CN114712350B - Application of DTTZ in the preparation of drugs for preventing and treating chemotherapy injuries - Google Patents

Abstract

The invention discloses an application of DTTZ in preparing a medicine for preventing and treating organism injury diseases caused by chemotherapy. DTTZ and pharmaceutically acceptable salts thereof can be used for preparing medicines for preventing and treating bone marrow suppression, gastrointestinal damage and hepatorenal toxicity caused by clinical chemotherapeutic agents. In particular, DTTZ exhibits outstanding high safety and definite pharmacological effects.

Description

Application of DTTZ in the preparation of drugs for preventing and treating chemotherapy injuries

Technical Field

The invention belongs to the technical field of biomedicine, and specifically discloses an application of DTTZ in the prevention and treatment of liver, intestinal and kidney diseases and chemotherapy-induced damage to organisms.

Background technique

Currently, chemotherapy is still the first choice for oncology treatment, mainly including platinum (cisplatin), alkylating agents (cyclophosphamide), antimetabolites (gemcitabine), anticancer antibiotics (doxorubicin), botanicals (paclitaxel), hormones, etc. These drugs can inhibit or kill tumor cells by acting on different links of tumor cell growth and reproduction, thereby achieving the purpose of eliminating tumors.

Cisplatin (cis-dichlorodiammineplatin) is a platinum-containing anticancer drug with the advantages of a broad anticancer spectrum, effective in hypoxic cells, and strong action. It is widely used in the treatment of various cancers. The mechanism of action of cisplatin is mainly to cause cross-linking by binding to DNA, thereby interfering with DNA repair and transcription. It is a cell-nonspecific chemotherapy drug. Cyclophosphamide does not have anticancer activity in vitro. It needs to be hydrolyzed by excessive phosphatidylinositol or phosphatase in the liver or tumor in vivo before it can be activated into phosphoramide nitrogen mustard with broad-spectrum antitumor effects. Although chemotherapy drugs have significant antitumor activity, due to their lack of tumor tissue specificity, chemotherapy drugs can also produce serious toxic side effects on normal tissues while eliminating tumors, thereby causing bone marrow suppression, hepatotoxicity, gastrointestinal reactions, neurotoxicity, etc. In clinical practice, cytoprotectants are administered before cisplatin treatment, but cisplatin-induced toxicity can still be observed. The accumulation of toxicity caused by these chemotherapy drugs or radiotherapy will limit their effectiveness in tumor treatment and prevent further tumor treatment plans. How to reverse the toxic side effects of chemotherapy or radiotherapy on normal tissues without interfering with tumor treatment is a difficult problem faced in tumor treatment.

Cytoprotectants are a class of protective drugs that do not have anti-tumor activity themselves, but when used in combination with radiotherapy and chemotherapy, they can selectively protect normal cells without interfering with the efficacy of radiotherapy and chemotherapy. An ideal cytoprotectant should have the following characteristics: it is non-toxic or less toxic to normal tissues; it can protect the body from multiple organ failure and reduce the toxic side effects of radiotherapy and chemotherapy while using chemotherapy drugs; it does not affect the anti-tumor efficacy of chemotherapy drugs; it is selective for normal cells and cancer cells. At present, Amifostine (also known as WR2721) is a cytoprotectant approved by the FDA for clinical use. It can be selectively dephosphorylated by alkaline phosphatase to an activated metabolite containing thiol groups, WR1065, through the difference in alkaline phosphatase content on the surface of normal tissues and cancerous tissues, thereby exerting a protective effect against chemotherapy. The world patent PCT/US1998/026096 of Stogoniv et al. discloses that amifostine and other aminothiol compounds are used to treat or reverse radiation or chemotherapy-induced damage. Our study found that DTTZ has a significant preventive and therapeutic effect on the renal toxicity and bone marrow suppression caused by cisplatin and cyclophosphamide in mice, and has a reversal and therapeutic effect on the bone marrow suppression and intestinal damage caused by γ-rays. More importantly, the previous toxicological study of DTTZ hydrochloride showed that its LD ₅₀ by intraperitoneal injection and oral administration in mice was 560±14 and 1092±22 mg/kg, respectively, which is higher than Amifostine's 321 mg/kg (intraperitoneal injection) and 842 mg/kg (oral). Therefore, compared with Amifostine, it is safer and more effective.

In 1979, Song Xiaoying et al. reported in Journal of Chinese Academy of Medical Sciences, 1979, 1(2): 147-53; Journal of Chinese Academy of Medical Sciences, 1980, 2(4): 241-5, etc. that DTTZ hydrochloride has a preventive effect on damage caused by acute ionizing radiation, especially when used before exposure to X-rays and gamma rays, it has a preventive effect on bone marrow suppression caused by lethal doses of ionizing radiation and improves the survival rate of mice. However, there is no mention of DTTZ's preventive and therapeutic effects on human organ damage caused by chemotherapy drugs.

The mechanism of damage caused by prevention and treatment of chemotherapy drugs is different from that of ionizing radiation damage. The prevention and treatment of damage caused by chemotherapy drugs is to promote the repair and regeneration of damage to various target organs. The main mechanism of the accumulation toxicity of platinum chemotherapy drugs in the body is: after hydrolysis and activation in cells, platinum is very easy to bind to amino acid side chains and proteins containing S donors in the body through coordination bonds (such as copper transporter CTRI, ATP7A/ATP7B, etc.), thereby affecting the cell functions controlled by these proteins. As a Lewis acid with weak acid properties, cisplatin has a high affinity for electron-donating atoms with weak base properties, so Pt(II) will preferentially bind to soft base compounds containing S donors. The thiol groups produced by the metabolism of DTTZ in the body can competitively release the coordination effect between cisplatin and proteins, helping to restore normal cell function.

The CAS number of DTTZ is 19351-18-9, and its structural formula is as follows:

The prevention mentioned in the present invention refers to implementation before chemotherapy; the treatment mentioned in the present invention refers to implementation one or more days after the occurrence of the one or more toxicities.

The organisms described in the present invention include humans and animals, wherein animals particularly include mammals.

Summary of the invention

The DTTZ of the present invention includes a DTTZ compound, and/or a pharmaceutically acceptable salt thereof, and/or a hydrate thereof.

The purpose of the present invention is to provide the use of DTTZ and/or its pharmaceutically acceptable salts and/or its hydrates in the preparation of drugs for preventing and treating damage to organisms caused by anti-tumor chemotherapy agents, including but not limited to the use in drugs for diseases such as liver, intestine, kidney and bone marrow suppression, especially in the process of tumor chemotherapy, to treat or prevent the toxic effects of normal tissues of the organism caused by treatment methods such as anti-tumor chemotherapy agents.

The anti-tumor chemotherapeutic agent of the present invention is selected from alkylating agent chemotherapeutic agents, antimetabolite chemotherapeutic agents, anti-tumor biotin chemotherapeutic agents, plant chemotherapeutic agents, hormone chemotherapeutic agents, platinum chemotherapeutic agents, and combinations of two or more thereof. The alkylating agent chemotherapeutic agents include nimustine, cyclophosphamide, and myleran; the antimetabolite chemotherapeutic agents include tadalafil and methotrexate; the anti-tumor biotin chemotherapeutic agents include dactinomycin, bleomycin, and doxorubicin hydrochloride; the plant chemotherapeutic agents include paclitaxel, vinblastine, and vincristine; the hormone chemotherapeutic agents include medroxyprogesterone and falotone; the platinum chemotherapeutic agents include cisplatin, carboplatin, levoplatin, oxaliplatin, and combinations of two or more thereof.

The organism damaging diseases described in the present invention include: kidney diseases related to anti-tumor chemotherapy, liver diseases related to anti-tumor chemotherapy, gastrointestinal diseases related to anti-tumor chemotherapy, mucosal damage related to anti-tumor chemotherapy, dry mouth related to anti-tumor chemotherapy, bone marrow suppression diseases related to anti-tumor chemotherapy, neurotoxicity related to anti-tumor chemotherapy, cardiotoxicity related to anti-tumor chemotherapy, ototoxicity related to anti-tumor chemotherapy, hair loss related to anti-tumor chemotherapy, and pulmonary fibrosis related to anti-tumor chemotherapy.

Another object of the present invention is to provide a drug containing DTTZ and a pharmaceutically acceptable salt thereof which has a selective therapeutic effect on damage caused by chemotherapy, and which reverses normal tissue damage while not affecting the inhibitory effect of chemotherapy drugs on tumors.

The effective therapeutic dose of DTTZ in the present invention is 10 mg/m ² -2000 mg/m ² based on body surface area.

The present invention provides a pharmaceutical composition of DTTZ or a pharmaceutically acceptable salt or hydrate thereof for use in the present invention. The present invention provides a safe and efficient preventive or therapeutic agent, wherein the active ingredient of the preventive or therapeutic agent is DTTZ, and DTTZ can be prepared into various pharmaceutical compositions with one or more pharmaceutically acceptable media carriers, adjuvants, auxiliary agents or diluents or other drugs, such as tablets, capsules, granules, injections and other solid and liquid preparations, etc.; in particular, tablets, capsules, injections, emulsions, nanoparticles, pills, inhalants, gels, powders, suppositories, suspensoids, creams, jellies, sprays, etc. The pharmaceutical composition of the present invention also includes.

The DTTZ used in the present invention can be used alone as an active ingredient or in combination with other drugs. Other drugs include drugs for anti-chemotherapeutic damage, liver, intestine, kidney and other organ protection drugs to form a pharmaceutical composition. They include representative drugs such as glutathione, metoclopramide, sodium mercaptosulfonate, etc., and can also be combined with other effective ingredients of traditional Chinese medicine to form various dosage forms, which are used to treat or reverse the toxic effects of liver, intestine, kidney diseases and bone marrow suppression caused by various treatment methods in normal tissues of the body through different administration routes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the neutral red detection method to investigate the selectivity of DTTZ to chemotherapy-damaged cells: A. Chemotherapy-damage caused by cisplatin; B. Chemotherapy-damage caused by doxorubicin (cells from left to right are: HIEC-6, CHO, MCF-7, A549, Hela);

Figure 2 is a diagram showing the protective effect of EdU staining on the DNA synthesis process of normal cells during DTTZ chemotherapy: A. HIEC-6; B. MCF-7 (the green area in the figure is the DNA synthesis period cells stained with EdU-488; the blue area is the cell nucleus stained with DAPI);

FIG3 shows the inhibitory effect of DTTZ and the positive control WR2721 on the clone formation of four cell types.

Figure 4 is an Annexin V-FITC apoptosis experiment to investigate the protective effect of DTTZ on cisplatin-induced normal cell apoptosis: A. HIEC-6; B. MCF-7 (lower left corner: normal cells; lower right corner: early apoptotic cells; upper right corner: late apoptotic cells; upper left corner: necrotic cells);

Figure 5 shows the protective effect of DTTZ on the hematopoietic system of mice induced by cyclophosphamide (CP): (A) average body weight (B) bone marrow DNA content (C) bone marrow nucleated cell number (D) spleen index (E) thymus index (F) spleen nodule number (G) lymphocyte single cell gel electrophoresis 0live tail moment and (H) tail DNA content (%);

Figure 6 shows that DTTZ does not affect the tumor inhibition effect of cisplatin on tumor-bearing mice: (A) average body weight (B) tumor volume (C) tumor inhibition rate (D) liver index (E) kidney index (F) colon length;

Figure 7 shows the protective effect of DTTZ on chemoradiotherapy damage: (A) Small intestinal crypt cell counts (B) HE staining results of small intestinal tissue, liver tissue and kidney tissue.

Figure 8 shows the 30-day survival rate results of DTTZ protecting mice from the toxicity of the chemotherapy drug cisplatin.

Detailed ways

The present invention will be further described below in conjunction with implementation cases:

Example 1 Neutral red assay to detect cell selectivity and optimal concentration of DTTZ hydrochloride chemotherapy protection

Five types of cells, including normal cells (HIEC-6), hamster ovary cells (CHO) and cancer cells (including human breast cancer (MCF-7), human non-small cell lung cancer (A549) and human cervical cancer (Hela), were selected to investigate the protective effect of DTTZ on normal cells induced by chemotherapeutic drugs cisplatin and doxorubicin. All cells were cultured in a 37°C, 5% CO ₂ incubator using DMEM culture medium containing 10% fetal bovine serum. Each cell was plated at a density of 5000 cells/well in a 96-well plate. After 24 hours, the cells were incubated with DTTZ or positive control WR2721 for 15 minutes in advance, and then chemotherapeutic drugs (cisplatin or doxorubicin) were added for continued incubation. After 24 hours, each well was washed twice with PBS, and the neutrophil proliferation and cytotoxicity detection kit was used to investigate the protective effect of DTTZ chemotherapy.

The results are shown in Figure 1. At a concentration of 0.1 mM, DTTZ exhibited clear cell selectivity against the cytotoxicity caused by cisplatin and doxorubicin, protecting HIEC-6 and CHO cells, but had little protective effect against MCF-7, A549 and Hela cancer cells.

Example 2 EdU staining to detect the protective effect of DTTZ hydrochloride on the DNA synthesis process of normal cells during chemotherapy protection

EdU (5-ethynyl-2'-deoxyuridine), also known as 5-ethynyl-2'-deoxyuridine in Chinese, is a new type of thymidine analog. EdU can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis. If DNA synthesis is inhibited, the incorporation of EdU is also inhibited. Therefore, the inhibitory effect of the drug on cell DNA synthesis can be judged based on the number of cells stained with EdU. HIEC-6 and MCF-7 were seeded in a 24-well plate at a density of 50,000 cells/well for 24 hours, and then DTTZ or positive control WR2721 was used to incubate the cells for 15 minutes in advance, and the chemotherapy drug cisplatin was added to continue incubation. After 24 hours, the cells were treated with the BeyoClickTM EdU-488 detection kit and photographed under an inverted fluorescence microscope.

The results are shown in Figure 2. DTTZ hydrochloride at a concentration of 0.1 mM has a protective effect on the inhibition of DNA synthesis in normal cells caused by cisplatin, but has no such protective effect on cancer cells.

Example 3: Clone formation experiment to investigate the cytotoxicity of DTTZ and positive control WR2721

The cell clone formation experiment is an important technical method used to detect cell proliferation ability, invasiveness, population dependence, etc. Compared with cell survival experiments (such as MTT assay), the clone formation rate reflects the effect of drugs on cell proliferation more sensitively. If the genetic material of the cell is interfered by the drug, a single cell will not be able to undergo mitosis to form a clone population. Therefore, the clone formation experiment can be used to examine the toxicity of drugs to cells.

Four types of cells, including normal cells (HIEC-6), human kidney epithelial cells (293T) and cancer cells (MCF-7), human cervical cancer (Hela), were selected to investigate the cytotoxicity of DTTZ and positive control WR2721. The cells were resuspended in 10% complete medium as a single cell suspension, inoculated into a 12-well plate at a cell density of 500 cells/well, and gently rotated to disperse the cells evenly, and cultured in an incubator for 24 hours. After the cells adhered to the wall, 0.1mM DTTZ or WR2721 was given respectively, and the cells were incubated for 24 hours. After carefully rinsing with PBS twice, the cells were cultured for 1-2 weeks. Observe frequently, and terminate the culture when visible clones appear in the culture dish. Discard the supernatant and carefully rinse twice with PBS. After adding 4% paraformaldehyde to fix the cells for 15 minutes, remove the fixative, add an appropriate amount of crystal violet staining solution for 10-30 minutes, then slowly wash away the staining solution with ddH20 and dry.

The results are shown in Figure 3. At a concentration of 0.1 mM, DTTZ had no cloning inhibitory effect on cells, while WR2721 significantly inhibited the formation of four cell clones. This result shows that DTTZ is safer than the positive control WR2721 at the effective concentration of this protective effect.

Example 4 Annexin V-FITC apoptosis experiment to investigate the protective effect of DTTZ on cisplatin-induced apoptosis of normal cells

Apoptosis is a type of programmed cell death, which is regulated by a series of genes and proteins. Phosphatidylserine is mainly distributed on the inner side of the cell membrane. In the early stage of apoptosis, different types of cells will turn phosphatidylserine to the cell surface, which is then selectively bound by Annexin V. Therefore, Annexin V is an early marker of apoptosis. Propidium iodide (PI) can stain necrotic cells or cells that lose cell membrane integrity in the late stage of apoptosis. HIEC-6 and MCF-7 were seeded in 12-well plates at a density of 200,000 cells/well for 24 hours, and then the cells were incubated with DTTZ or positive control WR2721 for 15 minutes in advance, and the chemotherapy drug cisplatin was added for further incubation. After 24 hours, the cells were treated with the Annexin V-FITC apoptosis detection kit, and the cell apoptosis ratio was detected by flow cytometry.

The results are shown in Figure 4. DTTZ at a concentration of 0.1 mM has a significant protective effect on normal cell apoptosis induced by cisplatin, but has no such protective effect on cancer cell MCF-7.

Example 5 DTTZ hydrochloride treats hematopoietic damage in mice induced by cyclophosphamide

After reaching the required weight of the experiment, C57BL/6 mice were randomly divided into 4 groups, 10 mice in each group, namely blank control group (control), cyclophosphamide group (100 mg/kg CP), drug group (250 mg/kg DTTZ hydrochloride + 100 mg/kg CP), positive control group (250 mg/kg WR2721 + 100 mg/kg CP). Mice in the cyclophosphamide group were given 100 mg/kg CP for three consecutive days; mice in the drug group were given 250 mg/kg DTTZ for 5 consecutive days, and DTTZ was given to mice 30 minutes after intraperitoneal injection of cyclophosphamide on the first three days; the drug administration method of the positive control group was the same as that of the drug group. Each reagent was administered intraperitoneally, with an injection volume of 0.2 mL per mouse; the control group was intraperitoneally injected with 0.2 mL of normal saline. Peripheral blood, bilateral femurs, liver, spleen and thymus of mice were collected three days after drug withdrawal. The number of bone marrow nucleated cells (BMNC) was detected by a hemocytometer, and the bone marrow DNA content was determined by an ultraviolet spectrophotometer.

① Blood cell parameters: Three days after drug withdrawal, mice in each group were weighed and data were recorded. After anesthesia, peripheral blood was collected from the eyeballs and placed in 1.5 mL anticoagulant tubes for separation of lymphocytes in the peripheral blood. Single cell gel electrophoresis experiment of lymphocytes: Lymphocytes were separated using a peripheral blood lymphocyte separation kit (as much as possible within 4 hours), and then analyzed by gel electrophoresis.

② Bone marrow nucleated cell count (BMNC): After removing the eyeballs and collecting peripheral blood, mice in each group were killed by cervical dislocation. The femurs on both sides of the lower limbs of each mouse were taken. One of them was taken, and the proximal and distal ends of the femur were cut off. The bone marrow cells were gently washed out with 10mL CaCl2, filtered with gauze, and BMNC was detected using a blood cell counter. The other femur was used for indicator ③.

③ Bone marrow DNA content: The bone marrow cells of the other femur in indicator ② were gently rinsed out with 1 mL of PBS, first kept at 4°C for half an hour, and then centrifuged to remove the supernatant and add 5 mL of freshly prepared perchloric acid, and then kept in a 90°C water bath for 15 minutes. After taking out the centrifuge tube and filtering, the filtrate was measured for ultraviolet absorption wavelength at 268 nm.

④ Organ index of spleen and liver: After the mice were killed by cervical dislocation, in addition to taking peripheral blood and bilateral femurs, the spleen and liver were also taken and weighed and recorded. Liver (or spleen) index = liver (or spleen) weight (mg) / total weight of the mouse (g)

⑤ Number of spleen nodules (CFU-S): The spleen in indicator ④ was weighed and placed in the prepared fixative (45 mL of picric acid, 3 mL of glacial acetic acid and 15 mL of formaldehyde). After 24 hours, it was rinsed clean and the number of nodules on the specimen was counted with the naked eye.

The results are shown in Figure 5. DTTZ treatment can increase the weight loss of mice caused by cyclophosphamide, increase the number of bone marrow cells in mice, and significantly reduce the degree of DNA damage, indicating that DTTZ can reduce cyclophosphamide-induced bone marrow cell apoptosis. The organ indexes of spleen and liver, which are related to hematopoiesis, were reduced in all mouse groups treated with cyclophosphamide, while the spleen index recovered to varying degrees in mice injected with DTTZ and the positive control WR2721. The number of spleen nodules is an important indicator of spleen hematopoietic function. Under normal circumstances, mice have almost no spleen nodules. When a stress response occurs, the current hematopoietic stem cells in the spleen migrate to the surface of the organ and proliferate into spleen nodules. Cyclophosphamide treatment of mice causes a stress response. After cyclophosphamide treatment, the number of spleen nodules treated with DTTZ and the positive control WR2721 further increases, indicating that the number of existing hematopoietic stem cells migrating to the spleen for proliferation and differentiation increases, proving that DTTZ can alleviate and restore hematopoietic system damage caused by chemotherapy drugs.

Example 6 DTTZ hydrochloride does not affect the tumor-suppressing effect of cisplatin on tumor-bearing mice, and has a therapeutic and reversal effect on the liver, kidney, and gastrointestinal toxicity caused by cisplatin

After the Balb/c nude mice reached the required mass for the experiment, 2x10 ⁷ MCF-7 cell suspension 0.2 mL was subcutaneously inoculated. When the tumor diameter was > 8 mm, the tumor-bearing mice were randomly divided into 4 groups, 6 in each group, namely blank control group (control), cisplatin group (10 mg/kg), drug group (250 mg/kg DTTZ + 10 mg/kg cisplatin), and positive control group (250 mg/kg WR2721 + 10 mg/kg cisplatin). The mice in the cisplatin group were given 10 mg/kg cisplatin for three consecutive days; the drug group was intraperitoneally injected with 10 mg/kg cisplatin for 3 consecutive days, and then 250 mg/kg DTTZ was given 30 minutes later; the positive control group was administered in the same way as the drug group. Each reagent was administered intraperitoneally, with an injection volume of 0.2 mL per mouse; the control group was intraperitoneally injected with 0.2 mL of normal saline. Each group of mice was weighed and the data was recorded. The short diameter (a) and long diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated: tumor volume (mm ³ ) = 0.52*a ² *b. Starting from the first day of administration, the tumor inhibition rate was calculated on the fifth day of initial administration: tumor inhibition rate = (control group - drug administration group) / control group * 100%. Organ index of kidney and liver = liver (or kidney) weight (mg) / total weight of the mouse (g)

As shown in Figure 6, after cisplatin administration, the weight of tumor-bearing mice decreased significantly (Figure A). DTTZ and the positive control WR2721 had a certain alleviating effect on the weight loss of mice, and the alleviating effect of DTTZ was more significant. At the same time, the tumor volume (Figure B) and the tumor inhibition rate on the 5th day of administration (Figure C) showed that the administration of DTTZ and WR2721 30 minutes after cisplatin did not affect the inhibitory effect of cisplatin on tumors. The cisplatin treatment group caused certain hepatorenal toxicity (Figures D and E), which was manifested as a decrease in organ index and a shortening of the colon length of mice. After DTTZ administration, the decrease in liver and kidney organ index caused by cisplatin was significantly alleviated, and the colon length of mice was restored, reducing the occurrence of colon inflammation. The results of organ HE staining showed (Figure 7) that after DTTZ administration, it had a significant protective effect on the intestinal, kidney and liver damage caused by chemotherapy, and significantly reduced the crypt cell damage caused by chemotherapy.

Example 7 Protective effect of DTTZ hydrochloride on chemotherapeutic drug toxicity in mice

C57BL/6J mice were adaptively raised for 2-3 days, and 36 mice were randomly divided into 3 groups (12 mice in each group), namely blank control group (control), simple cisplatin group (10 mg/kg/day), cisplatin + drug group (500 mg/kg/day DTTZ). The cisplatin group was gavaged with cisplatin for two consecutive days; the mice in the cisplatin + drug group were gavaged with DTTZ hydrochloride saline solution 30 minutes before and 24 hours after cisplatin administration, respectively, and the blank control group was only gavaged with 0.2 mL of saline.

The results are shown in Figure 8 , and DTTZ can significantly prolong the 30-day survival rate of chemotherapy mice.

Claims (3)

1. Use of dttz or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and treatment of damage to an organism by a cisplatin anti-tumor chemotherapeutic, wherein the damage to an organism is: renal disease related to anti-tumor chemotherapy, liver disease related to anti-tumor chemotherapy, and gastrointestinal disease related to anti-tumor chemotherapy.
2. 2. The use according to claim 1, wherein the organism is: a human or an animal.
3. 3. The use according to claim 1 or 2, wherein the DTTZ therapeutically effective dose is 10mg/m, calculated as body surface area ² —2000 mg/m ² 。