WO2013170637A1

WO2013170637A1 - Application of paeoniflorin compound in preparation of antitumor drug

Info

Publication number: WO2013170637A1
Application number: PCT/CN2013/000683
Authority: WO
Inventors: 李梢
Original assignee: 北京京朋汇药业研究发展有限公司
Priority date: 2012-05-15
Filing date: 2013-05-16
Publication date: 2013-11-21
Also published as: CN103417561A; CN104706647B; CN104706648B; CN104706647A; HK1191868A1; CN104706648A; CN103417561B

Abstract

An application of a paeoniflorin compound, or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer, or racemic mixture thereof in preparation of drugs for preventing and/or treating tumors. Preferably, the paeoniflorin compound is selected from albiflorin and/or paeoniflorin. The tumors are solid tumors.

Description

Use of paeoniflorin in the preparation of antitumor drugs CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the Japanese Patent Application Serial No. 2012. Technical field

The invention belongs to the field of anti-tumor medicines, and relates to the use of paeoniflorin compounds in preparing anti-tumor medicines, and particularly relates to the use of paeoniflorin and paeoniflorin in the preparation of anti-tumor drugs. Background technique

Tumors are the body's normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in the clonal dysplasia of new organisms, which appear as a mass. Tumor cells differ from normal cells in terms of external morphology, metabolism, and function, and exhibit persistent proliferation. According to effective statistics, tumors are the first of three major factors that threaten the health of human beings around the world. Therefore, the development of anti-tumor drugs has become a hot topic in recent years.

According to the traditional classification and research progress of anti-tumor drugs, commonly used anti-tumor drugs can be divided into the following categories: 1. Cytotoxic drugs, including drugs that destroy DNA structure and function (such as cyclophosphamide, irinotecan), Drugs that affect the biosynthesis of nucleic acids (such as 5-fluorouracil, cytarabine, methotrexate); Second, drugs that affect hormone balance, including anti-estrogen drugs (such as toremifene), anti-androgens Drugs (such as bicalutamide) and aromatase inhibitors (such as letrozole); Third, other and auxiliary drugs, including immune function regulators (such as interleukins, interferons), biological response modifiers (such as Ero Tini, gefitinib), cell differentiation inducers (such as retinoic acid, arsenic acid), antifolate preparations (Libitai), monoclonal antibodies (such as Avastin) and adjuvant analgesia, antiemetic, leukocyte elevation, etc. drug. However, these drugs have more or less side effects, such as cytotoxic drugs generally cause moderate to severe digestive system reactions (such as malignant vomiting, stomatitis), myelosuppression (such as leukopenia, thrombocytopenia) and organ toxicity (such as Neurotoxicity, liver and kidney toxicity), hormone balance interference drugs can cause mild to moderate gastrointestinal reactions, reproductive system damage and even depressive symptoms in patients, most regulators and inducers can also cause different degrees of skin reactions and Liver and kidney dysfunction. For example, Irinotecan Hydrochloride is commonly used in the treatment of adult metastatic colorectal cancer, but the effect is significant, but after administration, 20% of patients have gastrointestinal adverse reactions - severe diarrhea, 78.7% of patients have been neutral In neutropenia, 9% of patients develop transient acute cholinergic syndrome (Bret Wallace et al. Science, 330, 2010.; Sun Wei et al. Shuo Pharmacy, 18(35), 2007.; Wu Yuhong et al. Anhui Medicine, 14(10), 2010. ); Pemetrexed disodium (Alimta) is the first anti-pleural mesothelioma drug that causes bone marrow suppression in patients, including neutropenia, thrombocytopenia, anemia or each Hematopoietic reduction, liver and kidney dysfunction are hanged (Zheng Hang et al. Cancer Research, 34(4), 2007.; Wang Jianying et al. Chinese and Foreign Health Digest, 12, 201 1. ) ; Bevaciz b Avastin is a monoclonal antibody to vascular endothelial growth factor VEGF that binds to VEGF and prevents its binding to receptors on the surface of endothelial cells. , reduces microvascular production and inhibits the progression of metastatic lesions. However, its side effects are obvious, including gastrointestinal perforation, wound dehiscence syndrome, hemorrhage, hypertensive crisis, nephrotic syndrome and congestive heart failure (Eric O. Gamboa et al. Clinical Colorectal Cancer, 9( 1 ), 2010 A Mailliez et al. British Journal of Cancer, 103, 2010 Sanjaykumar Hapani et al. Oncology, 79, 2010).

In contrast, anti-tumor Chinese medicines have less damage to the body. The mechanism of anti-tumor Chinese medicine mainly involves cytotoxicity, improving immunity, inducing tumor cell apoptosis and differentiation, and inhibiting tumor angiogenesis. According to the efficacy and action characteristics of traditional Chinese medicine, anti-tumor Chinese medicine can be divided into the following categories: 1. Detoxification, such as Astragalus and Daqingye; 2. Blood-activating and stasis-like, such as rhubarb and Salvia; 3. Fuzheng Peiben Such as ginseng, astragalus, asparagus; four, phlegm and stagnation, such as Pinellia, melon, Tiannanxing; 5, Lishui and dampness, such as scorpion, psyllium, buckwheat; 6, topical drugs, such as Realgar, horse money. Some of the active ingredients in the above anti-tumor Chinese medicines, such as taxol in Taxus br _ev ifolic, are often used in anti-tumor research and have achieved good results (Tang Zhaohui et al. Chinese Clinical Rehabilitation, 27 , 2006.; Jiang Zhiwu et al. Heilongjiang Science and Technology Information, 7, 2010; Pluznilc, DH et al. Cancer Research, 54 (15), 1994). Therefore, the in-depth exploration of the active ingredients of anti-tumor Chinese medicine has important theoretical significance and clinical significance. Guiding significance.

However, due to the complexity of the compounds in most Chinese medicines, the difficulty in separation and purification, the limited quality control and the unclear efficacy and mechanism of action, it is very difficult to screen the anti-tumor active ingredients. Therefore, the research on the active ingredients of anti-tumor Chinese medicine is common. There are many shortcomings and limitations. For example, paclitaxel extracted from yew is a specific stabilizer for microtubules, which promotes the assembly of microtubules and maintains the stability of microtubules. The accumulation of these microtubules interferes with various functions of cells, especially the cell division stops at mitosis. Period, which blocks the normal division of cells and exerts an anti-cancer effect. However, paclitaxel has a significant side effect on patients. Common adverse reactions include allergic reactions (up to 39%), myelosuppression (47% for severe neutrophils, 5% for severe thrombocytopenia), and neurotoxicity. (The incidence of peripheral neuropathy was 62%), cardiovascular toxicity (55% incidence), gastrointestinal reactions (nausea, vomiting, diarrhea, and mucositis were 59% 43% and 39%, respectively) and hair loss ( The incidence rate is as high as 80% or more) (Ye Dongmei et al. Chinese Pharmacy, 29, 2011.; Ma Lianshun et al. Pharmaceutical World, 3, 2009 Deny, WB et al. Cancer Research, 58 (6 1998.).

Therefore, pharmaceutical researchers are still working on the development of new anti-tumor Chinese medicine active ingredients.

White peony is the root of Paeonia a piano ora Pa!! Known pharmacological effects include analgesia, sedation, anticonvulsant, anti-inflammatory, anti-pathogenic microorganisms and liver protection, and also on the immune system and smooth muscle. Function, clinically applicable to anti-epilepsy, epilepsy, detoxification, vertigo, treatment of rheumatoid arthritis, bacterial dysentery and enteritis, viral hepatitis and senile diseases, also anti-sulphate flocculation and dissolution of mucus One of the active ingredients of Radix Paeoniae Alba is a paeoniflorin compound, represented by paeoniflorin (English name: Albiflorin; English name Paeonilactone C; AL) and paeoniflorin (English name Paeoniflorin). It provides a new treatment for paeoniflorin compounds and provides new options for the prevention and treatment of clinical tumors. Summary of the invention

The object of the present invention is to provide a novel antitumor drug, paeoniflorin, which is derived from a traditional Chinese medicine and can be used for the prevention and/or treatment of various tumors in the clinic.

In order to achieve the above object, the present invention adopts the following technical solutions:

Use of a paeoniflorin compound or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer or racemic mixture thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.

Preferably, the paeoniflorin compound is selected from the group consisting of paeoniflorin of structural formula 1 and/or paeoniflorin of structural formula 2 -

In the use of the invention, the tumor is a solid tumor.

Preferably, the tumor is selected from the group consisting of colon cancer, lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex One or more of tumor, skin cancer, melanoma, ovarian cancer, bladder cancer, lymphoma, and gastric cancer.

More preferably, the tumor is selected from the group consisting of a colon cancer, a lung cancer, a neuroblastoma, a breast cancer, a cervical cancer, a rhabdomyosarcoma, a nasopharyngeal cancer, a pancreatic cancer, a laryngeal cancer, a prostate cancer, a renal cancer, and an adrenocortical tumor. Kind or more.

In the use of the present invention, a preferred embodiment is: the paeoniflorin compound is a paeoniflorin, and the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer. One or more.

In another aspect of the use of the present invention, the paeoniflorin compound is paeoniflorin, and the tumor is selected from one or more of laryngeal cancer, cervical cancer, nasopharyngeal cancer, and pancreatic cancer.

In the use of the present invention, the intestinal cancer is selected from one or more of colon cancer, rectal cancer, and colorectal cancer.

In the use of the present invention, the prevention of tumor refers to the treatment of precancerous lesions. Preferably, the preventing tumor refers to treating precancerous lesions of intestinal cancer and preventing the conversion of severe enteritis into intestinal cancer.

In the use of the invention, the medicament is in any of the clinically acceptable dosage forms. Preferably, the dosage form comprises a preparation for parenteral administration and a preparation for parenteral administration. More preferably, the preparation for parenteral administration is selected from the group consisting of a powder, a tablet, a granule, a capsule, a dropping tablet, an emulsion or a suspension; the parenteral preparation is selected as an injection, a spray Agent, suppository, infusion, patch or ointment.

In the use of the present invention, paeoniflorin and paeoniflorin can be used as the prophylactic and/or therapeutic agent for tumors, respectively. The sole active ingredient may also be used together as an active ingredient of the preventive and/or therapeutic tumor drug; the preventive and/or therapeutic tumor drug may also be prepared separately or together with other substances.

In vitro and in vivo experiments have shown that the paeoniflorin compounds of the present invention can significantly inhibit intestinal cancer, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal cancer, pancreatic cancer, laryngeal cancer, prostate cancer, lung cancer, neuroblastoma, renal cancer and A variety of tumors such as adrenal cortex have broad-spectrum anti-tumor function; Paeoniflorin can also treat precancerous lesions, effectively block the conversion of severe enteritis to intestinal cancer, treat precancerous lesions of intestinal cancer, and reduce intestinal cancer. The incidence rate is of great significance. Moreover, there are some differences in the dominant anti-tumor spectrum of paeoniflorin and paeoniflorin. Paeoniflorin can significantly inhibit intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer. Paeoniflorin can significantly inhibit laryngeal Cancer, cervical cancer, nasopharyngeal cancer and pancreatic cancer. Specifically,

1. Paeoniflorin inhibits tumor cell proliferation experiment

MTT test results showed that paeoniflorin on intestinal cancer cell line HT29, intestinal cancer cell line CT26, rhabdomyosarcoma cell line A204, nasopharyngeal carcinoma cell CNE-2Z, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1, prostate cancer cell DU145, cervical cancer The proliferation of cell Hela, breast cancer cell MDA231, breast cancer cell T47D and breast cancer cell MCF7 has a significant inhibitory effect. Among them, intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal cancer cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145 are particularly sensitive to paeoniflorin.

The results of MTT assay showed that paeoniflorin significantly inhibited the proliferation of nasopharyngeal carcinoma cells CNE-2Z, laryngeal carcinoma cells Hep2, pancreatic cancer cells PANC1, cervical cancer cells Hela and rhabdomyosarcoma cells A204. Among them, nasopharyngeal carcinoma CNE-2Z cells, laryngeal carcinoma Hep2 cells, pancreatic cancer PANC1 cells, and cervical cancer Hela cells are particularly sensitive to paeoniflorin.

2. Paeoniflorin inhibits tumor cell migration experiment

The results of tumor cell migration assay showed that paeoniflorin on intestinal cancer cell CT26, cervical cancer cell line Hela, neuroblastoma cell line SY5Y, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, rhabdomyosarcoma cell A204, Renal cancer Wilms cell G401, nasopharyngeal carcinoma cell CNE2Z, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, lung cancer cell A549, lung cancer cell LLC, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13 have significant inhibition. Among them, paeoniflorin inhibited the migration of cervical cancer cell line Hela, intestinal cancer cell line CT26, breast cancer cell line MDA231, nasopharyngeal carcinoma cell line CNE2Z and pancreatic cancer cell line PANC1.

The results of tumor cell migration assay showed that paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13, etc. The migration has a significant inhibitory effect. Among them, paeoniflorin ^: the inhibitory effect on intestinal cancer cell CT26, nasopharyngeal carcinoma cell CNE2Z, laryngeal carcinoma cell Hep2 and adrenal cortical tumor cell SW13 was particularly significant.

3, anti-tumor effect in vivo

In vivo experiments in nude mice showed that paeoniflorin can significantly inhibit the length and metastasis of CT26 intestinal cancer, LLC lung cancer, MDA231 breast cancer and Hela cervical cancer. In particular, the inhibition rate of paeoniflorin on CT26 intestinal cancer can reach 54.41%. The inhibition rate of Hela cervical cancer can reach 52.3%.

In vivo experiments in a nude mouse model showed that paeoniflorin significantly inhibited the growth and metastasis of CNE-2Z nasopharyngeal carcinoma, PANC1 pancreatic cancer, and Hela cervical cancer cells.

4, the role of prevention of tumorigenesis

The results of the experiment on the transformation of mouse enteritis to primary colorectal cancer induced by guanidinium lactone treatment of gene mutation agent AOM showed that paeoniflorin is effective in the treatment of precancerous lesions, prevention and blockade of intestinal cancer. Enteritis plays a significant role in the transformation of intestinal cancer. DRAWINGS

Figure 1: Effect of paeoniflorin on tumor cell migration (Transwell assay).

Figure 2: Effect of paeoniflorin on tumor cell migration (Scratch Analysis).

Figure 3: Effect of paeoniflorin on tumor cell migration (Transwell assay).

Figure 4: In vivo results of anti-tumor effects of paeoniflorin;

4A: tumor growth curve of CT26 intestinal cancer in nude mice;

4B: tumor growth curve of LLC lung cancer nude mice;

4C: tumor growth curve of MDA231 breast cancer nude mice;

4D: Tumor growth curve of Hela cervical cancer nude mice.

Figure 5: Blocking effect of paeoniflorin on enteritis-to-intestinal cancer transformation: Among them,

5A: photo of the colon of three groups of mice;

5B: Results of HE staining of colon tissue in three groups of mice.

Figure 6: Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay).

Figure 7: Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay). detailed description

The invention is described below with reference to specific embodiments. Those skilled in the art can understand that the examples are only intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

The paeoniflorin and paeoniflorin used in the following examples were purchased from Shanghai Dingrui Chemical Co., Ltd. (purity was 98% or more), and the total glucoside capsule (Pavlin) was purchased from Ningbo Lihua Pharmaceutical Co., Ltd. Example 1 Effect of Paeoniflorin on Tumor Cell Proliferation

1. Effect of paeoniflorin on tumor cell proliferation

Experimental protocol: Triazolium blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin treatment of rhabdomyosarcoma A204, Nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, intestinal cancer HT29, neuroblastoma cell SY5Y, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC 1, cervical cancer cell Hela, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, skin squamous cell carcinoma A431, renal cancer Wilms Cell G401 and prostate cancer cell DU145 were for about 24 hours; after MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.

Experimental results: Paeoniflorin was found to be pancreatic cancer cell PANC1, intestinal cancer cell line CT26, intestinal cancer cell line HT29, laryngeal cancer cell Hep2, cervical cancer cell line Hela, breast cancer cell MDA231, prostate cancer cell DU145, nasopharyngeal carcinoma cell CNE2Z, The proliferation of rhabdomyosarcoma cell A204, breast cancer cell T47D and breast cancer cell MCF7 cells all had significant inhibitory effects. Among them, paeoniflorin is most sensitive to the effects of intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal carcinoma cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145. The specific results are shown in Table 1.

Table 1 Effect of paeoniflorin on tumor cell proliferation (ic ₅₀ )

Tumor drug

A204 (rhabdomyosarcoma cells) 45 μ /ιυ1

CNE2Z (nasopharyngeal carcinoma cells) 43 μ§ ηύ

HT29 (intestinal cancer cells) 33 g/ml

CT26 (intestinal cancer cells) 59 g/ml

Hep2 (laryngeal cancer cell) 36 g/ml

PANC 1 (pancreatic cancer cells) 30 μ^πιΐ

SY5Y (neuroblastoma cells) 67 μ^ηιΐ

DU145 (prostate cancer cells) 41 μ^πιΐ

Hela (cervical cancer cells) 39 g/ml

MDA231 (breast cancer cells) 40 μ^η\\

T47D (breast cancer cells) 56 g/nil

MCF7 (breast cancer cells) 61 g/ml

A431 (skin squamous cell carcinoma) 92 g/ml

G401 (kidney cancer Wilms cell) 108 g/ml

2. Effect of paeoniflorin on tumor cell proliferation

Experimental protocol: thiazolyl blue (3-(4,5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, peony 3⁄4 ^: treatment of rhabdomyosarcoma cells A204, nasal Pharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, intestinal cancer cell HT29, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC 1, breast cancer cell MDA231, cervical cancer cell Hela, renal cancer Wilms cell G401 cell and prostate cancer cell DU 145 Approximately 24 hours, followed by MTT staining for 4 hours, dimethyl sulfoxide (DMSO) Dissolved, the absorbance photometric value was measured at 570 nm.

Experimental results: Paeoniflorin was found to have significant inhibitory effects on the proliferation of laryngeal carcinoma cell Hep2, cervical cancer Hela, nasopharyngeal carcinoma cell CNE-2Z, pancreatic cancer cell PANC1 and rhabdomyosarcoma cell A204 cells. The cells Hep2, cervical cancer cell Hda, nasopharyngeal carcinoma cell CNE-2Z and pancreatic cancer cell PANCl cells were most sensitive. The specific results are shown in Table 2. Table 2 Effect of paeoniflorin on tumor cell proliferation (IC ₅ o)

Cell type paeoniflorin

A204 cells (rhizomatous muscle cells) 67

CNE-2Z cells (nasopharynx cancer cells) 50 μ^πιΐ

CT26 cells (intestinal cancer cells) 82 πιΐ

Hep2 cells (laryngeal cancer cells) 44

Tumor cells _ώ

PANC1 cells (pancreatic cancer cells) 53 g/ml

Hela cells (cervical cancer cells) 49 μ^πιΐ

HT29 cells (intestinal cancer cells) 96 g/ml

MDA231 cells (breast cancer cells) \15μ^η\]

G401 cells (kidney cancer Wilms cells) 89 μ^ ιΐ

DU145 cells (prostate cancer cells) 97 g/ml Example 2 Effect of paeoniflorin compounds on tumor cell migration

1. Effect of paeoniflorin on tumor cell migration (Transwell assay and Scratch Analysis assay)

Experimental program:

1) Using the cell migration assay (Transwell) assay, add steroidal lactones to A549, LLC, Hela, SY5Y, CT26, T47D, MCF7, MDA23K A204, G401, CNE-2Z, DU145, Hep2, PANCl and SW13 tumor cells. After 24 hours (using a safe dose of 20 g/ml), anhydrous ice-cold methanol was fixed for 20 minutes, crystal violet stained for 15 minutes, and 100 times light microscopy was taken to detect the number of cell migration.

2) Using the Scratch Analysis test, add HeLa lactones to Hela, LLC, A549, SY5Y, CT26, MCF7, MDA231 and T47D cells for 6 hours and 24 hours (using a safe dose of 20)

), taking a 40x light microscope to detect the cell spacing.

Experimental results: 1) Transwell results showed that paeoniflorin (AL) can significantly inhibit lung cancer cell A549, lung cancer cell LLC, cervical cancer cell Hela, neuroblast cell SY5Y, intestinal cancer cell CT26, breast cancer T47D, breast Cancer MCF7, breast cancer MDA231, rhabdomyosarcoma cell A204, renal cancer Wilms cell G401, nasopharyngeal carcinoma The migration of CNE-2Z, prostate cancer cell line DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13 cells showed that the number of cells in the drug-administered group was significantly reduced compared with the control group, among them, paeoniflorin Inhibition of cervical cancer cell line Hela, intestinal cancer cell line CT26, breast cancer cell MDA231, nasopharyngeal carcinoma cell CNE2Z and pancreatic cancer cell PANC1 tumor cell migration is particularly significant. See Figure 1 for details.

2) The results of Scratch Analysis showed that compared with the control group, the scratch width of the paeoniflorin-administered group was significantly increased compared with the normal control group, which proved that paeoniflorin could inhibit the migration of the measured tumor cells. See Figure 2 for details.

In summary, the experimental results suggest that paeoniflorin has a broad and significant inhibitory effect on tumor cell migration.

2. Effect of paeoniflorin on tumor cell migration (Transwell assay)

Experimental protocol: Cell migration assay (Transwell) was used to add paeoniflorin to A204, CNE-2Z, CT26, DU145, Hep2, PANC1 and SW13 tumor cells for 24 hours (using a safe dose of 10 μΜ), fixed with anhydrous ice methanol. After 20 minutes, crystal violet staining for 15 minutes, photographing 100 times light microscopy and detecting the number of migrated cells.

Experimental results: Paeoniflorin can significantly inhibit the migration of rhabdomyosarcoma cell Α204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenal cortical tumor cell SW13. It was shown that the number of cells in the administration group was significantly reduced relative to the control group. Among them, paeoniflorin inhibited intestinal cancer cell line CT26, nasopharyngeal carcinoma cell line CNE2Z, laryngeal carcinoma cell line Hep2 and adrenocortical tumor cell line SW13. See Figure 3 for details. Example 3 Antitumor effect of paeoniflorin compounds in vivo

1. Antitumor effect of paeoniflorin in vivo

Experimental protocol: The tumor block inoculation technique was used to inoculate solid tumor blocks under the skin of the right axilla of nude mice, including CT26, LLC, MDA231 and Hela. Nude mice of each solid tumor transplantation model were randomly divided into 3 groups: control group (inoculated with tumor mass without administration), pre-dosing group of paeoniflorin (referring to the day of inoculation of tumor block) and paeoniflorin The drug-administered group (administered when the tumor-forming volume reached 70-100 mm ³ or more), 5-6 nude mice per group.

The pre-administration group of paeoniflorin was intraperitoneally administered on the day of inoculation of the tumor block at a dose of 200 _m g/kg, the frequency of administration was once every 2 days for 3 weeks, and the mice were sacrificed the day after the end of the administration. . In the paeoniflorin administration group, when the tumor volume reached 70-100 mm ³ or more, the paeoniflorin was administered intraperitoneally at a dose of 300 mg/kg, and the administration frequency was once every 2 days for 2 weeks. Mice were sacrificed the day after the end. Tumor volume and weight were measured and the tumor inhibition rate was calculated.

Experimental results: In the four different tumor animal models of colon cancer, lung cancer, breast cancer and cervical cancer, the pre-administration group of paeoniflorin and the paeoniflorin administration group can significantly inhibit the growth and metastasis of tumor cells. Especially for the CT26 intestinal cancer and Hela cervical cancer models, the effect is extremely significant. The specific anti-tumor results are shown in Table 3A, Table 3B, Table 3C and Table 3D.

1 CT26 colon cancer model experimental results (see Figure 4A):

A. CT26 tumor cattle. The length of the experiment is about 12 days, that is, the volume reaches 100mm ³ ; B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has significant preventive and therapeutic effects on intestinal cancer.

2 LLC Lung Cancer Model Experimental Results (Fig. 4B) _:

The growth rate of A. LLC tumor and CT26 tumor is equivalent, and the volume reaches 100 mm ^{3 and} is about 12 days;

B. Compared with the control group, the tumor volume and weight in the pre-administration group and the drug-administered group were significantly reduced, indicating that the paeoniflorin has certain preventive and therapeutic effects on lung cancer.

3 MDA231 breast cancer model experiment results (Figure 4C):

A. MDA231 cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm ^{3 and} is about 18 days.

B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has certain preventive and therapeutic effects on breast cancer.

4 Hela cervical cancer model experimental results (Figure 4D):

A. Hela cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm ^{3 and} is about 20 days.

B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has significant preventive and therapeutic effects on cervical cancer.

Experimental results of anti-tumor effect of paeoniflorin in vivo (CT26 colon cancer model)

Compared with the control group, P < 0.01. Experimental results of anti-tumor effect of paeoniflorin in vivo (LLC lung cancer model)

: compared with the control group, Ρ < 0.01 ;

*: Compared with the control group, Ρ < 0.05 Experimental results of anti-tumor effect of paeoniflorin in vivo (MDA231 breast cancer model)

Compared with the control group, P < 0.01. Experimental results of anti-tumor effect of paeoniflorin in vivo (Hela cervical cancer model)

Compared with the control group, P < 0.01.

2, anti-tumor effect of paeoniflorin in vivo

Experimental protocol: The dominant tumors of paeoniflorin in vitro, pancreatic cancer PANC1 cells and cervical cancer Hela cells, were selected to establish a subcutaneous tumor-bearing model in nude mice. After the tumor volume reached 70 mm ³ , paeoniflorin was intraperitoneally injected at a dose of 300 mg/kg, and the frequency of administration was once every 2 days for 2 weeks. The model group was used as a control. Mice were sacrificed the day after the end of dosing. Tumor volume and weight were measured and the tumor inhibition rate was calculated.

Experimental results: In the two animal models of pancreatic cancer and cervical cancer, the paeoniflorin administration group can significantly inhibit the growth and metastasis of tumor cells, indicating that paeoniflorin has significant inhibitory effects on pancreatic cancer and cervical cancer cells. The specific results are shown in Table 4A and Table 4B.

Table 4A Experimental results of anti-tumor effects of paeoniflorin in vivo (PANC 1 pancreatic cancer model)

Control group Paeoniflorin administration group dose (mg/kg) 300 mg/g

Body weight of nude mice (g) 20.58 ± 0.49 20.43 ± 0.51 tumor weight (g) 2.11 ± 0.47 1.36 ± 0.28 * Tumor inhibition rate (o/< 35.55)

Compared with the control group, P < 0.01 anti-tumor effect test results of paeoniflorin (Hela cervical cancer model)

Control group paeoniflorin administration group

U amount (mg/kg) 300 mg/Kg

Nude mouse weight (g) 20.94 ± 0.58 20.77 soil 0.32

Tumor weight (g) 1.78 ± 0.19 1.34 ± 0.25" tumor inhibition rate (%) 24.04

Compared with the control group, P < 0.05. Example 4 Therapeutic effect of paeoniflorin on precancerous lesions of colorectal cancer

Experimental protocol: BALB/C mice were divided into 3 groups: normal group, model group and paeoniflorin intervention group, 5 in each group. The model group and the intervention group were intraperitoneally injected with the gene mutation agent oxidized azomethane (AOM, 20 mg/g). The paeoniflorin dry group was administered intraperitoneally (100 mg/Kg) on the 2nd day of AOM injection, once every two days for 3 times. The model group did not have any drug treatment. One week after AOM administration, 3% sodium dextran sulfate (DSS) was used to replace normal drinking water. The mice were continuously consumed for 7 days; after that, they were returned to normal drinking water, and the mice were continuously consumed for 14 days. The cycle of AOM injection, drinking DSS water to drinking normal water for 14 days was repeated three times, and the paeoniflorin intervention group was normally administered in three cycles. Normal mice were fed and watered normally without any treatment. At the end of the third cycle, the mice were sacrificed, the intestinal tissues were dissected, pathological sections were prepared, and HE staining was performed.

Experimental results: The appearance showed that the colon of normal mice was uniform in thickness and the mucosa was not congested. Compared with normal mice, the mice in the model group had obvious colonic mucosal congestion, uneven intestinal thickness, and several tumors had been formed. However, the mice in the paeoniflorin intervention group had a uniform colon thickness and no obvious bleeding points. See tumor formation, see Figure 5A for details.

The HE staining results showed that the glandular structure of normal mouse gland was intact and distributed, no obvious inflammatory cell infiltration, no submucosal hemorrhage and ulceration. In the model group, mucosal ulcers appeared in the intestinal tissues of mice, multiple glandular deletions accompanied by inflammatory cell infiltration, and cancer cells infiltrated into the intestinal mucosa and submucosa, presenting typical features of intestinal cancer in situ. And the paeoniflorin ^: T pre-group mouse intestinal tissue showed focal inflammatory cell infiltration, individual gland loss, but no obvious cancer cell infiltration, showing the typical characteristics of enteritis, see Figure 5B.

The results showed that paeoniflorin played an important role in the transformation of ffi degree enteritis to intestinal cancer, which plays an important role in the treatment of precancerous lesions. Example 5 Comparison of the effects of paeoniflorin components and total glucosides of paeony on tumor suppression 1 > Comparison of effects of paeoniflorin and total glucosides of paeony on tumor cell proliferation (MTT)

Experimental protocol: thiazolyl blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin and paeoniflorin treatment A204, CNE-2Z, CT26, Hep2 and PANC l tumor cells were used for 24 hours, then MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.

Experimental results: It was found that paeoniflorin and total glucosides of paeony inhibited the proliferation of rhabdomyosarcoma cell line A204, nasopharyngeal carcinoma cell line CNE-2Z, intestinal cancer cell line CT26, laryngeal carcinoma cell line Hep2 and pancreatic cancer cell line PANCl. The IC ₅ o (half inhibition rate) values of lactones on these tumors were all lower than total glucosides of paeony, indicating that paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, colon cancer cell CT26, laryngeal carcinoma The inhibition of cell Hep2 and pancreatic cancer cell PANC l tumor cell proliferation is stronger than that of total glucosides of paeony. The specific results are shown in Table 5.

Table 5 Effect of paeoniflorin and total glucosides of paeony on tumor cell proliferation (IC ₅₀ )

Paeoniflorin

A204 (rhabdomyosarcoma cells) 45 g/ml 61 g/ml

CNE-2Z (Nasopharyngeal Carcinoma) 43 g/ml 64 μ^πιΐ

Mid-month tumor cell CT26 (intestinal cancer cells) 59

67 g/ml

Hep2 (laryngeal cancer cell) 36 g/ml 39 μ^ι \

PANC1 (pancreatic cancer cells) 30 μ§ πι\ 41 μ^πιΐ

2. Comparison of effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwdl)

Experimental protocol: Using the cell migration assay (Transwell) experiment, the A204, CNE-2Z, CT26, DU145. Hep2, PANCl and SW13 tumor cells were added with paeoniflorin and total glucosides for 24 hours (using a safe dose of 10 μΜ). ), anhydrous ice-methanol was fixed for 20 minutes, crystal violet stained for 15 minutes, photographed by a 100-fold light microscope and the number of migrated cells was detected.

Experimental results: Both paeoniflorin and total glucosides of paeoniflorin can significantly inhibit rhabdomyosarcoma cells 204, nasopharyngeal carcinoma cells CNE-2Z, intestinal cancer cells CT26, prostate cancer cells DU145, laryngeal cancer cells He _P 2, pancreatic cancer cells The migration of PANC l and adrenocortical tumor SW13 tumor cells showed that the number of cells in the drug-administered group was significantly lower than that of the control group; but the drug of paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer DU145, laryngeal carcinoma cells Hep2, inhibition of pancreatic cancer cell PANC 1 adrenal tumor cell migration and tumor SW13 stronger than all TGP ^¾:. See Figure 6 for details.

3. Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell proliferation (MTT)

Experimental protocol: Using thiazolyl blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin and total glucosides of paeony treated CNE- 2Z, Hela and HT29 tumor cells for 24 hours, MTT staining for 4 hours, two Methyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.

Experimental results: It was found that paeoniflorin and total glucosides of paeony on nasopharyngeal carcinoma cells CNE-2Z, cervical cancer cells Hela and intestinal cancer cells

The proliferation of HT29 has an inhibitory effect, but the IC ₅ o (half inhibition rate) of paeoniflorin on nasopharyngeal carcinoma cell line CNE2Z, cervical cancer cell line Hela and intestinal cancer cell line HT29 is lower than total glucosides of paeony, indicating that paeoniflorin The inhibition of tumor cell proliferation is stronger than that of total glucosides of paeony. The specific results are shown in Table 6.

Table 6 Effect of paeoniflorin and total glucosides of paeony on tumor cell proliferation (IC ₅₀ )

Mum. paeoniflorin

CNE-2Z (nasopharynx cancer cells) 50 μ≠η\ 64 μ^τ \

Tumor cell Hela (cervical cancer cell) 49 μ^ηιΐ 55 _μβ /ιη1

ΗΤ29 (intestinal cancer cells) 96 μ^πιΐ 137 g/ml

4. Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell)

Experimental protocol: Using the cell migration assay (Transwell) experiment, the HT29, DU145 and Hela tumor cells were added with paeoniflorin and total glucosides of paeony for 24 hours (using a safe dose of 10 μΜ), fixed with anhydrous ice methanol for 20 minutes, crystal violet The staining was performed for 15 minutes, and a 100-fold light microscope photograph was taken to detect the number of migrated cells.

Experimental results: Both paeoniflorin and total glucosides of paeony can significantly inhibit the migration of intestinal cancer cells ΗΤ29, prostate cancer cells DU145 and cervical cancer Hela tumor cells, which showed that the number of cells in the drug-administered group was significantly reduced compared with the control group; The inhibitory effect on the migration of intestinal cancer cells HT29, prostate cancer cells DUM5 and cervical cancer Hela tumor cells is stronger than that of total glucosides of paeony. See Figure 7 for details. ' Summary, paeoniflorin can effectively inhibit intestinal cancer, lung cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, prostate cancer, nasopharyngeal carcinoma, rhabdomyosarcoma, lung cancer, sputum mesothelioma, kidney cancer and adrenal cortex Tumor growth and metastasis can effectively inhibit the growth and metastasis of colorectal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer; paeoniflorin can effectively inhibit laryngeal cancer, cervical cancer, nasopharyngeal cancer, pancreatic cancer and The growth and metastasis of rhabdomyosarcoma is particularly effective in inhibiting the growth and metastasis of laryngeal, cervical, nasopharyngeal and pancreatic cancers. There are some differences in the dominant tumor species treated with paeoniflorin and paeoniflorin. Paeoniflorin can also effectively treat precancerous lesions of colorectal cancer and significantly block the conversion of severe ulcerative enteritis to intestinal cancer. Therefore, the results of in vitro and in vivo experiments show that paeoniflorin can effectively inhibit the growth and metastasis of a variety of tumors, and has a broad-spectrum anti-tumor effect; paeoniflorin compounds can also treat precancerous lesions of intestinal cancer, blocking severe The conversion of enteritis to intestinal cancer.

Claims

Rights request

A use of a paeoniflorin compound or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer or racemic mixture thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.

The use according to claim 1, wherein the paeoniflorin compound is selected from the group consisting of paeoniflorin of structural formula 1 and/or paeoniflorin of formula 2 -

3. Use according to claim 1 or 2, characterized in that the tumor is a solid tumor.

The use according to any one of claims 1 to 3, wherein the tumor is selected from the group consisting of colon cancer, lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, One or more of nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex, skin cancer, melanoma, ovarian cancer, bladder cancer, lymphoma, and gastric cancer; preferably, the tumor One or more selected from the group consisting of intestinal cancer, lung cancer, neuroblastoma, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal cancer, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, and adrenocortical tumor.

5. The use according to claim 1, wherein: the paeoniflorin compound is a paeoniflorin, and the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer. One or more.

The use according to any one of claims 1 to 5, characterized in that the intestinal cancer is selected from one or more of colon cancer, rectal cancer and colorectal cancer.

7. The use according to claim 1, wherein: the paeoniflorin compound is paeoniflorin, and the tumor is selected from one or more of laryngeal cancer, cervical cancer, nasopharyngeal cancer, and pancreatic cancer. .

The use according to any one of claims 1 to 3, characterized in that the prevention of tumor refers to the treatment of precancerous lesions; Preferably, the preventing tumor refers to treating precancerous lesions of intestinal cancer and preventing the conversion of severe enteritis into intestinal cancer.

Use according to any one of claims 1 to 8, characterized in that the medicament is in any clinically acceptable dosage form, including gastrointestinal administration and parenteral administration. Formulation

Preferably, the gastrointestinal administration preparation is selected from the group consisting of a powder, a tablet, a granule, a capsule, a dropping pill, an emulsion or a suspension;

Preferably, the parenteral administration preparation is selected from the group consisting of an injection, a spray, a suppository, a perfusion, a patch or an ointment.

The use according to any one of claims 1 to 9, characterized in that paeoniflorin and paeoniflorin are the sole active ingredients of the preventive and/or therapeutic tumor drug, respectively, or together as the prevention and / or treating the active ingredient of a tumor drug, or separately or together with other substances to prepare the drug for preventing and/or treating a tumor.