WO2023274316A1

WO2023274316A1 - Application of deuterated plinabulin in preparing drug for treating neutropenia

Info

Publication number: WO2023274316A1
Application number: PCT/CN2022/102466
Authority: WO
Inventors: 李文保; 丁忠鹏; 李飞飞; 彭超; 古敏晴; 徐赟; 王新文
Original assignee: 深圳华泓海洋生物医药有限公司
Priority date: 2021-06-29
Filing date: 2022-06-29
Publication date: 2023-01-05
Also published as: CN115531385A

Abstract

An application of deuterated plinabulin in preparing a drug for treating neutropenia. Disclosed is a method for treating and/or preventing neutropenia, comprising administering to a subject in need thereof a therapeutically effective amount of deuterated plinabulin as shown in formula (I), or a pharmaceutically acceptable salt thereof. The deuterated plinabulin as shown in formula (I) or a pharmaceutically acceptable salt thereof may be used to treat and/or prevent neutropenia, ameliorating toxic side effects of clinical tumor treatment.

Description

Application of deuterated Plinabulin in preparation of medicine for treating neutropenia

This application claims the priority of Chinese patent application 2021107264388 with the filing date of 2021/6/29. This application cites the full text of the above-mentioned Chinese patent application.

technical field

The invention relates to the fields of chemistry and medicine, in particular to the application of deuterated plinabulin in the preparation of medicines for treating neutropenia.

Background technique

Plinabulin (plInabulIn) is derived from the natural product Phenylahistin isolated from the marine fungus Aspergillus pylorus, and is a new type of 2,5-diketopiperazine (DKP) heterocyclic compound. It is a colchicine-like tubulin inhibitor that prevents microtubule assembly by affecting the dynamic cycle of microtubule depolymerization-polymerization, thereby interfering with cell division. At the same time, Plinabulin is also a novel vascular disruptor that selectively destroys the tumor vascular endothelial structure to achieve its anti-tumor activity. Due to the good effect of Plinabulin in the treatment of neutropenia, Plinabulin has obtained the breakthrough therapy certification in China and the United States at this stage, and has applied for a new drug NDA.

Neutropenia is a common hematological toxicity during chemotherapy. Accurate assessment, prevention and treatment are critical to the prognosis of cancer patients. Chemotherapy is one of the main treatments for malignant tumors. Myelosuppression is the most common dose-limiting toxicity of chemotherapy, among which neutropenia is very common. Neutropenia is a common and potentially life-threatening complication of cytotoxic myelosuppressive chemotherapy. Studies have shown that individuals with neutropenia are more susceptible to infection, and complications are likely to be life-threatening. The main method for the existing treatment of neutropenia is granulocyte colony-stimulating factor (G-CSF) drug therapy, G-CSF CSF is a biological drug, which has disadvantages such as high price, inconvenient clinical use, and limited effect in the first week after chemotherapy.

Contents of the invention

The technical problem to be solved by the present invention is to provide a kind of application of deuterated plinabulin in the preparation of medicine for the treatment of neutropenia in order to overcome the deficiency of the lack of drugs for effectively treating neutropenia in the prior art .

The present invention solves the above technical problems through the following technical solutions:

The present invention provides an application of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of medicines for treating and/or preventing neutropenia;

The present invention also provides a method for treating and/or preventing neutropenia, which comprises administering a therapeutically effective amount of deuterated plinabulin represented by formula (I) or its pharmaceutical preparation to an individual in need. acceptable salt;

In the above-mentioned application or the above-mentioned method:

In some embodiments, the pharmaceutically acceptable salt of deuterated plinabulin represented by formula (I) is:

In some embodiments, the neutropenia is induced by administering chemotherapy or by administering radiation therapy.

In some embodiments, the chemotherapy comprises administering a chemotherapeutic composition of one or more chemotherapeutic agents.

In some embodiments, the chemotherapeutic composition is docetaxel, paclitaxel, cyclophosphamide, "combination of docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinca combination of doxorubicin, doxorubicin, and cyclophosphamide” or “a combination of docetaxel and cyclophosphamide”. Among them, docetaxel, paclitaxel, cyclophosphamide and other drugs alone, "docetaxel, doxorubicin and cyclophosphamide (TAC)", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide Phosphamide" or "docetaxel and cyclophosphamide (TC)" and other combinations. For example, the chemotherapeutic composition is cyclophosphamide.

In some embodiments, the neutropenia is induced by chemotherapy or radiation therapy treating an individual with liver, pancreatic, lung, breast, colon, or prostate cancer.

In some embodiments, the individual in need thereof has liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, or prostate cancer.

In some embodiments, when the neutropenia is induced by administering chemotherapy, the method comprises administering a single dose of deuterated plinabulin represented by formula (I) or its pharmaceutically acceptable salt.

In some embodiments, when the neutropenia is induced by administering chemotherapy, the method comprises administering deuterated plinabulin as represented by formula (I) after administering the chemotherapeutic composition or a pharmaceutically acceptable salt thereof.

In some embodiments, when the neutropenia is induced by administration of cyclophosphamide, in each treatment cycle, the deuterated plinabulin represented by formula (I) or its pharmaceutically acceptable The administration dose of the salt is less than 60mg/kg.

In some embodiments, when the neutropenia is induced by administration of cyclophosphamide, the method comprises administration of deuterated prunab as represented by formula (I) within 24 hours after administration of cyclophosphamide Lin or a pharmaceutically acceptable salt thereof.

In some embodiments, when the neutropenia is induced by administration of cyclophosphamide, the method comprises administration of deutepurina represented by formula (I) within about 12 hours after administration of cyclophosphamide Bollin or a pharmaceutically acceptable salt thereof is administered, for example, 0.5 hours later.

In some embodiments, when said neutropenia is caused by administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide " or "docetaxel and cyclophosphamide" induction, in each treatment cycle, the administration dose of deuterated plinabulin or its pharmaceutically acceptable salt shown in formula (I) is less than 60mg/ kg.

In some embodiments, when said neutropenia is caused by administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide " or "docetaxel and cyclophosphamide" induction, the method includes administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or "docetaxel and cyclophosphamide" within 24 hours after administration of deuterated plinabulin or a pharmaceutically acceptable salt thereof as represented by formula (I).

In some embodiments, the deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof can be administered by any suitable route in the art, including oral administration, injection (such as intravenous, intramuscular , subcutaneous), etc., for example, by intravenous infusion.

In some embodiments, the deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof can be administered according to the body weight of the individual, and the non-limiting example range can be 0.5-60 mg/kg (single dose), such as 0.5-20 mg/kg (single dose), specifically, the administration dose may be 1.875 mg/kg, 3.75 mg/kg, 7.5 mg/kg or 10 mg/kg.

In some embodiments, the above dosage of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof can be administered at least once a week, for example, every 7 days 1 time.

In some embodiments, the method comprises simultaneously administering deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more G-CSF drugs.

In some embodiments, when the individual in need has liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, or prostate cancer, the method includes identifying a patient with said liver cancer, pancreatic cancer, lung cancer, breast cancer, A patient with colon cancer or prostate cancer; and administering deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof at a dose of about 0.5 mg/kg to about 60 mg/kg.

The present invention also provides a pharmaceutical composition, which comprises about 0.5 mg to about 60 mg of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical composition comprises about 0.5 mg to about 10 mg of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are hereby incorporated by reference in their entirety. If there are multiple definitions of a term herein, the definition in this section controls unless otherwise stated.

"Individual" as used herein refers to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate or bird, such as a chicken, and any other vertebrate or invertebrate.

As used herein, an "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent effective to the extent that it relieves one or more symptoms of a disease or condition or reduces the likelihood of its onset, and includes curing the disease or condition.

The term "prevention" refers to treating an individual who has not yet exhibited symptoms of a disease or condition, but is susceptible to or at risk for a particular disease or condition, whereby the treatment reduces the likelihood of the patient developing the disease or condition.

The term "treatment" refers to the treatment of an individual already suffering from a disease or condition.

The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compound and which is not biologically or otherwise undesirable for use in medicine. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Organic acids from which salts can be derived include, for example, acetic, propionic, glycolic, pyruvic, oxalic, maleic, malonic, succinic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic , Methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Inorganic and organic bases can also be used to form pharmaceutically acceptable salts. Inorganic bases from which salts can be derived include, for example, bases containing sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc.; particularly preferred are the ammonium, potassium, sodium, calcium, salt and magnesium salt. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, in particular isopropylamine, trimethylamine, di Ethylamine, Triethylamine, Tripropylamine, and Ethanolamine.

The positive progress effect of the present invention is: deuterated plinabulin or its pharmaceutically acceptable salt as shown in formula (I) can be used for the treatment and/or prevention of neutropenia, especially for the treatment of doxene Neutropenia caused by chemotherapeutic drugs such as taxel, doxorubicin and cyclophosphamide (TAC) or docetaxel and cyclophosphamide (TC), improve the toxic and side effects of clinical tumor treatment. Experiments have been carried out with thiopegfilgrastim (PEG-rhG-CSF) as a control group, and the deuterated plinabulin or its pharmaceutically acceptable salt has little damage to leukocytes and neutrophils, and has little effect on neutrophils. The therapeutic effect of neutropenia is better than that of plinabulin.

Description of drawings

Fig. 1 is the body weight change curve of male SD rats;

Fig. 2 is the body weight change curve of female SD rats;

Fig. 3 is the change curve of the white blood cell quantity of male SD rat;

Fig. 4 is the change curve of the number of white blood cells in female SD rats;

Fig. 5 is the change curve of the number of neutrophils in male SD rats;

Fig. 6 is the change curve of the number of neutrophils in female SD rats;

Fig. 7 is the thymus coefficient of male SD rat;

Fig. 8 is female SD rat thymus coefficient;

Fig. 9 is the body weight change curve of SD rats;

Figure 10 is the change curve of the number of white blood cells in SD rats;

Fig. 11 is the change curve of the number of neutrophils in SD rats;

Fig. 12 is SD rat thymus coefficient;

Fig. 13 is the body weight change curve of SD rats;

Fig. 14 is the body weight change curve of SD rats;

Figure 15 is the change curve of the number of white blood cells in SD rats;

Figure 16 is the change curve of the number of white blood cells in SD rats;

Fig. 17 is the change curve of the number of neutrophils in SD rats;

Fig. 18 is the change curve of the number of neutrophils in SD rats;

Fig. 19 is SD rat thymus coefficient;

Fig. 20 is SD rat thymus coefficient;

Fig. 21 is the body weight change curve of SD rats;

Figure 22 is the change curve of the number of white blood cells in SD rats;

Figure 23 is the change curve of the number of neutrophils in SD rats;

Figure 24 is SD rat thymus coefficient;

Fig. 25 is the body weight change curve of SD rats;

Figure 26 is the change curve of the number of white blood cells in SD rats;

Figure 27 is the change curve of the number of neutrophils in SD rats;

Figure 28 is SD rat thymus coefficient;

Fig. 29 is the body weight change curve of SD rats;

Figure 30 is the change curve of the number of white blood cells in SD rats;

Figure 31 is the change curve of the number of neutrophils in SD rats;

Figure 32 is the SD rat thymus coefficient.

detailed description

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples.

Deuterated plinabulin represented by formula (I) can be prepared and purified according to the methods and procedures detailed in Chinese invention patents ZL201510293269.8, ZL201610224082.7 and ZL201610664088.6, which are incorporated herein by reference in their entirety.

Preparation Example 1

(3Z,6Z)-3-Benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione (deutero Nabulin) synthesis

1) Synthesis of known compound 5-tert-butyl-1H-imidazole-4-carbaldehyde (aldehyde intermediate a):

The synthesis steps are as follows: the first step is to close the ring to form an oxazole ring; the second step is to heat treatment in formamide to form an imidazole ring; and then reduce and oxidize to obtain an imidazole aldehyde compound.

Its specific preparation process comprises the following steps:

(1) Dissolve ethyl isocyanoacetate (5.8mL, 53mmol) in 90mL dry tetrahydrofuran, add DBU (9.5mL, 63.6mmol), then slowly add trimethylacetic anhydride (12.9mL, 63.6mmol) , react overnight at room temperature, spin to dry the solvent, extract with ethyl acetate, rinse with 10% sodium carbonate solution, then rinse with 10% citric acid solution, and dry over anhydrous sodium sulfate. The solvent was spin-dried, separated and purified by column chromatography, petroleum ether: ethyl acetate = 15:1 as the eluent, and 10.3 g of the oxazole compound was obtained with a yield of 99%.

(2) Add oxazole compound (8.1g, 41mmol) into a 250mL round bottom flask, then add 60mL formamide, heat the reaction at 165°C, add 10% sodium carbonate to quench the reaction after 24h, extract with ethyl acetate, anhydrous Na2SO4 dried. The solvent was spin-dried, separated and purified by column chromatography, and dichloromethane:methanol=60:1 was used as the eluent to obtain 4.4 g of imidazole compound with a yield of 55%.

(3) Dissolve the imidazole compound (830 mg, 4.2 mmol) in dry tetrahydrofuran, add lithium tetrahydrogen aluminum (479 mg, 12.6 mmol) at -5°C, rise to room temperature for 4 h after 0.5 h, add water to quench, sand The core funnel was suction filtered, and the filtrate was spin-dried for the next reaction directly. Dissolve the spin-dried reduction product in 20 mL of dry acetone, add manganese dioxide (3.6 g, 42 mmol), react overnight at room temperature, filter with sand core funnel, and spin the solvent to obtain compound 5-tert-butyl-1H- Imidazole-4-carbaldehyde 351 mg, two-step yield 55%.

2) Preparation of intermediate 5-tert-butyl-1H-imidazole-4-deuteroformaldehyde (deuteroaldehyde compound b), the structural formula is as follows:

structural formula

synthetic route

The specific preparation process includes the following steps: Weigh 5-tert-butyl-1H-imidazole-4-carbaldehyde (304mg, 2mmol) into a 50mL dry single-necked bottle, and replace the nitrogen protection. Add 5 mL of dry ethanol and sodium borodeuteride (420 mg, 10 mmol) to the reaction bottle, replace the nitrogen protection, react overnight at room temperature, add 10 mL of water to quench the reaction, extract the organic phase with 30 mL of ethyl acetate, spin dry and put it directly into the next reaction , add 10mL of dry acetone and manganese dioxide (1.7g, 20mmol), react overnight at room temperature, filter with sand core funnel, and spin the filtrate to dry column chromatography to obtain 5-tert-butyl-1H-imidazole-4-deuterium Formaldehyde (199 mg, 1.3 mmol), a white solid, yield 65%.

¹ H NMR (400MHz,DMSO-d ₆ )δ6.97(s,1H),6.66(s,1H)1.05(s,9H); MS(ESI)m/z 154.10(M+H) ⁺ (calcd for C ₈ H ₁₂ DN ₂ O 154.10).

3) (Z)-1-acetyl-3-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione (deuterium-containing heterocyclic compound d) preparation, the structural formula is as follows:

Its preparation method is: 5-tert-butyl-1H-imidazole-4-deuteroformaldehyde (306mg, 2mmol), 1,4-diacetylpiperazine-2,5-dione (792mg, 4mmol), without Magnesium sulfate water (800mg), DMF (9mL), and cesium carbonate (977mg, 3mmol) were placed in a reaction flask, protected by nitrogen, and reacted at room temperature for 18 hours (the reaction was complete as detected by TLC). The reaction solution was poured into cold water, solids were precipitated, and filtered to obtain 306 mg of off-white solid, which was the (Z)-1-acetyl-3-((5-tert-butyl-1H-imidazol-4-yl) Deuterated methylene) piperazine-2,5-dione, the yield is 52.58%.

¹ H NMR (400MHz,DMSO-d ₆ )δ12.37(s,1H),12.00(s,1H),7.85(s,1H),4.31(s,2H),2.48(s,3H),1.39( s, 9H); MS (ESI) m/z 292.14 (M+H) ⁺ (calcd for C ₁₄ H ₁₈ DN ₄ O ₃ , 292.14).

4) Preparation of (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione

The structural formula of the compound is as follows:

Its preparation method is: (Z)-1-acetyl-3-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione (150mg , 0.51mmol), benzaldehyde (109mg, 1.03mmol), anhydrous magnesium sulfate (210mg, 1.74mmol), DMF (5mL), cesium carbonate (420mg, 1.29mmol) were placed in a reaction flask, nitrogen protection, 45 ° C reaction After 21 hours, TLC detected that the reaction was complete. The reaction solution was added to cold water, a yellow solid was precipitated, filtered, the filter cake was dissolved in a mixed solvent of absolute ethanol and ethyl acetate, the insoluble matter was filtered off, concentrated to dryness under reduced pressure, and 63 mg of a yellow solid was obtained, which was the compound 1, yield 36.65%.

¹ H NMR (400MHz,DMSO-d ₆ )δ12.31(s,1H),12.22(s,1H),10.00(s,1H),7.84(s,1H),7.52(d,J＝8Hz,2H ), 7.39(t, J=8Hz, 2H), 7.32(t, J=8Hz, 1H), 6.73(s, 1H), 1.37(s, 9H); MS(ESI) m/z 338.1715(M+H ) ⁺ (calcd for C ₁₉ H ₂₀ DN ₄ O ₂ , 338.1722).

Preparation Example 2

(3Z,6Z)-3-Benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione hydrochloride preparation

Its specific preparation process includes the following steps: take (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl) deuterated methylene) piperazine-2 , 5-diketone (100mg, 0.30mmol) was dissolved in 3ml of acetone, and hydrochloric acid (16mg, 0.44mmol) diluted with acetone was added dropwise. Stir the reaction at room temperature for 1.5 h, filter with suction, and wash the filter cake with acetone to obtain a white solid, which is salted with hydrochloric acid at a molar ratio of 1:1, with a yield of 72%. mp290-291℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ13.07(brs,1H),11.61(brs,1H),10.19(s,1H),8.36(brs,1H),7.51(d, J＝7.7Hz, 2H), 7.41(t, J＝7.6Hz, 2H), 7.31(t, J＝7.3Hz, 1H), 6.77(s, 1H), 1.35(s, 9H), by X-ray powder Diffraction test characterization, see patent ZL201610224082.7 for details.

Preparation Example 3

(3Z,6Z)-3-Benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione methanesulfonate preparation of

Its specific preparation process includes the following steps: take (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl) deuterated methylene) piperazine-2 , 5-diketone (200mg, 0.59mmol) was dissolved in 4ml of acetone at room temperature, and methanesulfonic acid (86mg, 0.89mmol) diluted with acetone was added dropwise. Stir the reaction at room temperature for 1.5 h, filter with suction, and wash the filter cake with acetone. 229 mg of white solid was obtained, the yield was 89%, and it was salted with methanesulfonic acid at a molar ratio of 1:1. mp303-305℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ12.92(brs,1H),11.69(brs,1H),10.20(s,1H),8.31(brs,1H),7.53(d, J＝7.6Hz, 2H), 7.43(t, J＝7.7Hz, 2H), 7.33(t, J＝7.3Hz, 1H), 6.78(s, 1H), 2.33(s, 3H), 1.37(s, 9H), characterized by X-ray powder diffraction test, see patent ZL201610224082.7 for details.

Preparation Example 4

(3Z,6Z)-3-Benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione oxalate preparation

Its specific preparation process includes the following steps: take (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl) deuterated methylene) piperazine-2 , 5-diketone (200mg, 0.59mmol) was dissolved in 4ml of acetone at room temperature, and oxalic acid (112mg, 0.89mmol) was slowly added. Stir the reaction at room temperature for 1.5 h, filter with suction, and wash the filter cake with acetone. 219 mg of white solid was obtained, the yield was 80%, and it was salted with oxalic acid at a molar ratio of 2:1. mp262-263℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ12.33(s, 1H), 12.23(s, 1H), 10.01(s, 1H), 7.86(s, 1H), 7.53(d, J＝7.6Hz, 2H), 7.42(t, J＝7.6Hz, 2H), 7.32(t, J＝7.4Hz, 1H), 6.75(s, 1H), 1.39(s, 9H), by X-ray powder Diffraction test characterization, see patent ZL201610224082.7 for details.

Effect Example 1 Single intraperitoneal injection of cyclophosphamide induces neutropenia model exploratory test in rats

1 Experimental materials

1.1 Experimental system

1.1.1 Experimental animals

Use SD rats, 6-8 weeks old, with individual body weight within ±20% of the average body weight, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011;

1.1.2 Feeding conditions

5 mice per cage, rearing temperature is room temperature 16-26 ℃ (diurnal temperature difference ≤ 4 ℃), 12/12 hours day and night alternating light and dark (turn off the light at 07:30, turn off the light at 19:30). Rats and mice were fed maintenance feed, which was purchased from Beijing Keao Xieli Feed Co., Ltd. Animals were free to ingest feed and drinking water for experimental animals.

1.2 Main reagents

Name: Cyclophosphamide for Injection

Manufacturer: Baxter Oncology GmbH

Batch number: OJ415A

Appearance: white crystal or crystalline powder

Specification: 0.2g/bottle

Storage conditions and matters needing attention: 5℃～25℃

Solvent: 0.9% Sodium Chloride Injection

Preparation method: Add an appropriate amount of sterile 0.9% sodium chloride injection into the drug bottle containing the drug powder, shake it slightly to make it fully dissolve and mix to form a solution of the corresponding concentration. If the dry powder cannot be completely dissolved immediately, the solution can be statically Leave it for a few minutes until it is completely clear (the whole process needs to be operated in a sterile environment).

1.3 Experimental Instruments

Name: Automatic five-differentiation blood analyzer;

Manufacturer: Siemens, Germany;

Model: ADVIA 2120i;

Instrument number: SEC-0002/SEC-0003.

2 Test methods and detection indicators

2.1 Animal grouping

There were 5 groups in the test, as shown in Table 1, which were normal control group, 12.5mg/kg cyclophosphamide group, 25mg/kg cyclophosphamide group, 50mg/kg cyclophosphamide group, and 100mg/kg cyclophosphamide group . There are 10 rats in each group, half male and half male, 50 rats in total. Rats with uniform levels of neutrophils were randomly divided into groups before modeling.

Table 1 Grouping of experimental animals

2.2 Dosage design and administration

In this test, 12.5mg/kg cyclophosphamide group, 25mg/kg cyclophosphamide group, 50mg/kg cyclophosphamide group, and 100mg/kg cyclophosphamide group were injected intraperitoneally at 12.5, 25, 50, and 100mg/kg respectively. The normal control group was given an equal volume of vehicle 0.9% sodium chloride by intraperitoneal injection; the specific dosage design is shown in Table 2.

Table 2 Dosage Design

2.3 Detection indicators

General state observation and weighing were performed once a day after administration, and hematological tests were performed once a day before and after administration. On the 15th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed and its organ coefficient (weight of organ per 10g body weight (mg)) was calculated.

3 Experimental results

3.1 General Observations

The general state of animals in each group was acceptable, their autonomous activities were normal, and their feces and urine were normal.

3.2 Weight

As shown in Figure 1 and Figure 2, as shown in Table 3 and Table 4, carry out weighing observation after the test begins, compared with normal control group, intraperitoneal injection of cyclophosphamide each group female and male rat body weight has a downward trend, but No significant difference.

Table 3 The effect of a single intraperitoneal injection of cyclophosphamide on the body weight of male SD rats (g/only)

Table 4 Effect of single intraperitoneal injection of cyclophosphamide on body weight of female SD rats (g/only)

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 3 and Table 5, compared with the normal control group, after the intraperitoneal injection of cyclophosphamide in male SD rats, the white blood cell count in the 12.5mg/kg cyclophosphamide group was significantly higher on the 1st, 2nd, 3rd, and 4th days. decreased (P≤0.05), and the white blood cell count increased significantly on the 13th day (P≤0.05). In the 25mg/kg cyclophosphamide group, the white blood cell count decreased significantly (P≤0.05) on the 1st to 7th day, and in the 50mg/kg cyclophosphamide group, the white blood cell count decreased significantly on the 1st to 7th, 10th, and 14th day (P≤0.05). In the 100mg/kg cyclophosphamide group, the white blood cell count decreased significantly (P≤0.05) on the 1st to 9th day, and the white blood cell count increased significantly on the 13th day (P≤0.05). There is a dose correlation between each dose group.

As shown in Fig. 4 and table 6, compare with normal control group, after female SD rat cyclophosphamide intraperitoneal injection model, 12.5mg/kg cyclophosphamide group is in the 1st, 2nd, 4 days leukocyte counts significantly decline ( P≤0.05), the 25mg/kg cyclophosphamide group significantly decreased the white blood cell count on the 1st to 5th and 7th day (P≤0.05), and the 50mg/kg cyclophosphamide group significantly decreased the white blood cell count on the 1st to 6th and 10th day ( P≤0.05), the white blood cell count in the 100mg/kg cyclophosphamide group decreased significantly from day 1 to 12 (P≤0.05), and there was a dose-related relationship between each dose group.

Table 5 Effect of single intraperitoneal injection of cyclophosphamide on white blood cell count in male SD rats (WBC×10 ⁹ /L)

Note: ^P≤0.05, 12.5mg/kg cyclophosphamide group vs normal control group; *P≤0.05, **P≤0.01, ***P≤0.001, 25mg/kg cyclophosphamide group vs normal control group; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001, 50mg/kg cyclophosphamide group vs normal control group; ^& P≤0.05, ^&& P≤0.01, ^&&& P≤0.001, 100mg/kg cyclophosphamide Amide group vs normal control group;

Table 6 Effect of single intraperitoneal injection of cyclophosphamide on white blood cell count in female SD rats (WBC×10 ⁹ /L)

Note: ^P≤0.05, ^P≤0.01, 12.5mg/kg cyclophosphamide group vs normal control group; *P≤0.05, **P≤0.01, ***P≤0.001, 25mg/kg cyclophosphamide group vs normal control group; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001, 50mg/kg cyclophosphamide group vs normal control group; ^& P≤0.05, ^&& P≤0.01, ^&&& P≤0.001, 100mg/kg cyclophosphamide group vs normal control group;

3.3.2 Results of neutrophil count (NEUT)

As shown in Table 7 and Figure 5, compared with the normal control group, male SD rats cyclophosphamide intraperitoneal injection model, 12.5mg/kg cyclophosphamide group in the first and second day neutrophil count significantly decreased ( P≤0.05), the 25mg/kg cyclophosphamide group significantly decreased the neutrophil count on the 1st to 4th day (P≤0.05), and the 50mg/kg cyclophosphamide group significantly decreased the neutrophil count on the 1st to 6th day decreased (P≤0.05), the 100mg/kg cyclophosphamide dose group significantly decreased the neutrophil count on the 2nd to 7th day (P≤0.05), and significantly increased the neutrophil count on the 10th to 14th day ( P≤0.05).

As shown in Table 8 and Figure 6, compared with the normal control group, female SD rats were intraperitoneally injected with cyclophosphamide for modeling, and the neutrophil count in the 12.5mg/kg cyclophosphamide group did not significantly decrease, and on the ninth day Significantly increased (P≤0.05). In the 25mg/kg cyclophosphamide group, the neutrophil count decreased significantly on the 2nd and 6th day (P≤0.05), and increased significantly on the 9th and 14th day (P≤0.05). In the 50mg/kg cyclophosphamide group, the neutrophil count decreased significantly on the 2nd and 4th day (P≤0.05), and increased significantly on the 8th, 9th, 11th, 12th and 14th day (P≤0.05). In the 100mg/kg cyclophosphamide dose group, the neutrophil count decreased significantly on the 2nd to 8th day (P≤0.05).

Table 7 Effect of single intraperitoneal injection of cyclophosphamide on neutrophil count in male SD rats (NEUT×10 ⁹ /L)

Note: ^P≤0.05, ^^P≤0.01, 12.5mg/kg cyclophosphamide group vs normal control group; *P≤0.05, **P≤0.01, ***P≤0.001, 25mg/kg cyclophosphamide group vs normal control group; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001, 50mg/kg cyclophosphamide group vs normal control group; ^&& P≤0.01, ^&&& P≤0.001, 100mg/kg cyclophosphamide group Phosphoramide group vs normal control group;

Table 8 Effect of single intraperitoneal injection of cyclophosphamide on neutrophil count in female SD rats (NEUT×10 ⁹ /L)

Note: ^P≤0.05, 12.5mg/kg cyclophosphamide group vs normal control group; *P≤0.05, **P≤0.01, 25mg/kg cyclophosphamide group vs normal control group; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001, 50mg/kg cyclophosphamide group vs normal control group; ^& P≤0.05, ^&& P≤0.01, 100mg/kg cyclophosphamide group vs normal control group

3.4 Thymus weight and thymus coefficient

The experimental data are shown in Table 9. On the 15th day of the experiment, the thymus was taken from each animal, weighed and the organ coefficient was calculated. The results show that, as shown in Figure 7 and Figure 8, compared with the normal control group, after the male SD rats were modeled with cyclophosphamide, there was no significant difference in the thymus coefficient of each dose group; after the female SD rats were modeled with cyclophosphamide, The thymus coefficient in the 25mg/kg cyclophosphamide group decreased significantly (P≤0.001), and there was no significant difference in the thymus coefficient in the other dosage groups.

Table 9 Effect of single intraperitoneal injection of cyclophosphamide on rat thymus coefficient

Note: **P≤0.01, 25mg/kg cyclophosphamide group vs normal control group.

Effect Example 2 Drug effect test of intravenous infusion of deuterated plinabulin on rat neutropenia induced by intraperitoneal injection of cyclophosphamide.

1 Experimental materials are the same as effect example 1

1.2 Main reagents

1.2.1 Name: Deuterated Plinabulin, prepared from Preparation Example 1;

Storage conditions and precautions: -15 ~ -40 ℃ storage;

Preparation method: Each preparation is prepared according to the actual use requirement and the proportion of the prescription. The specific preparation process is as follows:

1) Under strict light-proof conditions, take 3mL of 1,2-propylene glycol, add 20mg of deuterated plinabulin at 60°C to dissolve, keep stirring for 0.25-0.5h after the raw material drug is completely dissolved;

2) Continue to keep warm and stir, add 1mL HS-15, stir for 10min, then add 1mL HS-15;

3) Under the condition of heat preservation, after the medicinal liquid is sterilized by a 0.22 μm microporous membrane filter, it is prepared into a concentrated solution with a concentration of 4 mg/mL;

4) Take an appropriate amount of concentrated solution and dilute to the corresponding concentration with 5% glucose solution;

Preparation frequency: single preparation, injection diluent needs to be prepared on the day of administration (precipitation will occur when the concentration is slightly higher, please pay attention to observation, if precipitation occurs, it needs to be re-prepared), concentrated solution can be prepared once a week;

Temporary storage conditions and expiry date after preparation: store in a dry place in the dark at room temperature, transport it to the animal room in the dark, and use it immediately;

Temporary storage conditions and expiry date after preparation of subpackages: Complete administration in the dark within 6 hours after preparation of the injection diluent;

1.2.2 Name: Cyclophosphamide for Injection, with the same effect as Example 1.

1.2.3 Name: 1,2-propanediol

Manufacturer: Jiangxi Ipsen Pharmaceutical Co., Ltd.

Batch number: 20210502

Specification: 500g/bottle

Validity period: April 2023

Storage conditions and precautions: store at room temperature and avoid light;

1.2.4 Name: HS-15

Manufacturer: BASF

Batch number: 30744347G0

Specification: 1.0kg/bottle

Expiration date: September 07, 2022

Storage conditions and precautions: store at room temperature and avoid light;

1.3 Experimental equipment with the same effect as Example 1

2 Test methods and detection indicators

2.1 Animal grouping

The experiment consisted of 4 groups, each with 8 male SD rats, which were the normal control group, the deuterated plinabulin single administration group, the model control group, and the deuterated plinabulin treatment group. Rats with uniform levels of neutrophils were randomly divided into groups, and the specific group information is shown in Table 10.

Table 10 Experimental animal grouping table

2.2 Dosage design and administration

After the grouping in this experiment, except the normal control group and the deuterated plinabulin single-administration group were given normal saline, the animals in the other groups were given intraperitoneal injection of cyclophosphamide for modeling, the modeling dose was 100 mg/kg, and the administration volume 10mL/kg; after the non-modeling group animals were given normal saline for 30min, the normal control group was given 5% glucose solution, and the deuterated plinabulin single administration group was given the test product deuterated plinabulin at 7.5mg/kg , intravenous infusion for 15 minutes; the rest of the model-making groups were given modeling drugs for 30 minutes, the model control group was given 5% glucose solution by tail vein injection, and the deuterated plinabulin treatment group was given the test product deuterated at 7.5 mg/kg. Plinabulin, intravenous infusion 15min.

After blood sampling on the 14th day, the normal control group was injected with normal saline for 30 minutes and then injected with 5% glucose solution through the tail vein; the deuterium plinabulin alone group was given normal saline for 30 minutes after intraperitoneal injection with 7.5 mg/kg of deuterium Daprenabuline, intravenous infusion 15min. The model control group and the deuterated plinabulin treatment group were given intraperitoneal injection of cyclophosphamide for the second modeling, the modeling dose was 100 mg/kg, and the administration volume was 10 mL/kg; after 30 minutes of administration of the modeling drug, the model control The group was given 5% glucose solution by tail vein injection, and the deuterated plinabulin treatment group was given the test product deuterated plinabulin at 7.5 mg/kg, intravenously infused for 15 minutes.

The specific modeling and dosage design are shown in Tables 11 and 12.

Table 11 Modeling dose design table

Table 12 Dosage design table

2.3 Detection indicators

After the administration, the general state was observed once a day, the body weight was measured twice a week after the administration, and the blood test was performed once a day before and after the administration. On the 28th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed and its organ coefficient (the weight of the organ per 10 g body weight (mg)) was calculated.

3 Experimental results

3.1 General Observations

During the test, in addition to the normal control group and the deuterated plinabulin single administration group, the model control group and the deuterated plinabulin treatment group all had symptoms such as hypoactivity, sparse back hair, shapeless feces, perianal filth, etc. symptom.

3.2 Weight

As shown in Figure 9 and Table 13, from the administration to the end of the experiment, the body weights of the rats in the normal control group and the deuterated plinabulin alone administration group were equivalent, showing a gradual increase trend. Compared with the normal control group, the body weight of the model control group decreased significantly on the 5th to 12th and 16th to 23rd days after administration of the model (P≤0.05).

Compared with the model control group, the body weight of the deuterated plinabulin treatment group was significantly lower than that of the model control group on the 16th day after administration of the model (P≤0.05).

Table 13 Effect of intravenous infusion of deuterated plinabulin on body weight of rats induced by intraperitoneal injection of cyclophosphamide (g)

Note: *P≤0.05, ***P≤0.001, model control group vs normal control group; #P≤0.05, deuterated plinabulin treatment group vs model control group.

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 10 and Table 14, compared with the normal control group, the white blood cell count of the model control group was significantly lower than that of the normal control group on the 1st to 10th day and the 15th to 28th day after administration (P≤0.05). After one modeling, it decreased and then increased, and after another modeling, it decreased and rose to close to the normal level. The white blood cell counts in the deuterated plinabulin alone administration group were significantly lower than those in the normal control group on the 2nd, 3rd, 15th, and 16th days after administration (P≤0.05), and the other test days were comparable to the normal control group.

Compared with the model control group, the white blood cell counts in the deuterated plinabulin treatment group increased significantly on the 1st, 2nd, 14th, and 15th days after administration (P≤0.05).

Table 14 Effect of intravenous infusion of deuterated plinabulin on white blood cells of rats modeled with cyclophosphamide (WBC×10 ⁹ /L)

Note: ^P≤0.05, deuterated plinabulin single administration group vs normal control group; *P≤0.05, **P≤0.01, ***P≤0.001, model control group vs normal control group; ^# P ≤0.05, ^## P≤0.01, deuterated plinabulin treatment group vs model control group.

3.3.2 Neutrophil count results

As shown in Figure 11 and Table 15, compared with the normal control group, the number of neutrophils in the model control group decreased significantly (P≤0.05) after administration to the 2nd to 8th day and the 15th to 22nd day; On the 10th to 14th day and the 24th to 28th day after the administration, the neutrophil count in the model control group increased significantly (P≤0.05), and the overall trend was to decrease after the first modeling and then increase and return to normal level. The neutrophil count of the deuterated plinabulin alone administration group was significantly higher than that of the normal control group on the first day, and the other test days were comparable to the normal control group.

Compared with the model control group, the neutrophil counts in the deuterated plinabulin treatment group were significantly increased on

days

1, 2, 14, and 15 (P≤0.05).

Table 15 Effect of intravenous infusion of deuterated plinabulin on neutrophils in rats modeled with cyclophosphamide (NEUT×10 ⁹ /L)

Note: ^P≤0.05, deuterated plinabulin monotherapy group vs normal control group; *P≤0.05, **P≤0.01, ***P≤0.001, model control group vs normal control group; ^## P ≤0.01, deuterated plinabulin treatment group vs model control group.

3.4 Thymus coefficient

As shown in Figure 12 and Table 16, the thymus coefficient of the model control group was significantly lower than that of the normal control group (P≤0.05); the thymus coefficient of the deuterated plinabulin treatment group was significantly higher than that of the model control group (P≤0.05), and the remaining groups No statistical difference.

Table 16 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in rats induced by intraperitoneal injection of cyclophosphamide

Note: *P≤0.05, model control group vs normal control group; ^# P≤0.05, deuterated plinabulin treatment group vs model control group.

Effect Example 2A Drug effect test of intravenous infusion of deuterated plinabulin on rat neutropenia model induced by intraperitoneal injection of cyclophosphamide.

1 Experimental material is the same as effect embodiment 2.

1.2 The main reagents are the same as in the effect example 2.

1.2.1 Name: Plinabulin

Manufacturer: Qingdao Marine Biomedical Research Institute Co., Ltd.;

Batch number: 20210118;

Properties: yellow powder;

Configuration method: consistent with deuterated Plinabulin.

1.3 The experimental apparatus is the same as that in Example 2 for the effect.

2 Test methods and detection indicators

2.1 Animal grouping

There were 7 groups in the experiment, which were normal control group, model control group 1, model control group 2, Plinabulin group 1, Plinabulin group 2, deuterated plinabulin group 1, and deuterated plinabulin group 2. There were 8 male SD rats in each group, and rats with uniform levels of neutrophils were randomly divided into groups before modeling. The grouping situation is shown in Table 17.

Table 17 Experimental animal grouping table

2.2 Dosage design and administration

After the grouping of this experiment, except for the normal control group given normal saline, the animals in the other groups were given intraperitoneal injection of cyclophosphamide for modeling, the modeling dose was 100mg/kg, and the administration volume was 10mL/kg; Model control group 1 and group 2 were given 5% glucose solution by tail vein injection, Plinabulin group 1 and group 2 were given Plinabulin at 7.5 mg/kg, deuterated plinabulin group 1 and group 2 were given at 7.5 mg/kg. The test product deuterated plinabulin was administered intravenously for 15 minutes, with a single administration volume of 20mL/kg.

On the 8th day, the normal control group was given normal saline, and the model control group 2, Plinabulin 2 group and deuterated plinabulin 2 group were given intraperitoneal injection of cyclophosphamide for the second modeling, the modeling dose was 100mg/kg, The administration volume was 10mL/kg; 30 minutes after administration of the modeling drug, the model control group 2 was given 5% glucose solution by tail vein injection, the Plinabulin 2 group was given Plinabulin at 7.5 mg/kg, and the deuterated Plinabulin 2 group was given 7.5 mg/kg. Deuterated plinabulin was given per kg, intravenously infused for 15 minutes, single dose, and the dose volume was 20 mL/kg.

The specific dose design is shown in Table 18 and Table 19.

Table 18 Modeling dose design table

Table 19 Dosage design table

2.3 Detection indicators

After the administration, the general state was observed once a day, the weight was weighed once a day after the administration, the blood test was performed once a day before and after the administration, and the rats were dissected on the 14th and 24th days after the administration. Take the thymus from each animal, weigh it and calculate its organ coefficient (the weight of the organ per 10g body weight (mg)).

3 Experimental results

3.1 General Observations

Model control groups

1 and 2,

Plinabulin

1 and 2 groups, and

deuterated plinabulin

1 and 2 groups all had symptoms such as deformed stool, perianal secretions, and sparse back hair.

3.2 Weight

As shown in Figure 13, Figure 14, Table 20 and Table 21, the body weight of the animals in the normal control group showed a steady upward trend throughout the test. Compared with the normal control group, from the 2nd day to the 14th day after administration, the body weight of the

model control group

1 and 2 was significantly lower than the normal control group (P≤0.05); On the 2nd to 6th day after administration, the body weight of the Plinabulin 2 group decreased significantly (P≤0.05); compared with the model control group 2, the deuterated Plinabulin The body weight of the two groups decreased significantly (P≤0.05).

Table 20 Effect of intravenous infusion of deuterated plinabulin on body weight of rats induced by cyclophosphamide intraperitoneal injection (g)

Note: ^^^P≤0.001, model control group 1 vs normal control group;

Table 21 Effect of intravenous infusion of deuterated plinabulin on body weight of rats induced by intraperitoneal injection of cyclophosphamide (g)

Note: _a P≤0.05, _aa P≤0.01, _aaa P≤0.05, model control group 2 vs normal control group; ^bb P≤0.01, ^bbb P≤0.001, Plinabulin group 2 vs model control group 2; ^c P≤0.05, ^ccc P≤0.05, deuterated plinabulin 2 groups vs model control 2 groups.

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 15 and Table 22, intraperitoneal injection of cyclophosphamide was carried out on the first day for modeling and administration once. After administration to the 14th day, the white blood cell count in the model control group 1 was significantly lower on the 2nd to 9th day In the normal control group (P ≤ 0.05), then increased and then decreased to approach the normal level. On the 7th to 12th day after administration, the white blood cell count of the deuterated plinabulin 1 group was higher than that of the model control group 1, and on the 8th to 9th day after the administration, the white blood cell count of the deuterated plinabulin 1 group was higher than that of the model control group 1. The count was significantly higher than that of the model control group 1 (P≤0.05). At 7-13 days after administration, the white blood cell count in the deuterated plinabulin 1 group tended to be higher than that in the Plinabulin 1 group, and on the 7th and 8th days after administration, the white blood cell count in the deuterated plinabulin 1 group was significantly higher than that in the Plinabulin 1 group. Higher than Plinabulin 1 group.

Table 22 Effect of intravenous infusion of deuterated plinabulin on white blood cells of rats modeled with cyclophosphamide (WBC×10 ⁹ /L)

Note: ^^^P≤0.001, model control group 1 vs normal control group; ^& P≤0.05, deuterated plinabulin 1 group vs model control group 1; ^## P≤0.01, deuterated plinabulin 1 Group vs Plinabulin Group 1.

As shown in Figure 16 and Table 23, intraperitoneal injection of cyclophosphamide was used for modeling and administration on the 1st and 8th days, and until 24 days after administration, the model control group 2 was on the 2nd to 14th days, and the 21st day The white blood cell count on day 24 was significantly lower than that of the normal control group (P≤0.05), and then showed an upward and then downward trend; on the 8th to 10th day and the 17th to 24th day of administration, the white blood cell count of the deuterated plinabulin 2 group was significantly lower than that of the normal control group (P≤0.05). Compared with the model control group 2, it showed an increasing trend, and the deuterated plinabulin group 2 was significantly higher than the model control group 2 on the 8th to 9th day and the 18th to 20th day after administration (P≤0.05). On the 8th to 10th day and the 17th to 21st day of administration, the white blood cell count of the deuterated plinabulin 2 group showed a rising trend compared with that of the Plinabulin 2 group. On day 20, the deuterium plinabulin 2 group was significantly higher than that of the Plinabulin 2 group (P≤0.05).

Table 23 Effect of intravenous infusion of deuterated plinabulin on white blood cells of rats modeled with cyclophosphamide (WBC×10 ⁹ /L)

Note: aP≤0.05, aaaP≤0.001, model control group 2 vs normal control group; ^c P≤0.05, deuterated plinabulin group 2 vs model control group 2; ^d P≤0.05, deuterated plinabulin 2 Group vs Plinabulin 2 groups.

3.3.2 Neutrophil count results

As shown in Figure 17 and Table 24, the intraperitoneal injection of cyclophosphamide was carried out on the first day for modeling and administration once, and the neutrophil count in the model control group 1 was significantly lower than that in the normal control group on days 1 to 8 (P ≤0.05), then increased and then decreased to the normal level. On the 10th to 11th day, the neutrophil count in the model control group 1 was significantly higher than that in the normal control group (P≤0.05). Compared with the model control group 1, on the second day after administration, the neutrophil count in the deuterated plinabulin 1 group was significantly increased (P≤0.05); compared with the model control group 1, the deuterated plinabulin In the Bollinger 1 group, neutrophils showed an increasing trend on the 8th to 11th days after administration, but there was no statistical significance. Compared with the Plinabulin 1 group, on the second day after administration, the neutrophil count in the deuterated plinabulin 1 group was significantly increased (P≤0.05); compared with the Plinabulin 1 group, the deuterated plinabulin In group 1, neutrophils showed an increasing trend on the 9th to 11th day after administration, but there was no statistical significance.

Table 24 Effect of intravenous infusion of deuterated plinabulin on neutrophils in rats modeled with cyclophosphamide (NEUT×10 ⁹ /L)

Note: ^^^P≤0.001, model control group 1 vs normal control group; ^*** P≤0.001, Plinabulin group 1 vs model control group 1; ^& P≤0.05, deuterium plinabulin group 1 vs model control group Group 1;

As shown in Figure 18 and Table 25, the intraperitoneal injection of cyclophosphamide was carried out on the 1st and 8th day for modeling and administration, and the neutrophil count in the model control group 2 was significantly lower than that of the normal control group on the 2nd to 14th day (P ≤ 0.05), then increased and then decreased to normal levels, and was significantly higher than the normal control group (P ≤ 0.05) on the 16th to 22nd day. Compared with the model control group 2, the neutrophil count in the Plinabulin 2 group was significantly increased on the 2nd and 9th day (P≤0.05), and was significantly decreased on the 15th to 17th day (P≤0.05); The neutrophil counts in Nabulin 2 group increased significantly on the 2nd to 3rd day, the 8th to 10th day, and the 18th to 20th day (P≤0.05). Compared with the Plinabulin 2 group, the neutrophil count in the deuterated Plinabulin 2 group was significantly increased on the 2nd to 3rd day, the 8th to 10th day, and the 18th to 20th day (P≤0.05).

Table 25 Effect of intravenous infusion of deuterated plinabulin on neutrophils in rats modeled with cyclophosphamide (NEUT×10 ⁹ /L)

Note: aP≤0.05, aaP≤0.01, aaaP≤0.001, model control group 2 vs normal control group; ^b P≤0.05, ^bb P≤0.01, ^bbb P≤0.001, Plinabulin group 2 vs model control group 2; ^c P≤ 0.05, ^cc P≤0.01, deuterated plinabulin 2 groups vs Plinabulin 2 groups; ^d P≤0.05, ^d P≤0.01, deuterated plinabulin 2 groups vs Plinabulin 2 groups.

3.4 Thymus coefficient

As shown in Figure 19 and Figure 20 and Table 26 and Table 27, after one cyclophosphamide modeling and administration, the thymus coefficient of the model control group 1 was significantly lower than that of the normal control group (P≤0.05). After two cycles of cyclophosphamide modeling and administration, the thymus coefficient of the model control group 2 was significantly lower than that of the normal control group (P≤0.05).

Table 26 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in rats induced by intraperitoneal injection of cyclophosphamide

Note: ^P≤0.05, model control group 1 vs normal control group.

Table 27 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in rats induced by intraperitoneal injection of cyclophosphamide

Note: ^aa P≤0.01, model control group 2 vs normal control group.

4 Conclusion

Under the conditions of this experiment, neutrophils and leukocytes in the model control group decreased significantly about one week after cyclophosphamide modeling, and general clinical observations showed symptoms such as hypoactivity, diarrhea, and significant weight loss. Cyclophosphamide induced neutrophils Reduced model build succeeded.

After intraperitoneal injection of cyclophosphamide and administration of 1 and 2 times, the neutrophils in the Plinabulin group were significantly higher than those in the model control group at multiple time points; the neutrophils and The white blood cells were significantly higher than those in the model control group and Plinabulin group.

To sum up, under the conditions of this experiment, intravenous infusion of Plinabulin and deuterated plinabulin has a certain therapeutic effect on neutropenia induced by intraperitoneal injection of cyclophosphamide, and deuterated plinabulin has a certain therapeutic effect. Superior to Plinabulin.

Effect Example 2B Drug effect test of deuterated plinabulin intravenous infusion on cyclophosphamide-induced neutropenia model in rats.

1. Experimental materials 1.2 Main reagents 1.3 Experimental instruments Same effect as Example 2.

2 Test methods and detection indicators

2.1 Animal grouping

Three groups were set up in the experiment, which were normal control group, model control group, and deuterated plinabulin treatment group. There were 6 male SD rats in each group, and rats with uniform levels of neutrophils were randomly divided into groups before modeling. The grouping situation is shown in Table 28.

Table 28 Experimental animal grouping table

2.2 Dosage design and administration

After grouping in this experiment, except for the normal control group given normal saline, the animals in the other groups were intraperitoneally injected with cyclophosphamide for modeling, the modeling dose was 50 mg/kg, and the administration volume was 10 mL/kg; The normal control group and the model control group were given 5% glucose solution through tail vein injection, and the deuterated plinabulin treatment group was given 10 mg/kg deuterated plinabulin by intravenous infusion for 15 minutes, single administration. The design is shown in Table 29.

Table 29 Dose Design Table

2.3 Detection indicators

Observe the general state once a day after administration, weigh on the 1st, 2nd, 3rd, 7th, and 14th days after administration, and weigh before and after administration on the 2nd, 3rd, 5th, 7th, 9th, 11th, 14 days blood sampling for testing. On the 14th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed and calculated for its organ coefficient (the weight of the organ per 10 g of body weight (mg).

3 Experimental results

3.1 General Observations

After administration to the end of the experiment, the general clinical observations of the animals in each group were normal.

3.2 Weight

As shown in Figure 21 and Table 30, from the administration to the end of the experiment, the weights of the rats in the normal control group and the model control group were equivalent, showing a gradual increase trend. Compared with the model control group, the body weight of the rats in the deuterated plinabulin treatment group decreased significantly on the 2nd to 3rd day (P≤0.05), and there was no significant difference in the body weight of the rats in the model control group in the rest of the test days, showing a gradual increase trend.

Table 30 Effect of intravenous infusion of deuterated plinabulin on body weight of rats induced by intraperitoneal injection of cyclophosphamide (g)

Note: ^# P≤0.05, ^## P≤0.01, deuterated plinabulin treatment group vs model control group.

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 22 and Table 31, compared with the normal control group, the white blood cell count in the model control group decreased significantly on the 2nd to 7th day and the 14th day (P≤0.05), and the overall trend was to decrease after modeling and then rise to the normal level . Compared with the model control group, the white blood cell count in the deuterated plinabulin treatment group was significantly increased on the second day (P≤0.05), and the white blood cell count was significantly decreased on the ninth day (P≤0.05).

Table 31 Effect of intravenous infusion of deuterated plinabulin on white blood cells of rats modeled with cyclophosphamide (WBC×10 ⁹ /L)

Note: *P≤0.05, ***P≤0.001, model control group vs normal control group, ^# P≤0.05, deuterated plinabulin treatment group vs model control group.

3.3.2 Neutrophil count results

As shown in Figure 23 and Table 32, compared with the normal control group, the neutrophil count in the model control group decreased significantly (P≤0.05) on days 2 to 7, and increased significantly on day 9 (P≤0.05) , the overall trend was modeled and then decreased and then increased, and then dropped to the normal level.

Compared with the model control group, the neutrophil count in the deuterated plinabulin treatment group was significantly higher on the second day (P≤0.05), and the neutrophil count on the ninth day was significantly lower than that in the model control group (P≤ 0.05), but comparable to the normal control group.

Table 32 Effect of intravenous infusion of deuterated plinabulin on neutrophils in rats modeled with cyclophosphamide (NEUT×10 ⁹ /L)

Note: *P≤0.05, **P≤0.01, ***P≤0.001, model control group vs normal control group, ^# P≤0.05, deuterated plinabulin treatment group vs model control group.

3.4 Thymus coefficient

As shown in Figure 24 and Table 33, there was no obvious abnormal change in the thymus coefficients of the rats in the cyclophosphamide modeling and administration groups.

Table 33 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in rats induced by intraperitoneal injection of cyclophosphamide

Effect Example 3 Drug effect test of intravenous infusion of deuterated plinabulin on rat neutropenia induced by intravenous injection of cyclophosphamide.

2 Test methods and detection indicators

2.1 Animal grouping

Three groups were set up in the experiment, namely the normal control group, the model control group, and the deuterated plinabulin treatment group, with 8 male SD rats in each group, and the rats with uniform levels of neutrophils were randomly divided into groups before modeling . The grouping situation is shown in Table 34.

Table 34 Experimental animal grouping table

2.2 Dosage design and administration

After the grouping of this experiment, except for the normal control group given normal saline, the animals in the other groups were given cyclophosphamide by tail vein injection for modeling, the modeling dose was 50mg/kg, and the administration volume was 5mL/kg; , the normal control group and the model control group were given 5% glucose solution by tail vein injection, and the deuterated plinabulin treatment group was given the test product deuterated plinabulin at 7.5mg/kg, intravenous infusion for 15min, single administration , the administration volume is 20mL/kg. The design is shown in Table 35.

The specific dose design is shown in Table 35.

Table 35 Dose Design Table

2.3 Detection indicators

Observe the general state once a day after administration, weigh once a day after administration, conduct hematological testing once a day before and after administration, and dissect all rats on the 14th day after administration. The thymus of the animal was weighed and its organ coefficient (the weight of the organ per 10 g body weight (mg)) was calculated.

3 Experimental results

3.1 General Observations

During the test period, except for the normal control group, the rats in the other groups showed symptoms such as hypoactivity, sparse back hair, shapeless feces, and perianal filth.

3.2 Weight

As shown in Figure 25 and Table 36, the normal control group showed a steady upward trend after administration to the end of the test. Compared with the normal control group, there was no significant decrease in the model control group; compared with the model control group, there was a downward trend in the deuterated plinabulin treatment group, but there was no significant difference.

Table 36 Effect of intravenous infusion of deuterated plinabulin on body weight of rats induced by intravenous injection of cyclophosphamide (g)

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 26 and Table 37, the intravenous injection of cyclophosphamide was used for modeling and administration on the first day, and the white blood cell count in the model control group was significantly lower than that in the normal control group on the 2nd to 9th day after administration (P≤0.05 ), then increased and then decreased to normal levels. Compared with the model control group, on the 12th day after administration, the white blood cell count in the deuterated plinabulin treatment group was significantly higher (P≤0.05).

Table 37 Effect of intravenous infusion of deuterated plinabulin on white blood cell count in rats modeled with cyclophosphamide WBC (×10 ⁹ /L)

Note: ^P≤0.05, ^^^P≤0.001, model control group vs normal control group; ^* P≤0.05, deuterated plinabulin treatment group vs model control group.

3.3.2 Neutrophil count results

As shown in Figure 27 and Table 38, on the first day, intravenous injection of cyclophosphamide was administered once for modeling and administration, and the neutrophils in the model control group were significantly lower than those in the normal control group on days 2 to 8 (P≤0.05 ), then increased and then decreased to the normal level, which was significantly higher than that of the normal control group on the 1st, 9th and 11th days (P≤0.05). Compared with the model control group, the neutrophil count in the deuterated plinabulin treatment group tended to increase on the 2nd, 9th to 14th day, and was significantly increased on the 2nd day and the 12th day (P≤0.05) .

Table 38 Effect of intravenous infusion of deuterated plinabulin on neutrophil count in rats modeled with cyclophosphamide (NEUT×10 ⁹ /L)

Note: ^P≤0.05, ^^P≤0.01, ^^^P≤0.001, model control group vs normal control group; ^* P≤0.05, ^***- P≤0.05, deuterium plinabulin treatment group vs model control group.

3.4 Thymus coefficient

As shown in Figure 28 and Table 39, after intravenous injection of cyclophosphamide for modeling and administration, there was no significant difference among the groups.

Table 39 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in rats induced by intravenous injection of cyclophosphamide

4 Conclusion

Under the conditions of this experiment, neutrophils and leukocytes in the model control group decreased significantly about one week after intravenous injection of cyclophosphamide, and general clinical observations showed symptoms such as hypoactivity, diarrhea, and weight loss to a certain extent, indicating that intravenous injection Cyclophosphamide-induced neutropenia model was established successfully.

Intravenous injection of cyclophosphamide was administered once 30 minutes after modeling on the first day. The neutrophils in the deuterated plinabulin treatment group were significantly increased on the second day, and the white blood cells and neutrophils were significantly increased on the 12th day. raised.

In summary, under the conditions of this experiment, intravenous infusion of deuterated plinabulin has a certain therapeutic effect on neutropenia induced by intravenous injection of cyclophosphamide.

Effect Example 4 Effect of Intravenous Infusion of Deuterated Plinabulin on Neutrophils in Normal Rats

2 Test methods and detection indicators

2.1 Animal grouping

There were 5 groups in the experiment, which were normal control group, model control group, low-dose deuterated plinabulin group, middle-dose deuterated plinabulin group, and high-dose deuterated plinabulin group. There were 6 male SD rats in each group, and the rats with uniform levels of neutrophils were randomly divided into groups. The grouping situation is shown in Table 40.

Table 40 Experimental animal grouping table

2.2 Dosage design and administration

After the grouping in this experiment, the model control group was given intraperitoneal injection of cyclophosphamide for modeling, the modeling dose was 50 mg/kg, and the administration volume was 10 mL/kg. 1.875, 3.75, and 7.5 mg/kg deuterated plinabulin were given to the low, medium, and high doses of deuterated plinabulin alone, intravenous infusion for 15 minutes, single administration, normal control group and model control group A 5% glucose solution was injected into the tail vein. The design is shown in Table 41.

Table 41 Dose Design Table

2.3 Detection indicators

Observe the general state once a day after administration, measure body weight once before administration and on the 2nd, 3rd, 7th, 11th, 15th, and 18th days after administration, and measure body weight once before administration and on the 2nd, 3rd, and 18th days after administration. 5, 7, 9, 11, 13, 15, 18 days for a blood test. On the 18th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed and its organ coefficient (the weight of the organ per 10 g body weight (mg)) was calculated.

3 Experimental results

3.1 General Observations

During the period from the administration to the end of the experiment, the general clinical observations of the rats in each group were normal.

3.2 Weight

As shown in Figure 29 and Table 42, during the test period, the body weight of the rats in the low-dose deuterated plinabulin group was not different from that in the normal control group. The body weight of the model control group was significantly lower than that of the normal control group on the second day after administration (P≤0.05). Compared with the normal control group, the body weight of the rats in the middle and high dose groups of deuterated plinabulin decreased significantly from day 1 to day 7 (P≤0.05).

Table 42 Effect of intravenous infusion of deuterated plinabulin on body weight of rats (g)

Note: ^P≤0.05, model control group vs normal control group; ^# P≤0.05, deuterated plinabulin medium dose group vs normal control group; ^& P≤0.05, ^&& P≤0.01, deuterated plinabulin High-dose group vs normal control group.

3.3 Hematological testing

3.3.1 White blood cell (WBC) count results

As shown in Figure 30 and Table 43, during the test, compared with the normal control group, the white blood cell count in the model control group decreased significantly (P≤0.05) on days 2 to 9 and 18, and the overall trend was to increase after the model was established. to normal levels. There was no significant difference in white blood cell count between the low and middle dose groups of deuterated plinabulin compared with the normal control group. The white blood cell count in the high-dose deuterated plinabulin group was significantly lower than that in the normal control group on the second day after administration (P≤0.05), and then rose to the normal level.

Table 43 Effect of intravenous infusion of deuterated plinabulin on white blood cell count in normal rats (WBC×10 ⁹ /L)

Note: ^P≤0.05, ^^P≤0.01, model control group vs normal control group; ^& P≤0.05, deuterated plinabulin high-dose group vs normal control group.

3.3.2 Neutrophil count results

As shown in Figure 31 and Table 44, compared with the normal control group, the neutrophil count in the model control group decreased significantly (P≤0.05) on the 2nd to 5th day, and was higher than that of the normal control group on the 9th to 11th day, but No significant difference. Overall, it decreased after modeling and then increased, and then dropped to normal levels.

The trend of neutrophils in the low-dose deuterated plinabulin group was consistent with that in the normal control group. Compared with the normal control group, the neutrophils in the middle-dose group of deuterated plinabulin decreased significantly on the 2nd and 3rd day after administration (P≤0.05). In the high-dose deuterated plinabulin group, neutrophils decreased significantly on the 2nd, 3rd, and 9th day after administration (P≤0.05).

Table 44 Effect of intravenous infusion of deuterated plinabulin on neutrophil count in normal rats (NEUT×10 ⁹ /L)

Note: ^P≤0.05, ^^P≤0.01, model control group vs normal control group; ^# P≤0.05, deuterated plinabulin medium dose group vs normal control group; ^& P≤0.05, deuterated prunabulin Lin high-dose group vs normal control group.

3.4 Thymus coefficient

As shown in Figure 32 and Table 45, there was no significant difference in the thymus coefficients of the rats in each group.

Table 45 Effect of intravenous infusion of deuterated plinabulin on thymus coefficient in normal rats

4 Conclusion

Under the conditions of this experiment, the model control group had no abnormalities in the general clinical observation after intraperitoneal modeling with 50 mg/kg cyclophosphamide, and the body weight dropped first and then recovered after administration. The neutrophils and leukocytes decreased significantly about one week after modeling, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

During the experiment, the body weight of the rats in the middle and high doses of deuterated plinabulin group was significantly lower than that of the normal control group in the first week after administration, and the body weight of the rats in the high doses of deuterated plinabulin group was significantly lower than that of the normal control group in the first week after administration. The white blood cell count decreased significantly on the first day, the neutrophils in the middle-dose deuterated plinabulin group decreased significantly on the 2nd and 3rd day after administration (P≤0.05), and the high-dose deuterated plinabulin group decreased significantly on the 2nd and 3rd day after administration. On

days

2, 3, and 9, neutrophils decreased significantly (P ≤ 0.05), but the decrease in leukocytes and neutrophils in each group was not high, suggesting that deuterated plinabulin had a negative effect on white blood cells and neutrophils in normal rats. Minor damage effect.

Although the specific implementation of the present invention has been described above, those skilled in the art should understand that this is only an example, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principle and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

Application of a deuterated plinabulin as shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of medicines for treating and/or preventing neutropenia;
The application according to claim 1, wherein the pharmaceutically acceptable salt of deuterated plinabulin as shown in formula (I) is:
The use according to claim 1, characterized in that the neutropenia is induced by administration of chemotherapy or by administration of radiotherapy.
The use of claim 3, wherein the neutropenia is induced by chemotherapy or radiation therapy for the treatment of an individual suffering from liver, pancreatic, lung, breast, colon or prostate cancer.
The use according to claim 3 or 4, wherein said chemotherapy comprises administering a chemotherapeutic composition of one or more chemotherapeutic agents.
The application according to claim 5, characterized in that, the chemotherapeutic composition is docetaxel, paclitaxel, cyclophosphamide, "combination of docetaxel, doxorubicin and cyclophosphamide", "multiple The combination of cetaxel, paclitaxel, vinblastine, doxorubicin, and cyclophosphamide" or "the combination of docetaxel and cyclophosphamide".
The application according to claim 1, characterized in that, said deuterated plinabulin or its pharmaceutically acceptable salt as shown in formula (I) is administered orally or by injection (such as intravenous, intramuscular, subcutaneous) .
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises about 0.5 mg to about 60 mg of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof;
The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition comprises about 0.5 mg to about 10 mg of deuterated plinabulin or a pharmaceutically acceptable salt thereof as shown in formula (I) ;

And/or, the pharmaceutically acceptable salt of deuterated plinabulin as shown in formula (I) is:
The pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable excipient.
A method for treating and/or preventing neutropenia, characterized in that it comprises administering a therapeutically effective amount of deuterated plinabulin represented by formula (I) or a pharmaceutically effective amount thereof to an individual in need. acceptable salt;
The method according to claim 11, wherein the pharmaceutically acceptable salt of deuterated plinabulin as shown in formula (I) is:
The method according to claim 11, wherein the neutropenia is induced by administering chemotherapy or by administering radiation therapy, preferably, the neutropenia is induced by treating liver cancer, Chemotherapy or radiation therapy induction in individuals with pancreatic, lung, breast, colon or prostate cancer.
The method according to claim 13, wherein the chemotherapy comprises administering a chemotherapeutic composition of one or more chemotherapeutic agents, preferably, the chemotherapeutic composition is docetaxel, paclitaxel , cyclophosphamide, "combination of docetaxel, doxorubicin, and cyclophosphamide", "combination of docetaxel, paclitaxel, vinblastine, doxorubicin, and cyclophosphamide" or "docetaxel and Combinations of Cyclophosphamide".
The method according to claim 11, wherein the method satisfies one or more of the following conditions:

(1) The deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is administered orally or by injection;

(2) The administration amount of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is in the range of 0.5-60 mg/kg, such as 0.5-20 mg/kg;

(3) The deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is administered at least once a week;

(4) The method includes simultaneously administering deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more G-CSF drugs.
The method according to claim 15, wherein the method satisfies one or more of the following conditions:

(1) The deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is administered by intravenous infusion;

(2) The administration amount of the deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is 1.875 mg/kg, 3.75 mg/kg, 7.5 mg/kg or 10 mg/kg;

(3) The deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof is administered once every 7 days.
The method according to claim 13 or 14, wherein the method satisfies one or more of the following conditions:

(1) administering a single dose of deuterated plinabulin represented by formula (I) or a pharmaceutically acceptable salt thereof in a chemotherapy cycle;

(2) administering deuterated plinabulin or a pharmaceutically acceptable salt thereof as shown in formula (I) after administering the chemotherapeutic composition;

(3) When the neutropenia is induced by administering cyclophosphamide, within each treatment cycle, the deuterated plinabulin or its pharmaceutically acceptable salt as shown in formula (I) Administration dose is less than 60mg/kg;

(4) When the neutropenia is induced by administration of cyclophosphamide, within 24 hours after administration of cyclophosphamide, administration of deuterated plinabulin as shown in formula (I) or its pharmaceutically acceptable of salt;

(5) When the neutropenia is caused by administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or " When "docetaxel and cyclophosphamide" are induced, in each treatment cycle, the administration dose of deuterated plinabulin or its pharmaceutically acceptable salt shown in formula (I) is less than 60mg/kg;

(6) When the neutropenia is caused by administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or " Docetaxel and cyclophosphamide" induction, the method includes administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide amide" or "docetaxel and cyclophosphamide" within 24 hours after administration of deuterated plinabulin or a pharmaceutically acceptable salt thereof as shown in formula (I);

(7) When the individual in need suffers from liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, or prostate cancer, the method includes identifying a patient with the liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, or A patient with prostate cancer; and administering deuterated plinabulin or a pharmaceutically acceptable salt thereof as represented by formula (I) at a dose of about 0.5 mg/kg to about 60 mg/kg;

(8) The subject suffers from liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer or prostate cancer.
The method according to claim 17, wherein, when the neutropenia is induced by the administration of cyclophosphamide, the deuterium as shown in formula (I) is administered within 12 hours after administration of cyclophosphamide. Plinabulin or a pharmaceutically acceptable salt thereof is administered, for example, 0.5 hours later.