.

[0076] Example 13

[0077] Pharmacodynamic and safety evaluation of the CRT-γ cyclodextrin inclusion complex combined with paclitaxel injection for prostate cancer treatment according to Example 9: 1. Animal model: C57BL / 6 male mice (20±2g) were subcutaneously inoculated with RM-1 prostate cancer cells (1×10⁻⁶). 6 (each tumor cell / item), until the tumor volume reaches 100 mm. 3 Grouped later.

[0078] 2. Experimental groups (n=6) ① Normal control group (CON): Normal mice without tumors. On day 3, mice were given saline via intraperitoneal injection and oral gavage. The volume and frequency of intraperitoneal injection of saline were consistent with that of paclitaxel, and the volume and frequency of oral gavage of saline were consistent with that of crocin-γ-cyclodextrin inclusion complex. ② Model control group (MOD): Mice were given saline via intraperitoneal injection and oral gavage on the 3rd day after tumor inoculation. The volume and frequency of intraperitoneal injection of saline were consistent with that of paclitaxel, and the volume and frequency of oral gavage of saline were consistent with that of crocin-γ-cyclodextrin inclusion complex. ③ Paclitaxel treatment group (PTX): Mice were given the chemotherapy drug paclitaxel via intraperitoneal injection on the 3rd day after tumor inoculation. The injection dose was 10 mg / kg per mouse, and the injection frequency was once every 3 days. ④ Combined low-dose treatment group (CP-L): Mice were given paclitaxel via intraperitoneal injection and crocin-γ-cyclodextrin inclusion complex via oral gavage on the 3rd day after tumor inoculation. The paclitaxel injection dose was 10 mg / kg per mouse, and the injection frequency was once every 3 days. The oral dose of crocin-γ-cyclodextrin inclusion complex was 20 mg / kg based on crocin, and the administration frequency was once a day. ⑤ Combined drug medium-dose treatment group (CP-M): Mice were given paclitaxel by intraperitoneal injection and crocin-γ-cyclodextrin inclusion complex by oral gavage on the 3rd day after tumor inoculation. The paclitaxel injection dose was 10 mg / kg per mouse, and the injection frequency was once every 3 days. The oral dose of crocin-γ-cyclodextrin inclusion complex was 100 mg / kg based on crocin, and the administration frequency was once a day. ⑥ The high-dose combined treatment group (CP-H) was treated with paclitaxel via intraperitoneal injection on the 3rd day after tumor inoculation and with crocin-γ-cyclodextrin inclusion complex via oral gavage. The paclitaxel injection dose was 10 mg / kg per mouse, and the injection frequency was once every 3 days. The oral dose of crocin-γ-cyclodextrin inclusion complex was 200 mg / kg based on crocin, and the administration frequency was once a day.

[0079] Tumor volume was recorded every 3 days during the experiment in each group, measured along the largest axis (L) and the diameter (W) perpendicular to that axis. The calculation method was: Tumor volume = (L × W) 2 ) / 2.

[0080] When the tumor growth endpoint was reached, 0.5 mL of peripheral blood was collected in an anticoagulant tube for routine blood tests (WBC, RBC, PLT) to assess blood toxicity. Mice were then sacrificed, subcutaneous tumors were removed and weighed, and the tumor inhibition rate was calculated to assess antitumor efficacy. Tissues from the heart, liver, spleen, lungs, kidneys, brain, and testes were collected for H&E staining and pathological scoring to assess the toxicity to the major organs of tumor-bearing mice.

[0081] During the treatment period, mice were observed daily for their response to treatment, activity level, mental state, and hair loss. The significance of combined drug use was comprehensively evaluated based on the weight change curve, tumor inhibition rate, blood routine indicators, and pathological tissue sections of various organs.

[0082] 3. Experimental Results 3.1 Treatment response and weight changes in mice: During the administration period, the paclitaxel group experienced greater adverse reactions, with reduced activity and lethargy in mice. The model control group showed less lustrous fur and less activity compared to the paclitaxel-treated group. The combined treatment group showed smoother fur, better mental state, and higher activity than the paclitaxel-only group. Comparison of body weight trends showed that the body weight of mice in the model group and the combined treatment group increased gradually, while the weight gain in the paclitaxel group and the combined treatment group was slower. This may be related to the impact of paclitaxel accumulation on the mice's well-being in the later stages of administration. (See below) Figure 4 .

[0083] 3.2 Inhibitory effect on RM-1 in prostate cancer: Animals were euthanized by cervical dislocation after blood collection, and tumor tissue was quickly removed and weighed. The tumor inhibition rate was calculated using the following formula: Tumor inhibition rate = [1 - average tumor weight in the treatment group / average tumor weight in normal saline] × 100%.

[0084] like Figure 5 As shown in Figure A, compared with the MOD group, the tumor volume in each treatment group was significantly reduced, demonstrating that PTX and its combination with CRT can effectively reduce tumor volume. Figure 5 As shown in Figure B, in the initial stage of the experiment (day 0-1), there was almost no difference in tumor size and growth rate among the groups of mice. From day 2 to 7, the tumors in the model group grew rapidly, while the tumors in the drug-treated groups grew more slowly. At day 7 of drug administration, compared with the model group, the tumor volume in each drug-treated group was significantly reduced (…). P <0.001), and the tumor volume was smallest in the CP-H group. For example... Figure 5 As shown in Figure C, compared with the model group, the tumor weight in each treatment group was significantly reduced ( P <0.01, P <0.001), the CP-H group had the lowest tumor weight and it was significantly lower than that of the PTX group ( P <0.05). For example... Figure 5 As shown in Figure D, the tumor inhibition rates of the drug groups were: PTX group 49.18%, CP-L group 45.24%, CP-M group 44.08%, and CP-H group 66.01%, with the CP-H group showing the highest tumor inhibition rate. These results indicate that high-dose CRT combined with PTX achieves the best therapeutic effect for prostate cancer, and its efficacy is significantly superior to paclitaxel alone.

[0085] 3.3 Histopathological sections of RM-1 prostate cancer cells: like Figure 6 As shown in Figure A, after H&E staining, tumor tissue sections observed under a 40x light microscope revealed that the tumor cells in the model group were round, oval, or polygonal, relatively large, with deeply stained nuclei, large nuclear cytoplasm, frequent mitotic activity, and less nuclear pyknosis. In all treatment groups, partial necrosis of cells was observed in the tumor tissues, with uneven morphology and size, and increased infiltration of immune cells, indicating that each treatment group had a certain therapeutic effect on the tumor. In the CP-H group, tumor cells showed extensive necrosis, almost losing their cellular morphology, with blurred edges, loose arrangement, more nuclear fragmentation and pyknosis, crescent-shaped chromatin, reduced nuclear-cytoplasmic ratio, fewer cell division phases, and varying degrees of swelling, vacuolation, and necrosis in the tumor tissue. This indicates that high-dose crocin combined with paclitaxel has the best therapeutic effect on tumors in tumor-bearing mice. Figure 6 As shown in B-6C, representative images reveal a large number of brown positive signals in the tumor tissues of all treatment groups, while the model group shows fewer positive signals; TUNEL staining results show that the apoptosis index of each treatment group is significantly higher than that of the MOD group ( P <0.001); Meanwhile, compared with the PTX group, the apoptosis rate of tumor tissue in the CP-H group was significantly increased (9.34% vs 17.84%). P <0.01).

[0086] 3.4 Blood biochemistry analysis results: Myelosuppression is one of the side effects of PTX, mainly manifested as a decrease in neutrophils, platelets and red blood cells, with leukopenia being the most significant.

[0087] like Figure 7 As shown in A-7D, compared with the MOD group, the PTX group showed significantly decreased WBC, Mon, Gran, and PLT levels, while significantly increased MPV, indicating that PTX caused myelosuppression in prostate cancer-bearing mice. However, compared with the PTX group, the addition of crocin-γ-cyclodextrin inclusion complex significantly increased WBC, Mon, Gran, and PLT levels and decreased MPV, indicating that crocin can inhibit the myelosuppression caused by PTX in cancer mice and significantly increase the number of immune cells. The results indicate that the combined treatment of CRT and PTX can suppress the myelosuppression side effect caused by PTX treatment in prostate adenocarcinoma-bearing mice and increase the number of immune cells.

[0088] 3.5 Histopathological staining of major organs in tumor-bearing mice: Besides bone marrow suppression, paclitaxel also has neurotoxicity and other side effects such as damage to organs like the heart, lungs, liver, and kidneys. Based on a relatively ideal anti-tumor effect, the damage to major organs in mice was observed using H&E sections, and the ability of crocin to alleviate the toxic side effects of paclitaxel was evaluated.

[0089] like Figure 8As shown, in mouse brain slices, compared with the CON group, the MOD group showed mild neuronal necrosis in the hippocampus, manifested as nuclear pyknosis of nerve cells; in the PTX group, in addition to more cells showing nuclear pyknosis, more neurons showed atrophy and vacuolation, i.e., neuronal degeneration; in the combined treatment groups, all three groups showed improvement in neuronal necrosis and deformation in the PTX group, with the CP-H group showing the mildest hippocampal damage. In heart tissue, compared with the CON group, the PTX group showed slightly irregular arrangement of myocardial fibers, slight edema of the myocardial interstitium, and occasional slight condensation of individual nuclear chromatin; in the combined treatment groups, all three groups showed tightly and regularly arranged myocardial fibers, no interstitial edema, and uniform morphology of myocardial cell nuclei without abnormal condensation, indicating that the heart tissue morphology has returned to normal. In liver tissue, the PTX group showed irregular morphology of a few hepatocytes, slightly loose arrangement of hepatic cords, and occasional scattered lymphocyte infiltration; in the combined treatment groups, all three groups showed regular morphology of hepatocyte nuclei, neat arrangement of hepatic cords, and close resemblance to normal liver tissue, indicating that the liver has returned to normal tissue structure. In mouse spleen sections, compared to the CON group, both the MOD and PTX groups showed loss of the boundary between the red and white pulp of the spleen, indicating disruption of the normal spleen structure. However, in the combined treatment group, the boundary between the red and white pulp was restored, indicating recovery of the normal spleen structure. In mouse lung sections, compared to the CON group, the PTX group showed damage to the pseudostratified columnar epithelial cells of the bronchi. Compared to the PTX group, the bronchial damage in the combined treatment group significantly decreased with increasing crocin dosage until normal tissue structure was restored. In mouse kidney sections, compared to the CON group, the PTX group showed glomerular vacuolation and separation from the capsule. Compared to the PTX group, the combined treatment group showed restored glomerular separation from the capsule and reduced glomerular vacuolation. In mouse testicular sections, compared with the CON group, the PTX group showed more severe damage to the Sertoli cells, detachment of the basement membrane, disordered cell arrangement, and enlargement of the seminiferous tubule lumen. Compared with the PTX group, the combined drug group showed reduced testicular damage and increased sperm count.

[0090] Based on the above results, in this study we found that paclitaxel injection caused damage to the brain, heart, liver, spleen, lungs, kidneys and testes in mice with prostate cancer. Crocin was able to improve or even completely reverse the organ and tissue damage caused by paclitaxel.

[0091] 3.6 Survival curves of tumor-bearing mice: Survival rate is a core indicator that comprehensively reflects tumor progression, metastasis, host immune status, and treatment toxicity. For example... Figure 9As shown in Table 1, all treatment groups showed significant differences compared to the MOD group: PTX group (P=0.0016); CP-L group (P=0.0189); CP-M group (P<0.001); CP-H group (P<0.001); compared to the PTX group, only the CP-H group showed a significant difference (P=0.001). Compared to the MOD group, the median survival time of all treatment groups was prolonged, with the CP-H group having the longest median survival time. These results indicate that compared to PTX alone, the addition of crocin-γ-cyclodextrin inclusion complex can increase the survival time of prostate cancer-bearing mice, and high doses of crocin can significantly increase the survival time of mice, suggesting that the combination of CRT and PTX is a superior treatment option.

[0092] Table 1 Median survival time of tumor-bearing mice

[0093] Paclitaxel's adverse reactions, such as bone marrow suppression, allergies, and organ damage, severely impact chemotherapy efficacy and patients' quality of life. Synergistic sensitization to reduce paclitaxel toxicity and enhance chemotherapy efficacy is currently a hot research topic. In this study, we investigated whether the combined use of crocin and paclitaxel in an RM-1 prostate cancer mouse model offered a synergistic effect while simultaneously reducing toxicity.

[0094] Example 14

[0095] CRT pretreatment can improve PTX-induced HT22 cell damage: 1. Experimental Methods Mouse hippocampal neuronal HT22 cell line was routinely cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C, 5% CO2, and saturated humidity. Cells were seeded into 96-well plates, and after complete adhesion, the medium was changed to a drug-containing medium. Following treatment, the medium was replaced with 10% CCK8 reagent, and the cells were incubated for 1.5 hours under the aforementioned conditions. Measurements were then taken at 450 nm using a microplate reader, and data were processed using Graphpad Prism.

[0096] Apoptosis levels were assessed using the AV-PI apoptosis detection kit. Cells were loaded at 2 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates. After complete cell adhesion, the cells were treated with [drug name missing]. After treatment, the culture medium and adherent cells were collected, washed with PBS, resuspended in the buffer solution provided in the kit, and incubated with Annexin V reagent for 10 min at room temperature in the dark. PI reagent was added 5 min before flow cytometry analysis using a BD Biosciences flow cytometer. Apoptosis levels were quantified using FlowJo version 10 software.

[0097] 2. Experimental Results like Figure 10 As shown in Figure A, HT22 cells were treated with different concentrations of paclitaxel-containing culture medium for 24 hours, and the proliferation toxicity was detected and the results were statistically analyzed. The experimental results showed that the cell viability gradually decreased with increasing paclitaxel concentration. Significant cell damage was observed in the 100 nM paclitaxel treatment group, and this concentration of paclitaxel was used as the modeling condition in subsequent experiments. Figure 10 As shown in Figure B, HT22 cells were treated with different concentrations of crocin-containing culture media for 24 hours, and the proliferation toxicity was tested and the results were statistically analyzed. The experimental results showed that the cell survival rate in all treatment groups with crocin concentrations below 200 μM was greater than 80%, which proves the safety of crocin for HT22 cells; Figure 10 As shown in Figure C, HT22 cells were pretreated with different concentrations of crocin for 24 h, followed by treatment with 100 nM paclitaxel for 24 h, and the results were statistically analyzed. The experimental results show that pretreatment of HT22 cells with crocin can reduce cell damage caused by paclitaxel; Figure 10 As shown in Figure D, the cell density and morphology of each experimental group are observed under a microscope. Compared with the control group, the paclitaxel group (100 nM) showed decreased cell density, reduced polygonal tentacles, and significant cell damage. Compared with the paclitaxel group, the treatment groups (150 μM: CRT 150 μM + PTX 100 nM, 200 μM: CRT 200 μM + PTX 100 nM) showed improved cell state in a concentration-dependent manner.

[0098] For the HT22 cell line, such as Figure 10 Figure E shows representative results of apoptosis. The paclitaxel group showed a significant increase in both early and late apoptotic cells, while the crocin pretreatment group showed a decrease in apoptotic cells, demonstrating that crocin can alleviate paclitaxel-induced apoptosis. Figure 10F represents the statistical results of cell apoptosis rate. Compared with the control group, the cell apoptosis rate in the paclitaxel group was significantly increased, and the difference between the two groups was statistically significant. Compared with the paclitaxel group, the cell apoptosis rate in the treatment groups (Low: CRT 150μM + PTX 100nM, High: CRT 200μM + PTX 100nM) was reduced, and the high-dose group had a more significant effect on reducing cell apoptosis than the low-dose group. All results were statistically significant.

[0099] Example 15

[0100] CRT pretreatment can improve PTX-induced RSC96 cell damage: 1. Experimental Methods Rat Schwann RSC96 cell lines were routinely cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells adhered to the culture medium at 37°C, 5% CO2, and saturated humidity. Cells were seeded into 96-well plates and, after complete adhesion, were treated with drug-containing medium. Following treatment, the medium was changed to contain 10% CCK8 reagent, and the cells were incubated for 1.5 hours under the aforementioned conditions. Measurements were then taken at 450 nm using a microplate reader, and data were processed using Graphpad Prism.

[0101] Apoptosis levels were assessed using the AV-PI apoptosis detection kit. Cells were loaded at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. After complete cell adhesion, the cells were treated with [drug name missing]. After treatment, the culture medium and adherent cells were collected, washed with PBS, resuspended in the buffer solution provided in the kit, and incubated with Annexin V reagent for 10 min at room temperature in the dark. PI reagent was added 5 min before flow cytometry analysis using a BD Biosciences flow cytometer. Apoptosis levels were quantified using FlowJo version 10 software.

[0102] 2. Experimental Results like Figure 11 As shown in Figure A, RSC96 cells were treated with different concentrations of paclitaxel-containing culture medium for 24 hours, and the proliferation toxicity was detected and the results were statistically analyzed. The experimental results showed that the cell viability gradually decreased with increasing paclitaxel concentration. The 100 nM paclitaxel treatment group showed a significant decrease in cell viability, and this concentration of paclitaxel was used as the modeling condition in subsequent experiments. Figure 11As shown in Figure B, RSC96 cells were treated with different concentrations of crocin-containing culture medium for 24 hours, and the proliferation toxicity was tested and the results were statistically analyzed. The experimental results showed that the cell survival rate in all treatment groups with crocin concentrations below 200 μM was greater than 80%, which proves the safety of crocin for HT22 cells; Figure 11 As shown in Figure C, RSC96 cells were treated with different concentrations of crocin in combination with 100 nM paclitaxel for 24 h, and the results were statistically analyzed. The experimental results showed that the combined use of crocin could reduce the decrease in RSC96 cell viability caused by paclitaxel. Figure 11 As shown in Figure D, the cell density and morphology of each experimental group are shown under a microscope. Compared with the control group, the paclitaxel group (100 nM) showed decreased cell density, abnormal cell morphology, and significant cell damage. Compared with the paclitaxel group, the treatment groups (50 μM: CRT 50 μM + PTX 100 nM, 100 μM: CRT 100 μM + PTX 100 nM, 200 μM: CRT 200 μM + PTX 100 nM) showed restored cell state in a concentration-dependent manner.

[0103] For the RSC96 cell line, such as Figure 11 Figure E shows representative results of cell apoptosis. The paclitaxel group showed a significant increase in both early and late apoptotic cells, while the crocin-treated group showed a decrease in apoptotic cells, demonstrating that the combined use of crocin can alleviate paclitaxel-induced cell apoptosis. Figure 11 F represents the statistical results of cell apoptosis rate. Compared with the control group, the paclitaxel group showed a significant increase in cell apoptosis rate, with a statistically significant difference. Compared with the paclitaxel group, the treatment groups (Low: CRT 50μM + PTX 100nM, Medium: CRT 100μM + PTX 100nM, High: CRT 200μM + PTX 100nM) showed a decrease in cell apoptosis rate. Moreover, the high-dose group had a more significant effect on reducing cell apoptosis compared with the medium and low-dose groups, and all results showed statistically significant differences.

[0104] Example 16

[0105] CRT-γ cyclodextrin inclusion complex can improve PTX-induced neurological damage in mice: 1. Experimental Methods A nervous system injury model was established in mice by intraperitoneal injection of paclitaxel. Different doses of aqueous solutions of the CRT-γ cyclodextrin inclusion complex prepared according to Example 9 were used for treatment. After the model was established, a hot plate test was performed to check whether the model was successful. After the treatment, the hot plate test, new object recognition test and water maze test were performed to verify the therapeutic effect of the cyclodextrin inclusion complex.

[0106] Nervous system injury modeling protocol: Intraperitoneal injection of PTX at a dose of 6 mg / kg, every other day for two consecutive weeks.

[0107] Treatment protocol experimental groups (n=6): The CON group consisted of normal mice that had not undergone modeling. During the treatment phase, the mice were given saline orally by gavage. The volume and frequency of the saline orally administered saline were consistent with those of the crocin-γ-cyclodextrin inclusion complex. The PTX group consisted of mice with a nervous system injury model. During the treatment phase, they were given saline orally by gavage. The volume and frequency of the saline orally administered saline were consistent with those of the crocin-γ-cyclodextrin inclusion complex. The LOW group consisted of mice with a nervous system injury model. During the treatment phase, CRT-γ cyclodextrin inclusion complex was administered orally by gavage. The oral dose of CRT-γ cyclodextrin inclusion complex was 6 mg / kg based on CRT, and the administration frequency was once daily. The HIGH group consisted of mice with a nervous system injury model. During the treatment phase, CRT-γ cyclodextrin inclusion complex was administered orally by gavage. The oral dose of CRT-γ cyclodextrin inclusion complex was 20 mg / kg (calculated as CRT), and the administration frequency was once daily.

[0108] 2. Animal behavior After modeling and treatment, mice underwent a hot plate test. The hot plate temperature was set at 55±0.5℃, and the baseline pain threshold (5~30s was considered acceptable) was measured. Abnormal individuals were eliminated. During the experiment, the mouse was gently placed in the center of the hot plate, and the timer was started immediately. The reaction time was recorded when the mouse exhibited licking its hind paws or jumped off the hot plate.

[0109] After treatment, mice underwent a new object recognition experiment in a square open field (40×40×40cm). The experimental procedure included adaptation (5 min), familiarization (two identical objects, 5 min), and testing (a new object replacing the original object, 5 min), with an interval of 24 h. The time the mice's noses were in contact with the objects was recorded, and the recognition index (new object exploration time / total exploration time × 100%) was calculated. The experiment was conducted under low light and low noise conditions. The spontaneous exploration tendency of mice reflected their recognition and memory function. Data were statistically analyzed and compared to the difference in exploration time between new and old objects.

[0110] After treatment, mice underwent a water maze test to assess their spatial learning and memory abilities. The experimental setup consisted of a circular pool (120 cm in diameter and 60 cm in height) at a water temperature of 22 ± 1 °C. A platform (10 cm in diameter) was hidden 1 cm underwater, and visual cues were fixed to the pool wall. The experimental procedure included a 1-day visible platform period, a 5-day training period (4 times daily, maximum 60 seconds per session), and a 7-day exploration test (platform removed, 60 seconds). The time mice spent in the target quadrant, the number of times they crossed the original platform location, and the escape latency were recorded. The experiment was conducted under soundproof and temperature-controlled conditions. This paradigm reflects hippocampal-dependent memory function through the mouse's spatial navigation strategy.

[0111] 3. Experimental Results 3.1 General observation of animal behavior: Mice were observed for activity and mental state 1 hour after each drug administration. During the modeling period, compared with the control group, the modeling group showed reduced activity, lethargy, and decreased food intake; during the treatment period, the activity and food intake of the treatment group showed slight improvement.

[0112] 3.2 Hot plate test: After the mold is made, a hot plate test is performed, such as... Figure 12 As shown in Figure A, the heat-shrinking threshold in the paclitaxel group was significantly lower than that in the control group, proving that paclitaxel caused neurological damage in mice, and the model was established. After modeling, a hot plate test was performed, as shown in Figure A. Figure 12 As shown in Figure B, the heat shrinkage threshold was significantly lower in the paclitaxel group compared to the blank group, proving that paclitaxel caused neurological damage in mice. Compared to the model group, the heat shrinkage threshold was significantly increased in the treatment group, indicating that the crocin-γ-cyclodextrin inclusion complex prepared in Example 9 has a restorative and therapeutic effect on this damage.

[0113] 3.3 New Object Recognition Experiment Detection: After the experiment, mice were tested on recognizing new objects, such as... Figure 12 As shown in Figure C, the cognitive index was significantly lower in the paclitaxel group compared to the control group, while the cognitive index was increased in the treatment group compared to the model group, demonstrating the protective effect of crocin against paclitaxel-induced central nervous system damage.

[0114] 3.4 Water Maze Behavioral Experiment Testing: After treatment, mice underwent a water maze test for a total of 7 days, consisting of a 1-day plateau phase, a 5-day training phase, and a 1-day testing phase. Results were statistically analyzed after the experiment. Figure 13 As shown in Figure A, the escape latency of mice in all groups decreased with increasing training days. On the last day of the training period, the escape latency of the blank group mice was statistically different from that of the other groups. Figure 13Figure B shows representative trajectory diagrams of mice during the training and testing periods. During the training period, the control group performed best. Compared to the control group, the paclitaxel group exhibited chaotic movement trajectories. Compared to the paclitaxel group, the movement trajectories of the treatment groups showed a trend towards rationality. During the testing period, the control group mice had the clearest and most rational trajectories, crossing the original platform location the most times. Compared to the control group, the paclitaxel group mice had chaotic trajectories and crossed the original platform location fewer times. In contrast, the behavioral trajectories of the treatment groups showed a trend towards rationality, with trajectories denser in the target quadrant and more frequent crossings of the original platform location. Figure 13 As shown in C, the time mice spent in the target quadrant in each group was statistically analyzed. Compared with the control group, the time mice spent in the target quadrant was reduced in the paclitaxel group, while the time spent in the target quadrant was increased in each treatment group compared with the paclitaxel group. Figure 13 As shown in D, the number of times mice crossed the original platform location was counted in each group. Compared with the control group, the number of times mice crossed the original platform location was significantly reduced in the paclitaxel group. Compared with the paclitaxel group, this indicator was significantly increased in each treatment group, with a statistically significant difference between the high-dose treatment group and the paclitaxel group. Figure 13 Figure E shows the escape latency results for each group during the test period. Compared with the control group, the escape latency of the paclitaxel group was significantly increased, while the escape latency of each treatment group was significantly shortened compared with the paclitaxel group. The statistical results of the three indicators—time spent in the target quadrant, number of times the original platform was crossed, and escape latency—all demonstrate that paclitaxel chemotherapy causes cognitive impairment in the central nervous system of mice, and that crocin cyclodextrin inclusion complex can improve this damage.

[0115] 3.5 Serum NO level detection: The serum of mice in each group was tested using a NO assay kit. NO is a classic indicator of inflammation and oxidative stress, such as... Figure 14 As shown in Figure A, compared with the control group, the serum NO level in the paclitaxel group was significantly increased, proving that paclitaxel injection caused inflammation and oxidative damage in mice. Compared with the paclitaxel group, the serum NO level in each treatment group was reduced, proving that the prepared crocin cyclodextrin inclusion complex can improve the neurological damage caused by paclitaxel.

[0116] Serum NfL factor detection: The serum of mice in each group was measured using an NfL kit. NfL is a classic marker of neurological injury, such as... Figure 14 As shown in Figure B, the serum NfL level in the paclitaxel group was significantly increased compared with the control group, proving that paclitaxel injection caused neurological damage in mice. Compared with the paclitaxel group, the serum NfL level in each treatment group was decreased, proving that the prepared crocin cyclodextrin inclusion complex can improve the neurological damage caused by paclitaxel.

[0117] 3.6 Results of HE staining of hippocampus and spinal cord sections: HE-stained sections were prepared from nerve tissue samples taken from mice in each group. For hippocampal tissue, such as... Figure 15 As shown, the control group had a greater number of hippocampal neurons with a more spread-out morphology. In contrast, the paclitaxel group had a reduced number of hippocampal neurons with deeper nuclear staining, pyknosis, and a clear trend of neuronal apoptosis. For spinal cord tissue, such as... Figure 15 As shown, in the blank group, the boundary between gray and white matter was clear, and the neurons in the gray matter were clearly morphological and evenly distributed. In contrast, in the paclitaxel group, the morphological structure of the white matter was damaged and the number of neurons in the gray matter was reduced. Compared with the paclitaxel group, the low-dose treatment group did not improve the damage to the white matter, but the number of neurons in the gray matter increased, but the cell nuclei were stained more deeply, showing a state of nuclear condensation and a tendency for cell apoptosis. Compared with the paclitaxel group, the high-dose treatment group had a clear boundary between gray and white matter, the morphological structure of the white matter was intact, and the number of neurons in the gray matter increased.

[0118] 3.7 Results of LFB staining of hippocampus and spinal cord sections: LFB-stained sections were prepared from nerve tissue samples taken from mice in each group. For hippocampal tissue, such as... Figure 16 As shown, the hippocampal tissue in the blank group was densely and completely stained, with nerve fibers arranged neatly and tightly. In the paclitaxel group, nerve fibers were loosely arranged. Compared to the paclitaxel group, the treatment group showed denser staining, with nerve fibers exhibiting a trend towards tighter arrangement. For spinal cord tissue, such as… Figure 16 As shown, the nerve fibers in the blank group were tightly arranged, while those in the paclitaxel group were loosely and disordered, with blurred boundaries between gray and white matter. Compared with the paclitaxel group, the nerve fiber arrangement in each treatment group showed a trend of becoming more orderly and denser.

[0119] 3.8 Results of HE staining of sciatic nerve sections: For the sciatic nerve, such as Figure 17 As shown, the blank group had intact morphological structure. In terms of cross-sectional section results, the morphological structure of the paclitaxel group was severely damaged, the morphological structure of the low-dose treatment group was slightly improved, and the morphological structure of the high-dose group showed significant improvement. In terms of longitudinal section results, the nerve fibers in the paclitaxel group were obviously broken and disordered. The degree of nerve fiber breakage in the low-dose group was significantly improved, but the disordered nerve fiber arrangement still existed. In comparison, the high-dose group showed a significant therapeutic effect and significantly improved the nerve fiber breakage and disordered arrangement caused by paclitaxel.

[0120] 3.9 Transmission electron microscopy results of sciatic nerve sections: Transmission electron microscopy sections were prepared from the sciatic nerve tissue of each group of mice, such as... Figure 18As shown, the myelin sheath in the blank group was dense and intact, and the myelin sheaths were arranged neatly and tightly. In the paclitaxel group, the myelin sheath walls were loosely arranged and there was obvious tissue damage. Compared with the paclitaxel group, the myelin sheath walls in the treatment group were denser, and the arrangement of the myelin sheaths showed a trend of becoming tighter.

[0121] Conclusions: First, this invention efficiently and in large quantities prepared CRT cyclodextrin inclusion complexes. Infrared spectroscopy confirmed the successful preparation of the CRT cyclodextrin inclusion complexes, and in vitro dissolution experiments demonstrated that the CRT cyclodextrin inclusion complexes significantly improved the solubility of CRT. Second, the application of the CRT cyclodextrin inclusion complexes in combination with paclitaxel injection in cancer treatment was investigated. In the RM-1 mouse xenograft model of prostate cancer, the combination of crocin and paclitaxel significantly increased the tumor inhibition rate, demonstrating a significant synergistic effect. Meanwhile, in the safety assessment, the animals in the combination therapy group were in better condition than those in the paclitaxel monotherapy group; weight data showed that after combining crocin with paclitaxel, there was no further decrease in weight, demonstrating the advantage of reducing paclitaxel toxicity; in blood routine tests, the paclitaxel monotherapy group showed obvious bone marrow suppression, while the combination therapy group significantly improved the decrease in white blood cell count, neutrophil count, and platelet count, alleviating the bone marrow suppression caused by paclitaxel; in the H&E staining results of tissues and organs, the tissues and organs in the paclitaxel monotherapy group showed varying degrees of necrosis or apoptosis, indicating that paclitaxel has certain organ toxicity, and the combination therapy group alleviated the damage caused by paclitaxel.

[0122] Because tumor-bearing mouse models require consideration of animal ethics, euthanasia is necessary when the tumor volume exceeds a certain range. Therefore, building on the demonstration that CRT cyclodextrin inclusion complex can significantly improve organ toxicity caused by paclitaxel, we next investigated its effect on alleviating neurological damage induced by paclitaxel chemotherapy in normal mice without tumors. In vitro and in vivo results showed that crocin cyclodextrin inclusion complex can alleviate neurological damage induced by paclitaxel chemotherapy, significantly improving both central nervous system cognitive impairment and peripheral nervous system pain. This can reduce the likelihood of paclitaxel chemotherapy interruption due to severe neurological side effects, thereby enhancing the anti-tumor effect of paclitaxel in clinical applications and improving the quality of life for cancer patients.

[0123] In summary, the combined use of paclitaxel and crocin reduces the adverse reactions of paclitaxel, allowing more patients to benefit from its clinical use and demonstrating greater clinical value.

Claims

1. The use of crocin or a pharmaceutically acceptable salt thereof or a cyclodextrin inclusion complex thereof in the preparation of a medicament for use in combination with an antitumor chemotherapy drug, characterized in that, The combined administration is intended to: (a) enhance the antitumor efficacy of the antitumor chemotherapeutic agent; and / or (b) reduce the toxic side effects caused by the antitumor chemotherapeutic agent.

2. The application according to claim 1, characterized in that, Enhanced anti-tumor efficacy manifests as improved survival time and / or increased tumor inhibition rate in subjects.

3. The application according to claim 1 or 2, characterized in that, Reducing toxic side effects manifests as alleviating one or more of the following toxicities: bone marrow suppression, neurotoxicity, or other organ toxicity.

4. The application according to claim 2, characterized in that, The enhanced anti-tumor effects also include promoting the functional and morphological recovery of the spleen damaged by tumors or chemotherapy.

5. The application according to claim 1, characterized in that, The cyclodextrin inclusion complex of crocin is selected from any one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and α-cyclodextrin.

6. The application according to claim 1, 2, 4 or 5, characterized in that, The antitumor chemotherapy drug is selected from platinum-based drugs, taxanes, topoisomerase inhibitors, antimetabolites, alkylating agents, or plant alkaloids; preferably one or more of cisplatin, oxaliplatin, carboplatin, paclitaxel, docetaxel, irinotecan, topotecan, 5-fluorouracil, gemcitabine, cyclophosphamide, and doxorubicin; the targeted tumors are prostate cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, or ovarian cancer.

7. The application according to claim 1, 2, 4 or 5, characterized in that, In combination dosing regimens: Crocin or its pharmaceutically acceptable salts or its cyclodextrin inclusion complexes are prepared into pharmaceutical formulations; Preparation of antitumor chemotherapy drug formulations; The two preparations mentioned above can be used in combination.

8. The application according to claim 1, 2, 4 or 5, characterized in that, In the crocin cyclodextrin inclusion complex, the molar ratio of crocin to cyclodextrin is 1:1 to 1:15, preferably crocin to cyclodextrin = 1:

3.

9. The application of crocin or its pharmaceutically acceptable salt or its cyclodextrin inclusion complex in combination with antitumor chemotherapy drugs in the preparation of organ-protective drugs, characterized in that, It is used to restore the blurred boundary between the red and white pulp of the spleen caused by tumors to enhance anti-tumor effects and increase the tumor inhibition rate; it is used to improve the necrosis or apoptosis of hippocampal and spinal cord neurons and the disordered arrangement of nerve fibers to improve central nervous system cognitive impairment; it is used to improve sciatic nerve fiber rupture and demyelination to alleviate peripheral nervous system pain and reduce neurotoxicity; it is used to improve the decrease in white blood cell count, neutrophil count, and platelet count to reduce bone marrow suppression; it is used to improve the necrosis or apoptosis of seminiferous tubules in the testes to reduce testicular damage.

10. The application according to claim 9, characterized in that, Crocin or its pharmaceutically acceptable salts or cyclodextrin inclusion complexes, when used in combination with antitumor chemotherapy drugs, can simultaneously protect the central nervous system, peripheral nervous system, hematopoietic system, spleen, and testicular tissue.

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