USE OF BUTYLPHTHALIDE AND DERIVATIVE THEREOF

Abstract

The present application provides application of butylphthalide or optical isomer, prodrug, deuterium, metabolite, and ring-opening product or ring-opening product salt thereof in the preparation of a drug for preventing, alleviating, or treating peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy, and a use method of butylphthalide and a derivative thereof in preventing or treating peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy. In vivo and in vitro studies show that the drug of the present invention can effectively prevent, alleviate, or treat peripheral neuropathy caused by chemotherapy drugs without affecting the tumor-inhibiting efficacy and pharmacokinetic properties of the chemotherapy drugs.

Claims

1-14. (canceled)

15. A method for preventing, relieving, or treating a drug-induced peripheral neuropathy, including administering to a subject in need thereof a therapeutically effective amount of butylphthalide or an optical isomer, a prodrug, a deuterated product, a metabolite, a ring-opening product, or a salt of the ring-opening product thereof.

16. The method according to claim 15, wherein the peripheral neuropathy is a chemotherapeutic drug-induced peripheral neuropathy.

17. The method according to claim 15, wherein the peripheral neuropathy is chemotherapeutic drug-induced paresthesia or dyskinesia.

18. The method according to claim 15, wherein the peripheral neuropathy is paresthesia of pain and heat, or impaired motor coordination.

19. The method according to claim 15, wherein the drug is prepared into a clinically acceptable formulation.

20. The method according to claim 19, wherein the clinically acceptable formulation is an oral formulation, an injectable formulation, a topical formulation, or an external formulation.

21. The method according to claim 19, wherein the clinically acceptable formulation is a single dose form or a divided dose form.

22. The method according to claim 20, wherein the oral formulation comprises from about 1 mg to about 1,000 mg, or from about 1 mg to about 500 mg, or from about 1 mg to about 300 mg, or from about 1 mg to about 200 mg, or from about 5 mg to about 180 mg, or from about 10 mg to about 150 mg, or from about 30 mg to about 120 mg, or from about 50 mg to about 120 mg, or from about 80 mg to about 120 mg, or from about 90 mg to about 110 mg, or about 100 mg, of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof, on a basis of butylphthalide.

23. The method according to claim 20, wherein a daily dose of the oral formulation is from about 1 mg to about 10,000 mg, or from about 10 mg to about 5,000 mg, or from about 20 mg to about 3,000 mg, or from about 30 mg to about 2,000 mg, or from about 50 mg to about 1,500 mg, or from about 70 mg to about 1,200 mg, or from about 100 mg to about 1,000 mg, or from about 200 mg to about 900 mg, or from about 300 mg to about 800 mg, or from about 400 mg to about 700 mg, or from about 500 mg to about 600 mg, or from about 60 mg to about 800 mg, or from about 60 mg to about 600 mg, or from about 100 mg to about 800 mg, or from about 100 mg to about 600 mg, or from about 200 mg to about 600 mg, or from about 200 mg to about 800 mg, or from about 300 mg to about 600 mg, or from about 400 mg to about 600 mg, or from about 400 mg to about 800 mg, on a basis of butylphthalide.

24. The method according to claim 23, wherein the oral formulation is administered once per day by administering from about 60 mg to about 800 mg of the oral formulation of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof, or administering about 60 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg of the oral formulation each time, on a basis of butylphthalide.

25. The method according to claim 23, wherein the oral formulation is administered twice per day by administering from about 30 mg to about 400 mg of the oral formulation of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof each time, or administering about 30 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg of the oral formulation each time, on a basis of butylphthalide.

26. The method according to claim 23, wherein the oral formulation is administered thrice per day by administering from about 20 mg to about 300 mg of the oral formulation of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof, or administering about 20 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, or about 300 mg of the oral formulation each time, on a basis of butylphthalide.

27. The method according to claim 20, wherein the injectable formulation comprises from about 0.001 mg/mL to about 100 mg/mL, or from about 0.005 mg/mL to about 50 mg/mL, or from about 0.01 mg/mL to about 10 mg/mL, or from about 0.1 mg/mL to about 5 mg/mL, or from about 0.1 mg/mL to about 3 mg/mL, or from about 0.1 mg/mL to about 1 mg/mL, or from about 0.12 mg/mL to about 0.80 mg/mL, or from about 0.15 mg/mL to about 0.50 mg/mL, or from about 0.20 mg/mL to about 0.40 mg/mL, or from about 0.20 mg/mL to about 0.30 mg/mL, or about 0.25 mg/mL, of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof, on a basis of butylphthalide.

28. The method according to claim 20, wherein a daily dose of the injectable formulation is from about 1 mg to about 1,000 mg, or from about 5 mg to about 500 mg, or from about 10 mg to about 300 mg, or from about 15 mg to about 200 mg, or from about 20 mg to about 150 mg, or from about 25 mg to about 120 mg, or from about 30 mg to about 100 mg, or from about 35 mg to about 90 mg, or from about 40 mg to about 80 mg, or from about 45 mg to about 70 mg, or from about 50 mg to about 60 mg, or from about 1 mg to about 100 mg, or from about 2 mg to about 80 mg, or from about 5 mg to about 75 mg, or from about 10 mg to about 50 mg, or from about 15 mg to about 50 mg, or from about 20 mg to about 50 mg, or from about 25 mg to about 75 mg, or from about 25 mg to about 50 mg, on a basis of butylphthalide.

29. The method according to claim 28, wherein the injectable formulation is administered once per day by administering from about 1 mg to about 100 mg, or about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg, of the injectable formulation of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof each time, on a basis of butylphthalide.

30. The method according to claim 28, wherein the injectable formulation is administered twice per day by administering from about 1 mg to about 50 mg, or about 1 mg, about 2 mg, about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg, of butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof each time.

31. The method according to claim 16, wherein the chemotherapeutic drug includes one or more of: (1) a chemotherapeutic drug acting on a microtubule system or an anti-mitotic chemotherapeutic drug, including: a taxane drug, such as paclitaxel, docetaxel or the like; or a vinca alkaloid drug, such as vincristine, vinblastine or the like; or an epothilone drug, such as ixabepilone or the like; or a protease inhibitor drug, such as bortezomib or the like; or (2) a chemotherapeutic drug that interferes with DNA synthesis, including a platinum drug, such as cisplatin, carboplatin, oxaliplatin or the like; or (3) an immunomodulator drug, such as thalidomide, lenalidomide or the like.

32. The method according to claim 15, wherein the butylphthalide or the optical isomer, the prodrug, the deuterated product, the metabolite, the ring-opening product, or the salt of the ring-opening product thereof is used in combination with other drug for treating peripheral neuropathy.

33. The method according to claim 32, wherein the other drug for treating peripheral neuropathy is duloxetine or a salt thereof, or monosialotetrahexosyl ganglioside sodium.

34. A method for preventing, relieving, or treating a drug-induced peripheral neuropathy, including administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of butylphthalide or an optical isomer, a prodrug, a deuterated product, a metabolite, a ring-opening product, or a salt of the ring-opening product thereof, and a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0065] FIG. 1 shows a schematic diagram of a modeling method in Example 1.

[0066] FIG. 2 shows a diagram of the change in body weight of rats in each group from 0 to 16 days (D0-16) in Example 1. Compared with a model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0067] FIG. 3 shows a diagram of the change in thermal pain threshold of rats in each group from 0 to 13 days (D0-13) in Example 1. Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0068] FIG. 4 shows the comparison of thermal pain thresholds of rats in each group on days 7/10/13 (D7/D10/D13) in Example 1. Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0069] FIG. 5 shows a time axis of modeling and administration in Example 2.

[0070] FIG. 6 shows a curve diagram of the changing values of body weight of rats in Example 2 (mean±SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0071] FIG. 7 shows the change of thermal pain threshold of rats on days −1/7/14 in Example 2 (mean±SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0072] FIG. 8 shows the change of mechanical pain threshold of rats on days 0/6/13 in Example 2 (mean±SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0073] FIG. 9 shows the change of body weight of rats in each group in Example 3. Compared with a solvent group, *P≤0.05, **P≤0.01, and ***P≤0.001; and compared with an ab-PTX group, ##P<0.01.

[0074] FIG. 10 shows the change of body weight of rats in each group in Example 4. According to statistical analysis, compared with the solvent group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0075] FIG. 11 shows a time axis of modeling and administration in Example 6.

[0076] FIG. 12 shows the change of body weight of rats in each group on DO-D20 in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0077] FIG. 13 shows the change of thermal pain threshold of rats in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0078] FIG. 14 shows the change of mechanical pain threshold of rats in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, and ***P≤0.001.

[0079] FIG. 15 shows a curve diagram of the changing values of body weight of mice in each group in Example 8 (mean±SD, n=6).

[0080] FIG. 16 shows a curve diagram of the change of xenograft tumor volume of tumor-bearing mice in Example 8 (mean±SD, n=6; and compared with the solvent group, *P≤0.05, **P≤0.01, and ***P≤0.001).

[0081] Note: mpk in the figures is equivalent to mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

[0082] The examples listed below are provided to better illustrate the embodiments of the present disclosure, but the present disclosure is not limited to the listed examples. Those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above content of the disclosure, and the improved and adjusted embodiments still belong to the scope of the present disclosure.

Example 1

[0083] Study on in vivo efficacy of butylphthalide (intraperitoneal injection) on paclitaxel (albumin bound) chemotherapy-induced peripheral neuropathy in rats

[0084] 1. Experimental Animals

[0085] 59 male SD (Sprague Dawley) rats, 180-200 g.

[0086] 2. Drugs

[0087] Lyophilized powder of paclitaxel (albumin bound, ab-PTX), 100 mg/bottle, made by CSPC Ouyi Pharmaceutical Co., Ltd, batch number: B041909121, dissolved in normal saline immediately prior to use.

[0088] Butylphthalide (NBP) injection, 25 mg/5 mL, made by CSPC NBP Pharmaceutical Co., Ltd, batch number: Q27190401, diluted in normal saline to a target concentration immediately prior to use.

[0089] 3. Experimental Process

[0090] (1) Model Preparation and Administration Method

[0091] Male SD rats, ab-PTX 5 mg/kg, administration volume: 5 mL/kg, intraperitoneally injected (i.p.) on days 0, 2, 4, 6, 8, and 11 for modeling. NBP was administered at three doses (1.5, 5, and 15 mg/kg bid), starting on 1 day prior to the ab-PTX modeling, and during the ab-PTX administration and modeling, the first dose of NBP was administered by intraperitoneal injection 1 h prior to the ab-PTX administration. The second dose of NBP was administered with an interval of 4 h from the first dose, with an administration volume of 5 mL/kg, and was administered every day for 17 consecutive days. The normal group was administered with 0.9% normal saline (0.9% INJ NS) at the same frequency. The modeling method is shown in FIG. 1. The animal grouping, the administration method, and the doses are detailed in Table 3.

TABLE-US-00003 TABLE 3 Animal Grouping and Dose List in Example 1 Number of Dose Administration Group animals Drug (mg/kg) route Normal group 11 0.9% INJ NS — i.p. Model group 12 0.9% INJ NS/ab-PTX —/5 i.p./i.p. NBP 1.5 mg/kg/bid 12 NBP/ab-PTX 1.5/5  i.p./i.p. NBP 5 mg/kg/bid 12 NBP/ab-PTX  5/5 i.p./i.p. NBP 15 mg/kg/bid 12 NBP/ab-PTX 15/5 i.p./i.p. Note: (1) bid: twice per day; and (2) the three doses of NBP were converted into adult daily doses (based on 60 kg, oral administration) of 60 mg/d, 200 mg/d, and 600 mg/d, respectively.

[0092] (2) Grouping

[0093] Based on the thermal pain threshold baselines and the body weights of the rats, the rats with the baseline values ranging from 3 to 6 s were selected, and were equally grouped in accordance with the above Table 3.

[0094] (3) Observation Indexes

[0095] {circle around (1)} Body weight: all animals were weighed once prior to the experiment, and were weighed at set time every day after the commencement of administration, but only the body weight data on days −1, 0, 2, 4, 6, 8, 10, 12, 14, and 16 were statistically recorded.

[0096] {circle around (2)} Thermal pain threshold: IITC Life Science Electronic Von Frey Anesthesio meter was used and the intensity of the light source was adjusted to obtain a baseline of thermal pain threshold of about 3-6 s. In this case, the light intensity was 58%, and the cutoff value was set as 10 s. The rats were placed in a plastic compartment on a glass plate, to make the planta centers of the rats be exposed to the light source. The time of reflexive withdrawal, i.e., the thermal pain threshold, of the rat was recorded, and measured once for each of the left foot and the right foot of each rat, and the measurement was repeated three times with each interval therebetween ≥15 min, to compute a mean value of the 6 thermal pain thresholds of each rat.

[0097] 4. Experimental Results

[0098] (1) Body Weight

[0099] On days 4-16 (D4-16) of modeling, compared with the normal group, the body weights of the rats in the model group were significantly decreased (P<0.01); and compared with the model group, the body weights of the rats in the three dose groups of NBP (1.5, 5, and 15 mg/kg bid) were not significantly changed, as detailed in FIG. 2.

[0100] (2) Thermal Pain Threshold

[0101] Measurements were performed on days 7, 10, 13, and 17. On days 7-13 (D7-13), compared with the normal group, the thermal pain threshold of the model group was significantly increased (P<0.01); and compared with the model group, the increase of the thermal pain threshold of the model rats in the groups of NBP (1.5, 5, and 15 mg/kg bid) can be significantly decreased (P<0.05 or P<0.01), as detailed in FIG. 3 and FIG. 4.

[0102] On day 17 (D17), compared with the normal group, there was no statistical difference in the thermal pain threshold of the rats in the model group (D17 data was not shown), therefore the thermal pain threshold of the NBP therapy group was not detected, and the experiment was terminated.

[0103] 5. Conclusions

[0104] Butylphthalide can effectively alleviate paclitaxel (albumin bound)-induced abnormal thermal pain symptoms of peripheral neuropathy in rats.

Example 2

[0105] Study on in vivo efficacy of butylphthalide (intragastric administration) on paclitaxel (albumin bound) chemotherapy-induced peripheral neuropathy in rats

[0106] 1. Experimental animals

[0107] 72 male SD (Sprague Dawley) rats, 160-180 g.

[0108] 2. Drugs

[0109] Lyophilized powder of paclitaxel (albumin bound, ab-PTX), 100 mg/bottle, made by CSPC Ouyi Pharmaceutical Co., Ltd, batch number: B041909121, dissolved in normal saline immediately prior to use.

[0110] Butylphthalide (oral administration grade), 10 kg/bottle, made by CSPC NBP Pharmaceutical Co., Ltd, batch number: 518180803, diluted in a vegetable oil to a target concentration immediately prior to use.

[0111] Duloxetine hydrochloride, batch number: QRQYD-JN, TCI (Shanghai) Development Co., Ltd, prepared with normal saline containing 2% DMSO immediately prior to use.

[0112] 3. Experimental Process

[0113] 3.1 Model Preparation and Administration Method

[0114] In this experiment, male SD rats were administered with 5 mg/kg of ab-PTX with an administration volume of 5 mL/kg, and intraperitoneally injected (i.p.) on days 0, 2, 4, 6, 9, and 13 (the first dose of ab-PTX was administered on day 0 of the experiment) for modeling.

[0115] Three doses of NBP (3, 10, and 30 mg/kg bid) were used, and the positive control drug (duloxetine hydrochloride) was 15 mg/kg qd. Both NBP and duloxetine hydrochloride were administered 1 day prior to the ab-PTX modeling. During the ab-PTX administration and modeling, both NBP (first dose) and duloxetine hydrochloride were administered 1 h prior to intraperitoneal injection of ab-PTX. The second dose of NBP was administered with an interval of ≥4 h from the first dose, with an administration volume of 5 mL/kg, and was administered by intragastric administration for 14 consecutive days.

[0116] The model group was given vegetable oil at the same frequency and with the same administration method as NBP.

[0117] The normal group was given normal saline at the same frequency and with the same administration method as ab-PTX, and was given vegetable oil at the same frequency and with the same administration method as NBP.

[0118] The modeling method and the administration time are shown in FIG. 5. The animal grouping, the administration method, and the doses are detailed in Table 4.

TABLE-US-00004 TABLE 4 Animal Grouping and Dose List Number of Dose Administration Group animals Drug (mg/kg) route Normal group 12 Vegetable oil/normal saline — p.o./i.p. Model group 12 Vegetable oil/ab-PTX 5 p.o./i.p. NBP 3 mg/kg bid 12 NBP/ab-PTX  3/5 p.o./i.p. NBP 10 mg/kg bid 12 NBP/ab-PTX 10/5 p.o./i.p. NBP 30 mg/kg bid 12 NBP/ab-PTX 30/5 p.o./i.p. Duloxetine hydro- 12 Duloxetine hydrochloride/ab- 15/5 p.o./i.p. chloride 15 mg/kg qd PTX Note: (1) p.o.: intragastric administration; (2) i.p.: intraperitoneal injection; (3) the three doses of NBP were converted into adult daily doses (based on 60 kg, oral administration) of 60 mg/d, 200 mg/d, and 600 mg/d, respectively.

[0119] 3.2 Grouping

[0120] Based on the thermal pain threshold baseline values and the body weights of the rats, the rats with the baseline values ranging from 3 to 6 s were selected, and were equally grouped in accordance with Table 4 in 3.1.

[0121] 3.3 Observation Indexes

[0122] (1) Body weight: all animals were weighed once prior to the experiment, and were weighed at set time every day after the commencement of administration, but only the body weight data on days −1, 0, 2, 4, 6, 8, 10, 12, and 14 were statistically recorded.

[0123] (2) Thermal pain threshold: measurements were performed on days −1, 7, and 14.

[0124] Method: IITC Life Science Electronic Von Frey Anesthesio meter was used and the intensity of the light source was adjusted to obtain a baseline of thermal pain threshold of about 3-6 s. In this case, the light intensity was 58%, and the cutoff value was set as 10 s. The rats were placed in a plastic compartment on a glass plate, to make the planta centers of the rats be exposed to the light source. The time of reflexive withdrawal, i.e., the thermal pain threshold, of the rat was recorded, and was measured once for each of the left foot and the right foot of each rat, and the measurement was repeated three times with each interval therebetween ≥15 min, to compute a mean value of the 6 thermal pain thresholds of each rat.

[0125] (3) Mechanical stimulus pain threshold: measurements were performed on days 0, 6, and 13.

[0126] Method: the rats were placed in a plastic compartment on a wire mesh, left to stand still for 15 min, stimulated using a stimulation probe with a diameter of 0.4 mm of the IITC Life Science Electronic Von Frey Anesthesio meter at the planta centers of the rats by gradually increasing the force until reflexive withdrawal of the rats. The maximum value displayed by the instrument at this time, i.e., the mechanical pain threshold, was recorded. In each experiment, the left and right feet of each rat were measured three times with each interval therebetween ≥5 min, to compute a mean value of the 6 mechanical pain thresholds of each rat.

[0127] 4. Results

[0128] 4.1 Influence on Body Weight

[0129] On days 4-14 of the experiment, compared with the normal group, the body weights of the rats in the model group were obviously decreased (P<0.05); and throughout the administration period, compared with the model group, the body weights of the rats in the three dose groups of NBP (3, 10, and 30 mg/kg bid) were not obviously changed, and the positive drug duloxetine hydrochloride can significantly increase the body weights of the model rats (P<0.05), as detailed in FIG. 6.

[0130] 4.2 Influence on Thermal Pain Threshold

[0131] On day 7 of the experiment, compared with the normal group, the thermal pain threshold of the rats in the model group was significantly increased (P<0.01); and compared with the model group, the thermal pain threshold of the rats in the group of NBP (30 mg/kg bid) was significantly decreased (P<0.05).

[0132] On day 14 of the experiment, compared with the normal group, the thermal pain threshold of the rats in the model group was significantly increased (P<0.001); and compared with the model group, the thermal pain thresholds of the rats in the three dose groups of NBP (3, 10, and 30 mg/kg bid) and the group of positive drug duloxetine hydrochloride (15 mg/kg qd) were significantly decreased (P<0.05), as detailed in FIG. 7.

[0133] 4.3 Influence on Mechanical Pain Threshold

[0134] On day 6 of the experiment, compared with the normal group, the mechanical pain threshold of the rats in the model group was significantly increased (P<0.05); and compared with the model group, the mechanical pain threshold of the rats in the group of NBP (30 mg/kg bid) was significantly decreased (P<0.05).

[0135] On day 13 of the experiment, compared with the normal group, the mechanical pain threshold of the rats in the model group was significantly increased (P<0.001); and compared with the model group, the mechanical pain thresholds of the rats in the dose groups of NBP (10 and 30 mg/kg bid) and the group of positive drug duloxetine hydrochloride (15 mg/kg qd) were significantly decreased (P<0.05), and the mechanical pain threshold of the rats in the dose group of NBP (3 mg/kg bid) had a decreasing trend, but did not have statistical difference, as detailed in FIG. 8.

[0136] 6. Conclusions

[0137] Under the conditions of this experiment, NBP can dose-dependently effectively relieve the symptoms of peripheral neuropathy in an ab-PTX-induced PIPN (Paclitaxel-Induced Peripheral Neuropathy) model of rats.

Example 3

[0138] Influence of NBP on antitumor efficacy and PK of ab-PTX against B16/F10 xenograft tumor

[0139] 1. Experimental System

[0140] 1.1 Experimental Animals

[0141] 40 female C57BL/6N mice, 15-17 g.

[0142] 1.2 Cell strains

[0143] B16/F10 cells (mice melanoma cells): purchased from Shanghai Genechem Co., Ltd.

[0144] 2. Drugs

[0145] Lyophilized powder of paclitaxel (albumin bound), 100 mg/bottle, made by CSPC Ouyi Pharmaceutical Co., Ltd, batch number: B041909121, dissolved in normal saline immediately prior to use.

[0146] Butylphthalide, 25 mg/5 mL/bottle, made by CSPC NBP Pharmaceutical Co., Ltd, batch number: Q27190401, diluted in normal saline to a target concentration immediately prior to use.

[0147] 3. Experimental Process

[0148] 3.1 Model Preparation

[0149] After in vitro resuscitation and passage of B16/F10 cells to a sufficient amount, the cells were counted by a microscope, and diluted with a serum-free medium to adjust the cell count to about 1×10.sup.7 cells/mL. The cell suspension was kept in an ice-water bath.

[0150] The B16/F10 cell suspension was extracted by a sterile syringe, and inoculated into the subcutaneous tissue of the forelimb axilla of the C57BL/6N mice, with the inoculation volume of 0.1 mL/mouse containing about 1.0×10.sup.6 tumor cells, to prepare B16/F10 xenograft tumor model of the C57BL/6N mice.

[0151] 3.2 Administration Method

[0152] Therapy Group:

[0153] Monotherapy group of ab-PTX: intraperitoneal injection of ab-PTX at 25 mg/kg, biw (on days 1, 4, 8, 11, and 15). Administration volume: 10 mL/kg.

[0154] NBP/ab-PTX group: ab-PTX was administered by intraperitoneal injection at 25 mg/kg, biw (on days 1, 4, 8, 11, and 15); NBP was administered by intraperitoneal injection at 3 mg/kg, 10 mg/kg, or 30 mg/kg twice per day, bid (administration was commenced on day 0); and during the ab-PTX modeling, the first dose of NBP was administered by intraperitoneal injection 1 h prior to the ab-PTX administration, and the second dose of NBP was administered with an interval of more than 4 h from the first dose. The administration was performed for 15 consecutive days. The administration volume was 10 mL/kg.

[0155] Solvent group: the solvent (normal saline) was intraperitoneally injected at the same frequency and with the same administration volume as ab-PTX and NBP.

[0156] Note: the three doses of NBP were converted into adult daily doses (based on 60 kg, oral administration) of 60 mg/d, 200 mg/d, and 600 mg/d, respectively.

[0157] 3.3 Observation Indexes and Evaluation Indexes

[0158] 3.3.1 General Observation Indexes

[0159] (1) General state observation: all animals were observed once per day during the experimental period, and the abnormality and behavioral changes of their body parts were recorded.

[0160] (2) Body weight: all animals were weighed once prior to the experiment, and animals having suitable body weights were selected for the experiment. Animals were weighed at set time once per day after the commencement of administration.

[0161] (3) Death and near death: the death time of dead animal was recorded, and dying animal should be observed more frequently to determine the death time.

[0162] 3.3.2 Tumor Weight

[0163] At the end of the experiment, after the animals were euthanized by CO.sub.2 asphyxiation, the tumors were excised and weighed.

[0164] Tumor weight inhibition rate %=(1-tumor weight in therapy group/tumor weight in solvent group)×100%

[0165] 3.3.3 PK Blood Sampling and Detection

[0166] Before the administration of the last dose of ab-PTX (0 h), and 5 min, 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h after the administration, blood was sampled (as detailed in Table 5), anticoagulated with heparin, and centrifuged to separate plasma, which was stored at −80° C. for later use. The total amount of paclitaxel in the plasma was determined by protein precipitation-LC/MS/MS.

TABLE-US-00005 TABLE 5 List of Blood Sampling Time of Mice in Each Group Group Animal No. Blood sampling time Solvent group 1-8 0 h Therapy groups 1-4 0 h, 0.5 h, 8 h, 12 h Therapy groups 5-8 5 min, 1 h, 8 h, 24 h

[0167] 4. Results

[0168] 4.1 Influence on Body Weight

[0169] Compared with the solvent group, the body weight gain rate of mice in each therapy group was significantly decreased (P<0.01). Compared with the ab-PTX group, the body weight gain rate of the dose group of NBP/ab-PTX (10/25 mg/kg) was significantly decreased (P<0.01). Based on analysis of the tumor weight data, the decrease in the body weight gain rate of each therapy group should be caused by inhibition of the drug on the tumor weight, as detailed in FIG. 9.

[0170] 4.2 Influence on Tumor Weight

[0171] Compared with the solvent group, the tumor growth of each therapy group can be significantly inhibited (P<0.001); and compared with the ab-PTX group, the combined therapy of different doses of NBP and ab-PTX did not have statistical difference in tumor inhibition (P>0.05). The tumor weight of each group is detailed in Table 6.

TABLE-US-00006 TABLE 6 Influence on Tumor Weights of Mice (mean ± SD, n = 8) Number of animals Tumor weight Inhibition rate Group (surviving/total) (g) (%) Solvent group 8/8 5.2178 ± 1.5146   — NBP/ab-PTX 3/25 mg/kg 8/8 1.9635 ± 0.7882*** 62.4 NBP/ab-PTX 10/25 mg/kg 8/8 1.7374 ± 0.5333*** 66.7 NBP/ab-PTX 30/25 mg/kg 8/8 1.8226 ± 0.9578*** 65.1 ab-PTX 25 mg/kg 8/8 2.5024 ± 0.8133*** 52.0 Note: Compared with the solvent group: ***P < 0.001.

[0172] 4.3 Influence on PK

[0173] The total amount of paclitaxel in the plasma was determined by protein precipitation-LC/MS/MS. The results showed that there was no significant statistical difference in C.sub.max, AUC.sub.0-t, and other indexes in each therapy group, indicating that NBP did not affect the PK behavior of ab-PTX in mice, as detailed in FIG. 7.

TABLE-US-00007 TABLE 7 Influence on Pharmacokinetic Parameters C.sub.max AUC.sub.0-t AUC.sub.0-∞ T.sub.max t.sub.1/2 Group (ng/ml) (h*ng/ml) (h*ng/ml) (h) (h) NBP/ab-PTX 3/25 mg/kg 3157 8012 8065 0.5 1.55 NBP/ab-PTX 10/25 mg/kg 2315 7178 7226 0.5 1.72 NBP/ab-PTX 30/25 mg/kg 2210 6973 7130 1.0 2.31 ab-PTX 25 mg/kg 2443 8126 8178 0.5 1.55

[0174] 5. Conclusions

[0175] In this experiment, NBP did not have obvious influence on the antitumor efficacy of ab-PTX and the PK behavior of ab-PTX.

Example 4

[0176] Influence of NBP on antitumor efficacy and PK of ab-PTX against JIMT-1 xenograft tumor

[0177] 1. Experimental System

[0178] 1.1 Experimental Animals

[0179] 48 female Nu/Nu mice, 15-17 g.

[0180] 1.2 Cell Strains

[0181] JIMT-1 cells (human breast cancer cells): purchased from Nanjing Cobioer Biosciences Co., Ltd.

[0182] 2. Experimental Objective

[0183] JIMT-1 cell xenograft model of the Nu/Nu mice was used in this experiment to validate the influence of NBP on the antitumor efficacy and PK behavior of ab-PTX.

[0184] 3. Drugs

[0185] Lyophilized powder of paclitaxel (albumin bound), 100 mg/bottle, batch number: B042007410, made by CSPC Ouyi Pharmaceutical Co., Ltd, dissolved in normal saline immediately prior to use.

[0186] Butylphthalide (oral administration grade), 10 kg/bottle, batch number: 518180803, made by CSPC NBP Pharmaceutical Co., Ltd, diluted with vegetable oil to a target concentration immediately prior to use.

[0187] 4. Experimental Process

[0188] 4.1 Model Preparation

[0189] After in vitro resuscitation and passage of JIMT-1 cells to a sufficient amount, the cells were counted by a microscope, and diluted with a serum-free medium to adjust the cell count to about 1×10.sup.8 cells/mL. The cell suspension was kept in an ice-water bath.

[0190] The JIMT-1 cell suspension was extracted by a sterile syringe and inoculated into the subcutaneous tissue of the forelimb axilla of the Nu/Nu mice, with the inoculation volume of 0.1 mL/mouse containing about 1.0×10.sup.7 tumor cells, to prepare JIMT-1 xenograft tumor model of the Nu/Nu mice.

[0191] 4.2 Administration Method

[0192] Therapy Groups:

[0193] Monotherapy group of ab-PTX: intravenous injection of ab-PTX at 15 mg/kg once every week, qw (on days 0, 7, 14, 21, and 28). Administration period: 28 days. Administration volume: 10 mL/kg.

[0194] Monotherapy group of NBP: NBP was administered by transoral gavage at 60 mg/kg, bid (administration was commenced on day 0), the dosing interval should be longer than 4 h; and the administration period was 28 days. The administration volume was 10 mL/kg.

[0195] NBP/ab-PTX group: intravenous injection of ab-PTX at 15 mg/kg, qw (on days 0, 7, 14, 21, and 28). NBP was administered by transoral gavage at 6 mg/kg, 20 mg/kg, or 60 mg/kg twice per day, bid (administration was commenced on day 0), the dosing interval should be longer than 4 h; the first dose of NBP was administered 1 h prior to the ab-PTX administration. The administration period was 28 days. The administration volume was 10 mL/kg.

[0196] The solvent group was given normal saline at the same frequency and with the same method as ab-PTX, and was given vegetable oil at the same frequency and with the same method as NBP.

[0197] Note: The administration doses of NBP were converted into adult daily doses (based on 60 kg, oral administration) of 60 mg/d, 200 mg/d, and 600 mg/d, respectively.

[0198] 4.3 Observation Indexes and Evaluation Indexes

[0199] 4.3.1 General Observation Indexes

[0200] (1) General state observation: all animals were observed once per day during the experimental period, and the abnormality and behavioral changes of their body parts were recorded.

[0201] (2) Body weight: all animals were weighed once prior to the experiment, and animals having suitable body weights were selected for the experiment. Animals were weighed at set time once per day after the commencement of administration.

[0202] (3) Death and near death: the death time of dead animals was recorded, and dying animals should be observed more frequently to determine the death time.

[0203] 4.3.2 Tumor weight

[0204] At the end of the experiment, after the animals were euthanized by CO.sub.2 asphyxiation, the tumors were excised and weighed.

[0205] Tumor weight inhibition rate %=(1-tumor weight in therapy group/tumor weight in solvent group)×100%

[0206] 4.3.3 PK Blood Sampling and Detection

[0207] Before administration of the last dose of ab-PTX (0 h), and 5 min, 15 min, 0.5 h, 1 h, 4 h, 8 h, 24 h after the administration, blood was sampled (as detailed in Table 8), anticoagulated with heparin, and centrifuged to separate plasma, which was stored at −80° C. for later use. The total amount of paclitaxel in the plasma was determined by protein precipitation-LC/MS/MS.

TABLE-US-00008 TABLE 8 List of Blood Sampling Time of Mice in Each Group Group Animal No. Blood sampling time Solvent group 1-8 0 h Therapy groups 1-8 0 h, 5 min, 15 min, 0.5 h, 1 h, 4 h, 8 h, 24 h

[0208] 5. Results

[0209] 5.1 Influence on Body Weight

[0210] As can be seen from the body weight data, there was no significant difference in the body weights of the mice in each group, indicating that neither ab-PTX nor NBP had significant influence on the body weights of the mice, as detailed in FIG. 10.

[0211] 5.2 Influence on Tumor Weight

[0212] The tumor weight results showed that, compared with the solvent group, the tumor growth of the ab-PTX therapy group can be significantly inhibited (P<0.001), and the tumor growth of the mice in the NBP dose group of 60 mg/kg was not obviously affected; compared with the ab-PTX group of 15 mg/kg, the combined therapy of different doses of NBP and ab-PTX did not have statistical difference in tumor inhibition (P>0.05), as detailed in Table 9.

TABLE-US-00009 TABLE 9 Influence on Tumor Weights of Mice (mean ± SD, n = 8) Number of animals Tumor weight Inhibition rate Group (surviving/total) (g) (%) Solvent group 8/8 0.9969 ± 0.2426   — ab-PTX 15 mg/kg 8/8 0.2789 ± 0.0858*** 72.0 NBP/ab-PTX 6/15 mg/kg 8/8 0.2466 ± 0.1651*** 75.3 NBP/ab-PTX 20/15 mg/kg 8/8 0.2807 ± 0.1309*** 71.8 NBP/ab-PTX 60/15 mg/kg 8/8 0.3140 ± 0.1724*** 68.5 NBP 60 mg/kg 8/8 0.9570 ± 0.2529   4.0 Note: Compared with the solvent group: ***P < 0.001.

[0213] 5.4 Influence on PK

[0214] The results showed that the main pharmacokinetic parameters of paclitaxel in the plasma of the monotherapy group of ab-PTX and the combined therapy group of NBP and ab-PTX were substantially consistent without significant difference, indicating that combined therapy of butylphthalide and ab-PTX did not affect the pharmacokinetic behavior of paclitaxel in the plasma, as detailed in Table 10.

TABLE-US-00010 TABLE 10 Main Pharmacokinetic Parameters of Total Amount of Paclitaxel in Therapy Groups Mean ± SD (n = 8) NBP/ab-PTX NBP/ab-PTX NBP/ab-PTX Parameters (unit) ab-PTX 6/15 mg/kg 20/15 mg/kg 60/15 mg/kg C.sub.max (ng/mL) 2981 ± 718  2510 ± 290  2208 ± 382  3083 ± 498  AUC.sub.0-t(h*ng/mL) 2526 ± 963  2565 ± 371  2467 ± 633  2732 ± 376  AUC.sub.0-∞(h*ng/mL) 2575 ± 992  2600 ± 369  2491 ± 659  2762 ± 383  t.sub.1/2(h) 1.28 ± 0.28 1.16 ± 0.14 1.08 ± 0.13 1.09 ± 0.21 Vss (L/kg) 7.39 ± 1.64 6.64 ± 0.63 6.91 ± 1.73 5.70 ± 0.94 CI(L/h/kg) 6.61 ± 2.40 5.87 ± 0.81 6.43 ± 1.84 5.53 ± 0.82 MRT.sub.0-t(h) 1.05 ± 0.24 1.04 ± 0.13 1.01 ± 0.14 0.956 ± 0.140

[0215] 6. Conclusions

[0216] In this experiment, NBP did not have obvious influence on the antitumor efficacy of ab-PTX and the PK behavior of ab-PTX.

Example 5

[0217] Influence of NBP on in vitro efficacy of ab-PTX

[0218] 1. Cell and Culture Conditions

TABLE-US-00011 TABLE 11 Cell and Essential Culture Medium Histological Cell strains Source source Culture medium MDA-MB-231 Cell Culture Center, Peking Union Human breast RPMI Medium 1640 Medical College cancer cells containing 10% FBS JIMT-1 Nanjing Cobioer Biosciences Co., Human breast DMEM containing 10% Ltd cancer cells FBS SK-OV-3 Shanghai Institutes for Biological Human ovarian McCoy's 5A containing Sciences, Chinese Academy of cancer cells 10% FBS Sciences HT29 Shanghai Institutes for Biological Human colon McCoy's 5A containing Sciences, Chinese Academy of cancer cells 10% FBS Sciences A549 ATCC Human lung RPMI Medium 1640 cancer cells containing 10% FBS A375 Cell Culture Center, Peking Union Human DMEM containing 10% Medical College melanoma cells FBS

[0219] The above cell culture conditions were 37° C. and 5% CO.sub.2.

[0220] 2. Experimental Objective

[0221] To investigate whether NBP affects the function of ab-PTX in the in vitro inhibition of tumor cell proliferation.

[0222] 3. Drugs

[0223] Lyophilized powder of paclitaxel (albumin bound), 100 mg/bottle, batch number: B041909121, made by CSPC Ouyi Pharmaceutical Co., Ltd.

[0224] Butylphthalide (oral administration), 10 kg/bottle, batch number: 518180803, made by CSPC NBP Pharmaceutical Co., Ltd.

[0225] 4. Experimental Design

[0226] This experiment includes a monotherapy group of ab-PTX, a combined therapy group (ab-PTX/NBP), and an NBP group. Different drug concentration gradients were set for each group.

[0227] Monotherapy group of ab-PTX: {circle around (1)} MDA-MB-231, JIMT-1, SK-OV-3, and A549 cell strains: ab-PTX was 3× serially diluted from an initial concentration of 200 nM to 8 concentrations: 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, and 0.09 nM, respectively. {circle around (2)} A375 and HT29 cell strains: in the first experiment, ab-PTX was 3× serially diluted from an initial concentration of 100 nM to 8 concentrations: 100, 33.33, 11.11, 3.70, 1.24, 0.41, 0.14, and 0.05 nM, respectively; and in the repeated experiment, ab-PTX was 2× serially diluted from an initial concentration of 100 nM to 8 concentrations: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 nM, respectively.

[0228] ab-PTX/NBP group: the final concentration of NBP was 30 μM, and the concentration gradient of ab-PTX was the same as that of the monotherapy group.

[0229] Monotherapy group of NBP: NBP was 2× serially diluted from an initial concentration of 480 μM to 8 concentrations: 480, 240, 120, 60, 30, 15, 7.5, and 3.75 μM, respectively.

[0230] The action time of the drug was 72 h.

[0231] 5. Experimental Process

[0232] A certain number of conventionally cultured cells in logarithmic growth phase were inoculated in a 96-well plate with 100 μL per well. 24 h after adherence, 100 μL of the culture solution containing different concentration gradients of ab-PTX was added to each well of the monotherapy group of ab-PTX; 100 μL of the culture solution containing different concentration gradients of ab-PTX and 30 μM NBP was added to each well of the combined therapy group of ab-PTX/NBP; 100 μL of the culture solution containing different concentration gradients of NBP was added to each well of the monotherapy group of NBP; 3 repeated wells were provided for each concentration of each drug, and the blank wells (only culture medium was present) and the normal wells (drug concentration was 0) were provided. The drugs functioned for 72 h, then MTT working solution (5 mg/mL) was added at 20 μL per well; after functioning at 37° C. for 4 h, the supernatant was removed, 150 μL of DMSO (analytically pure) was added; the mixture was fully mixed using a microplate oscillator, the plate was wiped clean, and the optical density (OD) at 550 nm was measured using a microplate reader.

[0233] The cell growth inhibition rate was computed using the following equation:

Inhibition rate (%)=(OD value.sub.normal well−OD value.sub.administration well)/(OD value.sub.normal well−OD value.sub.blank well)×100%

[0234] Based on the inhibition rate of each concentration, the median inhibitory concentration IC.sub.50 of the drug was computed using SPSS19.0.

[0235] The experiment was repeated three times, and the IC.sub.50 value was expressed as the mean±SD of the results of the 3 experiments.

[0236] 6. Results

[0237] 6.1 Influence of NBP on IC.sub.50 Value of Ab-PTX

[0238] The results of the monotherapy group of ab-PTX showed that ab-PTX had obvious inhibitory effect on the in vitro proliferation of human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), and human melanoma (A375), and the mean IC.sub.50 value was in the range of 5-50 nM. NBP at a particular concentration (30 μM) was added for the combined therapy. Compared with the monotherapy of ab-PTX, the mean IC.sub.50 of ab-PTX of cells in the combined therapy group was not obviously changed, and remained in the range of 5-50 nM. The result showed that NBP did not significantly affect the function of ab-PTX in the in vitro inhibition of tumor cell proliferation.

[0239] IC.sub.50 values of the monotherapy of ab-PTX and the combined therapy of ab-PTX and NBP in inhibiting the in vitro proliferation of 6 tumor cell strains are shown in Table 12.

TABLE-US-00012 TABLE 12 IC.sub.50 Values (nM) of Monotherapy of ab-PTX and Combined Therapy of ab-PTX and NBP in Inhibiting the in vitro Proliferation of 6 Tumor Cell Strains IC.sub.50 (nM) Monotherapy of Combined therapy of Cell type ab-PTX ab-PTX/NBP MDA-MB-231 37.11 ± 11.78 47.60 ± 9.05  JIMT-1 5.61 ± 2.40 7.24 ± 0.96 SK-OV-3 20.82 ± 8.20  27.66 ± 15.05 HT29 5.03 ± 1.39 5.95 ± 1.83 A549 31.20 ± 16.72 46.23 ± 21.37 A375 12.18 ± 7.49  17.89 ± 7.51 

[0240] 6.2 Influence of NBP on the In Vitro Proliferation of Tumor Cells

[0241] The NBP alone did not have obvious inhibitory effect on the in vitro proliferation of human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), and human melanoma (A375). The inhibition rate of 30 μM NBP on the proliferation of 6 cell strains is shown in Table 13.

TABLE-US-00013 TABLE 13 Inhibition Rate (%) of 30 μM NBP Alone on 6 Cell Strains Cell type Inhibition rate (%, 30 μM NBP) MDA-MB-231  3.43 ± 3.95 JIMT-1  7.39 ± 13.23 SK-OV-3 12.37 ± 7.41 HT29 10.07 ± 7.94 A549  5.12 ± 7.36 A375 10.92 ± 0.57

[0242] 7. Conclusions

[0243] NBP does not affect the inhibitory effect of ab-PTX on the in vitro proliferation of human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), and human melanoma cells (A375); and the NBP alone did not have obvious inhibitory effect on the in vitro proliferation of human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), and human melanoma (A375).

Example 6

[0244] Study on in vivo efficacy of butylphthalide on bortezomib chemotherapy-induced peripheral neuropathy

[0245] 1. Experimental Animals

[0246] 72 male SD (Sprague Dawley) rats, 180-200 g.

[0247] 2. Drugs

[0248] Bortezomib: batch number: 20191102, made by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd, dissolved in normal saline containing 2% DMSO immediately prior to use.

[0249] Butylphthalide (oral administration grade), 10 kg/bottle, batch number: 518180803, made by CSPC NBP Pharmaceutical Co., Ltd, diluted in vegetable oil to a target concentration immediately prior to use.

[0250] Duloxetine hydrochloride, batch number: QRQYD-JN, made by TCI (Shanghai) Development Co., Ltd, prepared with normal saline containing 2% DMSO immediately prior to use.

[0251] 3. Experimental Process

[0252] 3.1 Model Preparation and Administration Method

[0253] In this experiment, male SD rats were administered with bortezomib at a dose of 0.3 mg/kg with an administration volume of 5 mL/kg, and were intraperitoneally injected on days 0, 2, 4, 6, 8, 10, and 13 for modeling.

[0254] Three doses (3, 10, and 30 mg/kg bid) of NBP were used. The first dose of NBP was administered by transoral gavage 1 day prior to the Bortezomib modeling. During the Bortezomib administration and modeling, the first dose of NBP was administered 1 h prior to the Bortezomib administration every day. The second dose of NBP was administered with an interval of ≥4 h from the first dose, with an administration volume of 10 mL/kg, and was administered for 21 consecutive days.

[0255] The normal group was given normal saline containing 2% DMSO at the same frequency and with the same administration method as Bortezomib, and was given vegetable oil at the same frequency and with the same administration method as NBP. (The modeling and administration time are shown in FIG. 11. The animal grouping, the administration method, and the doses are detailed in Table 14).

TABLE-US-00014 TABLE 14 Animal Grouping and Dose List Number of Dose Administration Group animals Drug (mg/kg) route Normal control 12 Vegetable oil/normal 0/0  p.o./i.p. group saline containing 2% DMSO Model group 12 Vegetable  0/0.3 p.o./i.p. Oil/Bortezomib Low-dose group of 12 NBP/Bortezomib  3/0.3 p.o./i.p. NBP Moderate-dose 12 NBP/Bortezomib 10/0.3 p.o./i.p. group of NBP High-dose group of 12 NBP/Bortezomib 30/0.3 p.o./i.p. NBP Positive drug 12 Duloxetin/Bortezomib 15/0.3 p.o./i.p. duloxetine group

[0256] 3.2 Grouping

[0257] Based on the thermal pain threshold baseline values and the body weights of the rats, the rats with the baseline values ranging from 3 to 6 s were selected, and were equally grouped in accordance with Table 14 in 3.1.

[0258] 3.3 Observation Indexes

[0259] (1) Body weight: all animals were weighed once prior to the experiment, and were weighed at set time every day after the commencement of administration.

[0260] (2) Thermal pain threshold: measurements were performed on days 0, 7, 14, and 21.

[0261] Method: IITC Life Science Electronic Von Frey Anesthesio meter was used and the intensity of the light source was adjusted to obtain a thermal pain threshold baseline of about 3-6 s. In this case, the light intensity was 58%, and the cutoff value was set as 10 s. The rats were placed in a plastic compartment on a glass plate, to make the planta centers of the rats be exposed to the light source. The time of reflexive withdrawal, i.e., the thermal pain threshold, of the rats was recorded, and was measured once for each of the left foot and the right foot of each rat, the measurement was repeated three times with each interval therebetween ≥15 min, to compute a mean value of the 6 thermal pain thresholds of each rat.

[0262] (3) Mechanical Stimulus Pain Threshold: Measurements were Performed on Days −1, 6, 13, and 20.

[0263] Method: the rats were placed in a plastic compartment on a wire mesh, left to stand still for 15 min, stimulated using a stimulation probe with a diameter of 0.4 mm of the IITC Life Science Electronic Von Frey Anesthesio meter at the planta centers of the rats by gradually increasing the force until reflexive withdrawal of the rats. The maximum value displayed by the instrument at this time, i.e., the mechanical pain threshold, was recorded. In each experiment, the left and right feet of each rat were measured three times with each interval therebetween ≥5 min, to compute a mean value of the 6 mechanical pain thresholds of each rat.

[0264] 4. Results

[0265] 4.1 Influence on Body Weight

[0266] On day 21 of the experiment, compared with the normal control group, the body weights of the rats in the model group were significantly decreased (P<0.05); and compared with the model group, the body weights of the rats in the three dose groups of NBP (3, 10, and 30 mg/kg bid) were not obviously changed, as detailed in FIG. 12.

[0267] 4.2 Influence on Thermal Pain Threshold

[0268] On day 21 of the experiment, compared with the normal control group, the thermal pain threshold of the model group was significantly decreased; and compared with the model group, the decrease of the thermal pain threshold of the model rats in the group of NBP 3, 10, and 30 mg/kg bid can be significantly reversed, as detailed in FIG. 13.

[0269] 4.3 Influence on Mechanical Stimulus Pain Threshold

[0270] On day 20 of the experiment, compared with the normal control group, the mechanical stimulus pain threshold of the model group was significantly decreased; and compared with the model group, the decrease of the mechanical stimulus pain threshold of the model rats in the group of NBP 10 and 30 mg/kg bid can be significantly reversed, as detailed in FIG. 14.

[0271] 5. Conclusions

[0272] Under the conditions of this experiment, NBP can dose-dependently effectively relieve the symptoms of Bortezomib-induced peripheral neuropathy in the CIPN model of rat.

Example 7

[0273] Experiment for influence of NBP on in vitro anti-tumor activity of Bortezomib

[0274] 1. Cell and Culture Conditions

TABLE-US-00015 TABLE 15 Cell and Essential Culture Medium Cell strains Source Histological source Culture medium MM.1S Nanjing Cobioer Biosciences Human multiple RPMI Medium 1640 Co., Ltd myeloma cells containing 10% FBS Jeko-1 Shanghai Institutes for Human mantle cell RPMI Medium 1640 Biological Sciences, Chinese lymphoma cells containing 20% FBS Academy of Sciences HT29 Shanghai Institutes for Human colon cancer McCoy's 5A Biological Sciences, Chinese cells containing 10% Academy of Sciences FBS A549 ATCC Human non-small cell RPMI Medium 1640 lung cancer cells containing 10% FBS DU145 Institute of Basic Medical Human prostate cancer RPMI Medium 1640 Sciences, Chinese Academy of cells containing 10% FBS Medical Sciences

[0275] The above cell culture conditions were 37° C. and 5% CO.sub.2.

[0276] 2. Experimental Objective

[0277] To investigate whether NBP affects the function of Bortezomib in the in vitro inhibition of tumor cell proliferation.

[0278] 3. Drugs

[0279] Bortezomib (bulk drug), made by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd., batch number: 20190802

[0280] Butylphthalide (oral administration grade), 10 kg/bottle, made by CSPC NBP Pharmaceutical Co., Ltd., batch number: 518180803

[0281] 4. Experimental Design

[0282] This experiment includes a monotherapy group of Bortezomib, a combined therapy group (Bortezomib/NBP), and a monotherapy group of NBP. Different drug concentration gradients were set for each group.

[0283] Monotherapy group of Bortezomib: {circle around (1)} MM.1S, Jeko-1, and HT29 cells: Bortezomib was 1.5× serially diluted from an initial concentration of 20 nM to 8 concentrations: 20, 13.33, 8.89, 5.93, 3.95, 2.63, 1.76, and 1.17 nM, respectively; {circle around (2)} A549 cells: Bortezomib was 2×serially diluted from an initial concentration of 200 nM to 8 concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 nM, respectively; and {circle around (3)} DU145 cells: Bortezomib was 2× serially diluted from an initial concentration of 100 nM to 8 concentrations: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 nM, respectively.

[0284] Bortezomib/NBP group: the final concentration of NBP was 30 μM, and the concentration gradient of Bortezomib was the same as the final concentration of the monotherapy group of Bortezomib.

[0285] Monotherapy group of NBP: NBP was 2× serially diluted from an initial concentration of 480 μM to 8 concentrations: 480, 240, 120, 60, 30, 15, 7.5, and 3.75 μM, respectively.

[0286] The action time of the drug was 72 h.

[0287] 5. Experimental Process

[0288] A certain number of conventionally cultured cells in logarithmic growth phase were inoculated in a 96-well plate with 100 μL per well. For suspension cells MM.1S and Jeko-1, on the day of inoculation, 100 μL of a culture solution containing different concentration gradients of Bortezomib was added into each well of the Bortezomib group, 100 μL of a culture solution containing different concentration gradients of Bortezomib and 30 μM NBP was added into each well of the combined therapy group of Bortezomib/NBP, and 100 μL of a culture solution containing different concentration gradients of NBP was added into each well of the monotherapy group of NBP. For adherent cells HT29, A549, and DU145, the above drugs were added 24 h after adherence. 3 replicate wells were set for each concentration of each drug, and blank wells (only culture medium was present, and no tumor cells were inoculated) and normal wells (a culture medium inoculated with tumor cells, drug concentration was 0) were set. The drugs functioned for 72 h, then MTT working solution (5 mg/mL) was added at 20 μL per well; after functioning at 37° C. for 4 h, the supernatant was removed, 150 μL of DMSO (analytically pure) was added; the mixture was fully mixed using a microplate oscillator, the plate was wiped clean, and the optical density (OD) at 550 nm was measured using a microplate reader.

[0289] The cell growth inhibition rate was computed using the following equation:

Inhibition rate (%)=(OD value.sub.normal well−OD value.sub.administration well)/(OD value.sub.normal well−OD value.sub.blank well)×100%

[0290] Based on the inhibition rate of each concentration, the median inhibitory concentration IC.sub.50 of the drug was computed using SPSS19.0.

[0291] The experiment was repeated three times, and the IC.sub.50 value was expressed as the mean±standard deviation of the results of the 3 experiments.

[0292] 6. Results

[0293] The results showed that both the monotherapy group of Bortezomib and the combined therapy group of Bortezomib/NBP had obvious inhibitory effect on the in vitro proliferation of MM.1S, Jeko-1, HT29, A549, and DU145, and mean IC.sub.50 values thereof were in the range from 1 to 20 nM, suggesting that NBP did not affect the function of Bortezomib in the in vitro inhibition of tumor cell proliferation, as detailed in FIG. 16.

TABLE-US-00016 TABLE 16 IC.sub.50 Values (nM) of Bortezomib and Combined Therapy of Bortezomib/NBP for 5 Cell Strains Bortezomib/NBP Bortezomib 1st 2nd 3rd 1st dose 2nd dose 3rd dose Mean ± SD dose dose dose Mean ± SD MM.1S 2.55 2.94 2.99  2.83 ± 0.24 2.55 3.25 2.99  2.93 ± 0.35 Jeko-l 4.83 3.51 4.47  4.27 ± 0.68 5.82 3.45 4.60  4.62 ± 1.19 HT29 4.43 4.97 5.65  5.02 ± 0.61 4.67 5.56 5.72  5.32 ± 0.56 A549 11.82 13.31 10.56 11.89 ± 1.38 9.01 11.60 10.92 10.51 ± 1.34 DU145 12.79 11.88 17.13 13.94 ± 2.80 9.59 10.39 15.32 11.77 ± 3.10

[0294] The monotherapy of NBP does not have obvious inhibitory effect on the in vitro proliferation of human multiple myeloma cells (MM.1S), human mantle cell lymphoma cells (Jeko-1), human colon cancer cells (HT29), human lung cancer cells (A549), and human prostate cancer cells (DU145). The inhibition rate of 30 μM NBP alone on the proliferation of 5 cell strains is detailed in Table 17.

TABLE-US-00017 TABLE 17 Inhibition Rate (%) of 30 μM NBP Alone on Proliferation of 5 Cell Strains Inhibition rate (%, 30 μM NBP) Cell type First dose Second dose Third dose Mean ± SD MM.1S 0.16 14.08 22.08 12.11 ± 11.09 Jeko-1 −7.67 −18.83 −8.11 −11.54 ± 6.32  HT29 9.98 8.27 13.73 10.66 ± 2.79  A549 −11.08 9.68 4.74  1.11 ± 10.84 DU145 −2.22 9.44 4.87 4.03 ± 5.88

[0295] Conclusions: NBP does not affect the inhibitory effect of Bortezomib on the in vitro proliferation of human multiple myeloma cells (MM.1S), human mantle cell lymphoma cells (Jeko-1), human colon cancer cells (HT29), human lung cancer cells (A549), and human prostate cancer cells (DU145).

Example 8

[0296] Experiment for influence of NBP on in vivo anti-tumor efficacy of Bortezomib

[0297] 1. Experimental System

[0298] 1.1 Experimental Animals

[0299] 42 female Nu/Nu mice, 18-20 g.

[0300] 1.2 Cell Strains

[0301] MM.1S cells (human multiple myeloma cells), purchased from Nanjing Cobioer Biosciences Co., Ltd.

[0302] 2. Experimental Objective

[0303] A xenograft model of MM.1S cells of the Nu/Nu mice was used in this experiment to validate the influence of NBP on the antitumor efficacy and PK behavior of Bortezomib.

[0304] 3. Drugs

[0305] Bortezomib (bulk drug): manufacturer: CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd, batch number: 20191102; dissolved in normal saline containing 2% DMSO immediately prior to use.

[0306] Butylphthalide (oral administration grade), 10 kg/bottle, manufacturer: CSPC NBP Pharmaceutical Co., Ltd, batch number: 518180803, diluted in vegetable oil to a target concentration immediately prior to use.

[0307] Palbociclib (bulk drug), manufacturer: Shanghai Fangnan Biotechnology Co., Ltd, batch number: AL-0127-API-1811001; prepared with a solution of propylene glycol and 20% hydroxypropyl-β-cyclodextrin at a ratio of 5:95 to a solution having a target concentration immediately prior to use.

[0308] 4. Experimental Process

[0309] 4.1 Model Preparation

[0310] After in vitro resuscitation and passage of MM.1S cells to a sufficient amount, the cells were counted by a microscope, and diluted with a serum-free medium to adjust the cell count to about 1×10.sup.8 cells/mL. The cell suspension was kept in an ice-water bath.

[0311] The MM.1S cell suspension was extracted by a sterile syringe and inoculated into the subcutaneous tissue of the forelimb axilla of the Nu/Nu mice. The inoculation volume was 0.1 mL/mouse, containing about 1.0×10.sup.7 tumor cells, to prepare MM.1S xenograft tumor model of the Nu/Nu mice.

[0312] 4.2 Administration Method

[0313] Monotherapy group of Bortezomib: administered by intraperitoneal injection at 0.5 mg/kg, biw (on days 0, 3, 7, 10, and 15);

[0314] Monotherapy group of NBP: administered by transoral gavage at 60 mg/kg, bid (the administration was commenced on day 0), and the second dose of NBP was administered with an interval from the first dose of ≥4 h;

[0315] Monotherapy group of Palbociclib: intragastric administration at 70 mg/kg, qd (the administration was commenced on day 0);

[0316] Combined therapy group of NBP/Bortezomib: the administration frequency and dose were the same as the frequency and dose of the monotherapy group of NBP and the monotherapy group of Bortezomib, respectively.

[0317] Combined therapy group of Bortezomib/Palbociclib: the administration frequency and dose were the same as the frequency and dose of the monotherapy group of Bortezomib and the monotherapy group of Palbociclib, respectively.

[0318] Combined therapy group of NBP/Bortezomib/Palbociclib: the administration frequency and dose were the same as the frequency and dose of the monotherapy group of NBP, the monotherapy group of Bortezomib, and the monotherapy group of Palbociclib, respectively.

[0319] The solvent group was given the solvent at the same frequency and with the same administration method as Bortezomib and NBP.

[0320] The administration period was 16 days, and the administration volume was 10 mL/kg.

[0321] 4.3 Observation Indexes and Evaluation Indexes

[0322] 4.3.1 Observation Indexes

[0323] (1) General state observation: all animals were observed once per day during the experimental period, and the abnormality and behavioral changes of their body parts were recorded.

[0324] (2) Body weight: all animals were weighed once prior to the experiment, and animals having suitable body weights were selected for the experiment. Animals were weighed at set time once per day after the commencement of administration.

[0325] (3) Death and near death: the death time of dead animals was recorded, and dying animals should be observed more frequently to determine the death time.

[0326] 4.3.2 Tumor Volume Evaluation

[0327] After animals were grouped, the long diameter and the short diameter of the tumor were measured twice every week.

[0328] (1) Tumor volume: V=½×A×B.sup.2

[0329] (2) Relative tumor volume:

[00001]$\mathrm{RTV}=\frac{\mathrm{TVn}}{\mathrm{TV}0d}$

[0330] (3) Relative tumor volume proliferation rate:

[00002]$T/C\%=\frac{\mathrm{Therapy}\mathrm{group}\mathrm{RTVxnd}}{\mathrm{Solvent}\mathrm{group}\mathrm{RTVxnd}}\times 100\%$

[0331] (4) Tumor growth inhibition rate:

[00003]$\mathrm{TGI}\%=\left[1-\frac{({\mathrm{TV}}_{\mathrm{xn}}-{\mathrm{TV}}_{x0}}{\left({\mathrm{TV}}_{\mathrm{Mn}}-{\mathrm{TV}}_{M0}\right)}\right]\times 100\%$

[0332] Note: V: tumor volume; A: tumor length; B: tumor width; RTV: relative tumor volume; TV.sub.nd: tumor volume on day n; TV.sub.0d: tumor volume on day 0; RTV.sub.xnd: mean relative tumor volume on day n; TV.sub.Xn: mean tumor volume of the therapy group on day n; TV.sub.X0: mean tumor volume of the therapy group on day 0; TV.sub.Mn: mean tumor volume of the solvent group on day n; and TV.sub.M0: mean tumor volume of the solvent group on day 0

[0333] 4.3.3 Tumor Weight

[0334] At the end of the experiment, after the animals were euthanized by CO.sub.2 asphyxiation, the tumors were excised and weighed.

[0335] Tumor weight inhibition rate %=(1-tumor weight in therapy group/tumor weight in solvent group)×100%

[0336] 4.3.4 PK Blood Sampling and Detection

[0337] Before administration of the last dose of Bortezomib (0 h), and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h after the administration, blood was sampled (as detailed in Table 8) (except that blood was sampled from the mice in the solvent group only at 0 h), anticoagulated with heparin, and centrifuged to separate plasma, which was stored at −80° C. for later use. Bortezomib and butyphthalide in the plasma were determined by protein precipitation-LC/MS/MS.

TABLE-US-00018 TABLE 18 List of Blood Sampling Time of Mice in Each Group Group Animal No. Blood sampling time Solvent group 1-6 0 h Therapy groups 1-6 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h

[0338] 5. Experimental Results

[0339] 5.1 Influence on Body Weight

[0340] As can be seen from the body weight data, there was no significant difference in the body weights of the mice in each group (P>0.05), indicating that none of Bortezomib, Palbociclib, and NBP had influence on the body weights of the mice, as detailed in FIG. 15.

[0341] 5.2 Influence on Tumor Volume

[0342] At the end of the experiment, compared with the monotherapy group of Bortezomib, during the combined therapy of NBP/Bortezomib, NBP did not have obvious negative influence on the tumor inhibition efficacy of Bortezomib; and compared with the combined therapy group of Bortezomib/Palbociclib, in the combined therapy group of NBP/Bortezomib/Palbociclib, NBP did not have obvious negative influence on the antitumor efficacy of combined therapy of Bortezomib/Palbociclib, either. The tumor volume of each group is detailed in FIG. 16, and the body weight, TV, RTV, TGI %, T/C %, tumor weight, etc. at the end of the experiment (day 14) are detailed in Table 19.

TABLE-US-00019 TABLE 19 Summary of Various Index Parameters of NU/NU Mice on Day 14 of the Experiment (mean ± SD, n = 6) Bor/Pal NBP/Bor/Pa NBP/Bor Bor Pal 0.5/70 1 60/0.5/70 60/0.5 NBP 60 Index Solvent 0.5 mg/kg 70 mg/kg mg/kg mg/kg mg/kg mg/kg Body 24.4 ± 24.7 ± 23.9 ± 24.0 ± 24.1 ± 23.8 ± 24.9 ± weight(g) 1.8 1.2 1.7 0.9 2.1 1.3 1.2 TV (mm.sup.3) 1220.4 ± 1124.8 ± 605.4 ± 436.7 ± 420.6 ± 876.9 ± 938.1 ± 625.3 260.5 388.7 178.3 178.9 300.8 451.9 RTV 9.8 ± 9.6 ± 4.3 ± 3.4 ± 3.3 ± 7.2 ± 7.2 ± 2.1 2.8 1.5 0.9 0.6 2.8 2.0 TGI % — 8.7 56.3 71.7 73.1 31.3 25.8 T/C % 98.0 44.4 34.9 34.2 73.9 73.8 Tumor weight 1.648 ± 1.282 ± 0.550 ± 0.391 ± 0.413 ± 1.086 ± 1.040 ± 0.678 0.206 0.392 0.176 0.227 0.341 0.398 Tumor weight — 22.2 66.6 76.3 75.0 34.1 36.9 inhibition rate (%) Note: Bor represents Bortezomib, and Pal represents Palbociclib.

[0343] Conclusions: in this experiment, NBP did not have obvious influence on the antitumor efficacy of Bortezomib and of the combined therapy of Bortezomib/Palbociclib.

[0344] 5.3 Influence on PK

[0345] The results showed that the Bortezomib exposure doses in the bodies of the mice in the Bortezomib group, the NBP/Bortezomib group, the Bortezomib/Palbociclib group, and the NBP/Bortezomib/Palbociclib group were substantially consistent, suggesting that NBP did not have obvious influence on the in vivo pharmacokinetic behaviors of the monotherapy of Bortezomib and the combined therapy of Bortezomib/Palbociclib. The main pharmacokinetic parameters are detailed in Table 20, Table 21, and Table 22.

TABLE-US-00020 TABLE 20 Summary of Main Pharmacokinetic Parameters of Bortezomib in Plasma of NU/NU Mice (mean ± SD, n = 6) Bor Bor/Pal NBP/Bor/Pal NBP/Bor Parameters (unit) 0.5 mg/kg 0.5/70 mg/kg 60/0.5/70 mg/kg 60/0.5 mg/kg C.sub.max (ng/mL) 113 ± 8  114 ± 17 128 ± 16  126 ± 37  AUC.sub.0-t (h*ng/mL) 290 ± 104 322 ± 57 234 ± 66  264 ± 75  t.sub.1/2(h) 56.6 ± 1.16  40.2 ± 14.8 65.6 ± 66.2 59.1 ± 63.5 Vd (L/kg) 14.2 ± 3.2  24.2 ± 6.4 37.2 ± 11.6 24.2 ± 7.1  CI(L/h/kg) 1.81 ± 0.56  0.44 ± 0.09 0.86 ± 0.67 0.97 ± 0.91 MRT.sub.0-t(h) 5.9 ± 0.8 12.7 ± 2.2 10.5 ± 0.9  9.5 ± 4.2

TABLE-US-00021 TABLE 21 Summary of Main Pharmacokinetic Parameters of NBP in Plasma of NU/NU Mice (mean ± SD, n = 6) NBP/Bor/Pal NBP/Bor NBP Parameters (unit) 60/0.5/70 mg/kg 60/0.5 mg/kg 60 mg/kg C.sub.max (ng/mL) 145 ± 108 125 ± 74 117 ± 88 AUC.sub.0-t(h*ng/mL) 242 ± 259  433 ± 166  236 ± 100 t.sub.1/2(h) 4.91 ± 2.52 10.9 ± 3.2 10.1 ± 9.7 Vd (L/kg) 1979 ± 1486 1723 ± 699 1445 ± 298 CI(L/h/kg) 258 ± 124 112 ± 36  173 ± 104 MRT.sub.0-t(h) 2.1 ± 0.7  7.5 ± 1.2  3.1 ± 0.6

[0346] Note: In Tables 20 and 21, Bor represents Bortezomib, Pal represents Palbociclib, C.sub.max represents a maximum blood concentration, AUC.sub.0-t represents the area under the drug-time curve from 0 to t, t.sub.1/2 represents the half-life, Vd represents the apparent volume of distribution, CI represents the clearance rate, and MRT.sub.0-t represents the mean residence time from 0 to t.

TABLE-US-00022 TABLE 22 Results of Comparison between Mean Plasma Exposures of Bortezomib and NBP in Combined Therapy Group and Monotherapy Group Group C.sub.max AUC.sub.0-t LN(C.sub.max) LN(AUC.sub.0-t) Ratio to monotherapy group of Bortezomib Bor/Pal 0.5/70 mg/kg 1.01 1.11 1.00 1.02 NBP/Bor/Pal 60/0.5/70 mg/kg 1.13 0.81 1.05 0.96 NBP/Bor 60/0.5 mg/kg 1.12 0.91 1.02 0.98 Ratio to monotherapy group of NBP NBP/Bor/Pal 60/0.5/70 mg/kg 1.24 1.03 1.05 1.00 NBP/Bor 60/0.5 mg/kg 1.08 1.83 1.02 1.11 Note: Bor represents Bortezomib, and Pal represents Palbociclib.

[0347] The ratios of C.sub.max and AUC.sub.0-t of Bortezomib in the combined therapy groups of Bortezomib/Palbociclib, the combined therapy groups of NBP/Bortezomib/Palbociclib, and the combined therapy groups of NBP/Bortezomib to those in the monotherapy group of Bortezomib were 1.01-1.13 and 0.81-1.11, respectively. Upon logarithmic transformation of C.sub.max and AUC.sub.0-t, the ratios of the combined therapy groups of Bortezomib/Palbociclib, the combined therapy groups of NBP/Bortezomib/Palbociclib, and the combined therapy groups of NBP/Bortezomib to the monotherapy group of Bortezomib were 1.00-1.05 (LNC.sub.max) and 0.96-1.02 (LNAUC.sub.0-t), respectively, therefore the in vivo exposures of Bortezomib in the monotherapy group and the combined therapy groups were substantially consistent.

[0348] The ratios of C.sub.max and AUC.sub.0-t of NBP in the combined therapy groups of NBP/Bortezomib/Palbociclib and the combined therapy groups of NBP/Bortezomib to those in the monotherapy group of NBP were 1.08-1.24 and 1.03-1.83, respectively. Upon logarithmic transformation of C.sub.max and AUC.sub.0-t, the ratios of the combined therapy groups of NBP/Bortezomib/Palbociclib and the combined therapy groups of NBP/Bortezomib to the monotherapy group of NBP were 1.02-1.05 (LNC.sub.max) and 1.00-1.11 (LNAUC.sub.0-t), respectively. Due to the obvious individual differences in mice after intragastric administration of NBP, a small number of mice were arranged in each experimental group. The ratio of the exposure level of the combined therapy group to the exposure level of the monotherapy group after logarithmic transformation showed that the in vivo exposure of the monotherapy group of NBP was not obviously different from the in vivo exposure of the combined therapy group.

[0349] Conclusions: in this experiment, NBP substantially did not affect the pharmacokinetic behavior of Bortezomib in mice, and Bortezomib substantially did not affect the pharmacokinetic behavior of NBP in mice.