WO2020073642A1 - 靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途 - Google Patents

靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途 Download PDF

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WO2020073642A1
WO2020073642A1 PCT/CN2019/084873 CN2019084873W WO2020073642A1 WO 2020073642 A1 WO2020073642 A1 WO 2020073642A1 CN 2019084873 W CN2019084873 W CN 2019084873W WO 2020073642 A1 WO2020073642 A1 WO 2020073642A1
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indirubin
bortezomib
multiple myeloma
btz
cell
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PCT/CN2019/084873
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French (fr)
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郝牧
于珍
邱录贵
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中国医学科学院血液病医院（血液学研究所）
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the use of indirubin compounds and bortezomib in the preparation of drugs for treating multiple myeloma, and belongs to the field of medicine and pharmacy.
  • MM Multiple myeloma
  • proteasome inhibitors bovine serum, plasma, and many others.
  • immunomodulators thalidomide, lenalidomide
  • the median overall survival period was extended from less than 3 years to 5 to 7 years.
  • most of the clinically used anti-myeloma drugs such as proteasome inhibitors and immunomodulators are patented products of foreign pharmaceutical companies, which are expensive.
  • the chemotherapy based on them has significantly benefited patients and brought huge medical treatment. Cost; The excessive medical burden has seriously affected the treatment process of patients.
  • the first generation proteasome inhibitor bortezomib is currently one of the domestic skeleton drugs for MM treatment, which can specifically inhibit the activity of proteasome, inhibit the expression of genes related to the proliferation of MM cells, and reduce interleukin 6 (IL-6)
  • IL-6 interleukin 6
  • the secretion of MM cell growth factors and the expression of adhesion factors eventually lead to tumor cell apoptosis.
  • bortezomib is expensive.
  • the price of a foreign imported bortezomib (3.5 mg) is about 7,000 yuan.
  • Even domestic bortezomib (3.5 mg) is about 5,000 yuan.
  • the technical problem to be solved by the present invention is to provide the use of indirubin compounds and bortezomib in the preparation of drugs for treating multiple myeloma, especially in the preparation of drugs for multiple myeloma resistant to bortezomib the use of.
  • Another technical problem to be solved by the present invention is to provide a drug for treating multiple myeloma resistant to bortezomib.
  • the present invention adopts the following technical solutions:
  • the indirubin compound is indirubin and / or indirubin-3'-oxime (Indirubin-3'-oxime, ID3, Id3).
  • Indirubin is a new type of double indole antitumor drug that has a therapeutic effect on chronic myeloid leukemia (slow granule) found in Chinese herbal medicine Qingdai. It is the first new type of anti-leukemia drug in China.
  • the CAS number of indirubin is 479-41-4, and the structural formula is as follows:
  • the indirubin-3'-oxime (ID3, Id3) has the structural formula as follows:
  • ID3 (Id3) and its derivatives are a class of bis-indole heterocyclic planar structure compounds.
  • Studies on the anticancer mechanism of ID3 (Id3) and its derivatives by foreign researchers in recent years have shown that ID3 (Id3) and its derivatives have an inhibitory effect on the cell cycle-dependent protein kinase (CDK) that is widespread in cells, leading to cancer cells The cell cycle is stagnant, thereby inhibiting tumor cell proliferation.
  • CDK cell cycle-dependent protein kinase
  • the multiple myeloma refers to a common multiple myeloma and / or multiple myeloma that is resistant to bortezomib.
  • the treatment refers to inhibiting the growth of multiple myeloma tumor volume and / or inhibiting the growth of tumor mass.
  • Indirubin compound ID3 single drug or small dose combined with bortezomib (BTZ) can significantly inhibit MM cell proliferation and promote MM cell apoptosis. While reducing the dose of BTZ, it can achieve the same curative effect as BTZ, and significantly reduce the economic cost of medication for patients.
  • Indirubin-like compound ID3 (Id3) can overcome bortezomib resistance by continuously inducing endoplasmic reticulum stress response and weakening unfolded protein response (UPR). It can significantly inhibit the proliferation of BTZ-resistant MM cells and promote their apoptosis.
  • a pharmaceutical composition for treating multiple myeloma the active ingredients are indirubin compounds and bortezomib.
  • the indirubin compound is indirubin (Indirubin, CAS No. 479-41-4) and / or indirubin-3'-oxime (Indirubin-3'-oxime, ID3, Id3, CAS number: 160807 -49-8).
  • the multiple myeloma refers to a common multiple myeloma and / or multiple myeloma that is resistant to bortezomib.
  • the treatment refers to inhibiting the growth of multiple myeloma tumor volume and / or inhibiting the growth of tumor mass.
  • the pharmaceutical composition of the present invention has a good effect of inhibiting the growth of myeloma, and can solve the problem that multiple myeloma is resistant to existing drugs.
  • composition of the present invention can be administered by any route, for example, oral, nasal, parenteral, intravenous, intradermal, subcutaneous, or topical administration in liquid or solid form.
  • the active ingredient can be placed in a solution or suspension.
  • Solutions and suspensions may also contain the following ingredients: sterile diluents for injection, for example, water for injection: liposomal particle suspensions, in which a stable active drug is contained in the center of the particle, the particle has a predetermined pH value and protected internal environment; liposome particle suspension, in which the active drug is externally attached to the particle surface or any of the two membranes; saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other Synthetic solvents; antibacterial agents, such as benzyl alcohol; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraethyl buffer, such as acetic acid (salt), citric acid (salt), and tonicity adjustment Substances, such as sodium chloride or glucose.
  • Preparations for parenteral administration can be enclosed in ampoules, disposable syringes
  • the advantage of the present invention is that the present invention finds for the first time that the indirubin-like compound ID3 (Id3) single drug is effective for patients resistant to bortezomib.
  • ID3 (Id3) and bortezomib is less toxic, significantly reduces the amount of bortezomib, and can significantly reduce the cost of treatment for patients.
  • Figure 1A is the results of the inhibition rate of the combination of two drugs on the proliferation of MM cells in ANBL6 cells treated with 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M ID3 (Id3) combined with 2.5nM BTZ for 24h.
  • FIG. 1B is a graph of the synergy index (CI) of the combination of two drugs of different concentrations in FIG. 1A obtained by using CompuSyn software.
  • Figure 1C shows the results of the inhibition rate of MM cell proliferation by U266 cells treated with 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M ID3 (Id3) combined with 2.5nM or 10nM BTZ for 48h.
  • FIG. 1D is a graph of the synergy index (CI) of the combination of different concentrations of the two drugs in FIG. 1C obtained by using CompuSyn software.
  • Figure 1E shows the results of ARP1 cells treated with 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M ID3 (Id3) combined with 5nM BTZ for 24h, the inhibition rate of MM cell proliferation by the combination of the two drugs.
  • Fig. 1F is a graph of the synergy index (CI) of the combination of different concentrations of the two drugs in Fig. 1E obtained by using CompuSyn software.
  • Figure 2A shows the results of the ID3 (Id3) combined with BTZ can synergistically promote the apoptosis of MM cells (ANBL6).
  • Fig. 2B is the result of the experiment that ID3 (Id3) combined with BTZ can synergistically promote the apoptosis of MM cells (U266).
  • Figure 2C shows the results of ID3 (Id3) combined with BTZ can synergistically promote the apoptosis of MM cells (ARP1).
  • Fig. 3A is the test result of the combination of ID3 (Id3) and BTZ can significantly inhibit the growth of tumor cells in mice.
  • Figure 3B is a comparison of the inhibitory effect of the combination of low-dose ID3 (Id3) and BTZ on tumor growth in mice compared with high-dose ID3 (Id3) and high-dose BTZ single drug application and the control group.
  • Figure 3C is a comparison of the inhibitory effect of the combination of low-dose ID3 (Id3) and BTZ on tumor growth in mice compared with low-dose ID3 (Id3) and low-dose BTZ single drug application and the control group.
  • Figure 4 shows the test results of ID3 (Id3) combined with BTZ can synergistically inhibit the endoplasmic reticulum stress response.
  • Figure 5 shows the test results of the different sensitivity of the two MM cell lines ANBL6 and ANBL6-BR to BTZ.
  • Figure 6 shows the results of ID3 (Id3) can promote the apoptosis of MM cells by reducing the proportion of SBL of ANBL6-BR cells.
  • Figure 7 shows the results of the sensitivity test of ANBL6 and ANBL6-BR to ID3 (Id3).
  • Figure 8A shows the results of ID3 (Id3) reversible BTZ resistance in MM cells.
  • Fig. 9 is a graph showing the results of ID3 (Id3) combined with BTZ can synergistically promote the apoptosis of MM cells (ANBL6-BR) resistant to BTZ.
  • Figure 10 is a graph showing the endoplasmic reticulum stress response of IDBL (Id3) inhibiting BTZ-resistant ANBL6-BR cells.
  • FIG. 11A is an experimental result diagram of the combined use of ID3 (Id3) and BTZ to synergistically inhibit the activity of chymotrypsin-like proteasome in multiple myeloma cell lines (ANBL6, ANBL6BR, ARP1, U266).
  • 11B is a graph showing the experimental results of the combined use of ID3 (Id3) and BTZ to synergistically inhibit the activity of trypsin-like proteasomes in multiple myeloma cell lines (ANBL6, ANBL6BR, ARP1, U266).
  • FIG. 12 is an experimental result diagram of the combined use of ID3 (Id3) and BTZ to synergistically inhibit the levels of intracellular free ubiquitin and ubiquitinated protein in multiple myeloma cell line ARP1.
  • Fig. 13 is a graph showing the results of an experiment in which the combination of ID3 (Id3) and BTZ can synergistically inhibit the level of multiple myeloma cell line ARP1 proteasome activation complex subunit 4 (PSME4) and ubiquitin dissociase (USP7).
  • ID3 ID3
  • BTZ can synergistically inhibit the level of multiple myeloma cell line ARP1 proteasome activation complex subunit 4 (PSME4) and ubiquitin dissociase (USP7).
  • Example 1 ID3 (Id3) combined with BTZ can synergistically inhibit MM cell proliferation
  • ANBL6 cell line Harvard Medical School
  • U266 cell line purchased from ATCC
  • ARP1 cell line University of Iowa, USA
  • BTZ Bortezomib
  • the CCK8 method is a method for measuring the number of cells using colorimetry.
  • the CCK8 kit contains WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfobenzene)- 2H-tetrazole monosodium salt ⁇ . It is reduced by the dehydrogenase in the cell to a highly water-soluble yellow formazan product (Formazan) under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS) dye). The amount of formazan produced is proportional to the number of living cells. Therefore, this feature can be used directly for cell proliferation and toxicity analysis.
  • the 96-well plate was connected with ANBL6, U266 and ARP1 of MM cell line 2 ⁇ 10 4 / 100ul / well. Set up a control group and a sample addition group. The control group did not add drugs, and the loading group added 1640 medium (HyClone RPMI-1640) containing different concentrations of ID3 (Id3) or / and BTZ.
  • 1640 medium HyClone RPMI-1640
  • ANBL6, U266, ARP1 cell lines were treated with 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M ID3 (Id3) single drug or (and) 2.5nM, 5nM, 10nM BTZ treatment for 24h or 48h, each concentration repeated three wells, CCK8 detection
  • the cells proliferated, and the CompuSyn software was used to calculate the two-drug synergy index CI (Combination Index), (CI ⁇ 1 is the two-drug synergy).
  • CI Combination Index
  • Different combinations of drugs have different CI values. The smaller the synergy index, the stronger the synergy between the two drugs.
  • ID3 Id3
  • BTZ BTZ in the ANBL6, U266, and ARP1 cell lines with a concentration of CI ⁇ 1 is as follows:
  • the combination of the two drugs can significantly inhibit the proliferation of MM cells.
  • Figure 1B, Figure 1D and Figure 1F are graphs of the synergy index (CI) of the combination of different concentrations of two drugs obtained using CompuSyn software.
  • Example 2 ID3 (Id3) combined with BTZ can synergistically promote MM cell apoptosis.
  • ANBL6 cell line same as Example 1
  • ARP1 cell line same as Example 1
  • Flow cytometer BD company Canto II, 7AAD / PE Annexin V Apoptosis Detection Kit: BD company 559763
  • FIG. 1 shows the test results.
  • MM cell lines ANBL6, U266 and ARP1 were treated with a combination of ID3 (Id3) and BTZ concentration with the smallest CI index for 24h or 48h.
  • Flow cytometry was used to detect changes in the proportion of apoptotic (AnnexinV / 7AAD) MM cells.
  • Figure 2A shows that after treating the ANBL6 cell line with the two-drug combination group (5 ⁇ M ID3 (Id3), 2.5nM BTZ) for 24 hours, the proportion of AnnexinV-positive cells was significantly higher than that of the two drugs alone.
  • the proportion of AnnexinV positive cells in the combination group was 22.6%; while for 5 ⁇ M ID3 (Id3) and 2.5nM BTZ, the control group was 17.9%, 16.2% and 6.4%, respectively.
  • Figure 2B shows that after 1.25 ⁇ M ID3 (Id3) and 2.5nM BTZ combined treatment of U266 cell line for 48h, the proportion of AnnexinV positive cells was 29.6%; and 1.25 ⁇ M ID3 (Id3), 2.5nM BTZ, control group were 18.1%, 23.8 % And 10.7%.
  • Figure 2C shows that after treating the ARP1 cell line with the two-drug combination group (5 ⁇ M ID3 (Id3), 5nM BTZ) for 24 hours, the proportion of AnnexinV positive cells was also significantly higher than that of the two drugs alone.
  • the proportion of AnnexinV positive cells in the combination group was 34.7%; while 5 ⁇ M ID3 (Id3), 5nM BTZ and control group were 28.1%, 25.1% and 7.6%, respectively.
  • Example 3 In vivo animal model confirms the anti-MM activity of ID3 (Id3) combined with BTZ
  • NOD-SCID mice purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • BTZ Bortezomib
  • Purpose and principle Use tumor cells and immune-deficient mice cultured in vitro to establish experimental animal tumor experimental models, and observe the killing effect or proliferation inhibition effect of drugs on tumor cells.
  • ARP1 cells with good growth in vitro were injected subcutaneously at 5-6 weeks old, about 20g female NOD-SCID mice subcutaneously in the left lower abdomen to prepare xenograft MM mouse model, each injection 10 6 / 100ul. Ten days after the injection, the tumor tissue of the mouse was visible.
  • Control group PBS was injected subcutaneously twice a week.
  • BTZ Bortezomib group
  • ID3 (Id3) group ID3 (Id3) was intraperitoneally injected every other day.
  • Figure 3A is the experimental results of the combination of ID3 (Id3) and BTZ can significantly inhibit the growth of tumor cells in mice.
  • Figure 3B is a comparison of the inhibitory effect of the combination of low-dose ID3 (Id3) and BTZ on tumor growth in mice compared with high-dose ID3 (Id3) and high-dose BTZ single drug application and the control group. Use, can significantly reduce the amount of bortezomib.
  • Figure 3C is a comparison of the inhibitory effect of low-dose ID3 (Id3) and BTZ on tumor growth in mice compared with low-dose ID3 (Id3) and low-dose BTZ single drug application and the control group
  • ID3 (Id3) (6mg / kg) can significantly inhibit the growth of tumor cells in mice, which is significantly different from the control group (p ⁇ 0.001), and no significant difference from the bortezomib 1mg / kg group. There was no significant difference between bortezomib (0.5 mg / kg) group and ID3 (Id3) (1.25 mg / kg group) compared with the control group.
  • the tumor volume of the bortezomib (0.5mg / kg) ID3 (Id3) (1.25mg / kg) combination medication group was significantly different from that of the control group and bortezomib (1mg / kg group), respectively.
  • Example 4 The combination of ID3 (Id3) and BTZ can activate the JNK apoptosis pathway by inhibiting the endoplasmic reticulum stress response, resulting in MM cell apoptosis
  • ARP1 cell line same as Example 1
  • BTZ Bortezomib
  • RIPA protein lysate purchased from Biyuntian Biotechnology Institute
  • Biorad wet film transfer instrument Biorad
  • PVDF membrane Millipore
  • ECL chemiluminescent mixture Thermo scientific, Pierce TM ECL Western Blotting Substrate 32106
  • Western blotting distinguishes different components by electrophoresis and transfers them to a solid support, using antibodies as probes to detect the target protein.
  • anti-iRE1a anti-XBP-1s
  • anti-p-JNK anti-GAPDH
  • antibodies are all diluted 1: 1000), horizontal shaker at 4 °C overnight;
  • the PVDF membrane was placed in an ECL chemiluminescence mixture (1: 1) and incubated at room temperature with shaking for 1 min. It was placed in an ImageQuant LAS-4010 fluorescence / chemiluminescence imaging analyzer and developed according to the instrument operation steps. 3. Results and evaluation:
  • Figure 4 shows the results of ID3 (Id3) combined with BTZ can synergistically inhibit endoplasmic reticulum stress response.
  • the combination of 2.5nM BTZ and 2.5 ⁇ M ID3 (Id3) can activate the expression of CHOP, promote the phosphorylation of JNK, and significantly inhibit the expression of iRE1a and its phosphorylation level, and at the same time upregulate the expression of the shear type XBP-1.
  • Example 5 Verify the sensitivity of ANBL6 and ANBL6-BR to BTZ
  • BTZ-sensitive MM cell line ANBL6 and BTZ-resistant MM cell line ANBL6-BR Harvard University, USA
  • BTZ Bortezomib
  • Cells ANBL6 and ANBL6-BR were treated with BTZ series concentrations (1.25nM, 2.5nM, 5nM, and 10nM), respectively, and 72h later (with blank control group added), CCK8 method was used to detect cell proliferation.
  • Figure 5 shows that the two MM cell lines ANBL6 and ANBL6-BR have different sensitivity to BTZ.
  • Example 6 ID3 (Id3) can promote MM cell apoptosis by reducing the S-phase ratio of the BTZ-resistant MM cell line ANBL6-BR.
  • BTZ Bortezomib
  • PI can combine with intracellular DNA and RNA. After digesting RNA with RNA inhibitor, the fluorescence intensity of PI bound to DNA detected by flow cytometry directly reflects the amount of DNA in the cell. .
  • the DNA content of each phase of the cell cycle is different.
  • the G1 / G0 phase of normal cells has the DNA content of diploid cells (2N), while the G2 / M phase has the DNA content of tetraploid cells (4N), while the S phase
  • the DNA content is between diploid and tetraploid. Therefore, the flow cytometry PI staining method can be used to detect the DNA content in the cells to distinguish G1 / G0, S and G2 / M cells.
  • ID3 (Id3) can promote boron by regulating the cell cycle Tezomib-resistant MM cells apoptosis.
  • Figure 6 shows that ID3 (Id3) can promote the apoptosis of MM cells by reducing the S-phase ratio of the BTZ-resistant MM cell line ANBL6-BR.
  • Example 7 The killing effect of ID3 (Id3) on the BTZ-resistant cell line ANBL6-BR is concentration-dependent
  • BTZ-sensitive MM cell line ANBL6 and BTZ-resistant MM cell line ANBL6-BR Harvard University, USA
  • BTZ Bortezomib
  • Figure 7 shows that the sensitivity of the two cell lines to ID3 (Id3) is significantly different, and the killing effect of ID3 (Id3) on the BTZ-resistant cell line ANBL6-BR is concentration-dependent.
  • Example 8 ID3 (Id3) combined with BTZ can synergistically inhibit the proliferation of BTZ-resistant MM cells.
  • BTZ Bortezomib
  • CCK8 method was used to detect the cell proliferation activity of the control group and the medication group.
  • 96-well plate was connected with ANTZ6-BR, a BTZ-resistant MM cell line. Set up a control group and a sample addition group. In the control group, no drug was added, and the sample-adding group was added with culture medium containing different concentrations of ID3 (Id3) or / and BTZ. Each concentration was repeated for three wells. After 24h or 48h, CCK8 detected cell proliferation and calculated the synergy between the two drugs. Index CI (CI ⁇ 1 is synergy between two drugs). Different combinations of drugs have different CI values. The smaller the synergy index, the stronger the synergy between the two drugs.
  • Figure 8 shows that the ANBL6-BR cells treated with 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M ID3 (Id3) combined with 10nM BTZ for 24h, the inhibition rate of MM cell proliferation was significantly higher than that of the two drugs Single use (CI ⁇ 1) shows that ID3 (Id3) can overcome the resistance of bortezomib.
  • Example 9 ID3 (Id3) combined with BTZ can synergistically promote the apoptosis of BTZ-resistant MM cells (ANBL6-BR)
  • the MM cell line ANBL6-BR was treated with a combination of ID3 (Id3) with the smallest CI index and BTZ concentration for 24 hours, and flow cytometry was used to detect changes in the proportion of apoptotic (AnnexinV / 7AAD) MM cells.
  • Figure 9 shows that after treating the ANBL6-BR cell line with the two-drug combination group (1.25 ⁇ M ID3 (Id3), 10nM BTZ) for 24h, the proportion of AnnexinV-positive cells was significantly higher than that of the two drugs alone. Among them, the proportion of AnnexinV positive cells in the combination group was 16.6%; while for 1.25 ⁇ M ID3 (Id3) and 10 nM BTZ, the control group was 7.4%, 7.8% and 4.8%, respectively.
  • ID3 (Id3) can activate P53, inhibit the phosphorylation of checkpoint 1 (checkpoint 1), inhibit endoplasmic reticulum stress response, activate JNK-mediated apoptosis pathway and other multiple signaling pathways, and promote BTZ Drug-resistant MM cells apoptosis and overcome bortezomib resistance.
  • ANBL6-BR cell line (BTZ resistance): Harvard University, USA
  • ANBL6 cell line (BTZ sensitive): Harvard University, USA
  • BTZ Bortezomib
  • RIPA protein lysate purchased from Biyuntian Biotechnology Institute
  • Biorad wet film transfer instrument Biorad
  • PVDF membrane Millipore
  • ECL chemiluminescent mixture Thermo scientific, Pierce TM ECL Western Blotting Substrate 32106
  • Method MM cell line ANBL6-BR and its control cell line ANBL6 were inserted into the six-well plate. Set up the control group and the loading group separately. No drug was added to the control group, and 10 ⁇ M ID3 (Id3) was added to the medium in the sample addition group. After 24 hours of drug action, cells were collected to prepare protein lysate, and Western blot method was used to detect the expression of the target protein.
  • Figure 10 shows that compared to bortezomib-sensitive cell lines, bortezomib-resistant cell lines were activated by endoplasmic reticulum stress-associated JNK apoptosis pathway after 24h of ID3 (Id3) treatment.
  • the stress response is suppressed.
  • BCL2 and p-chk1 protein expression was significantly suppressed, and P53 protein expression increased. It indicates that ID3 (Id3) can kill bortezomib-resistant cells by acting on multiple signaling pathways.
  • Indirubin monooxime combined with BTZ can synergistically inhibit the activity of chymotrypsin-like and trypsin-like proteasomes in multiple myeloma cell lines (ANBL6, ANBL6BR, ARP1, U266)
  • ID3 (Id3) purchased from MCE
  • BTZ Bortezomib
  • Proteasome-Glo TM Chymotrypsin-Like Cell-Based Assay kit Promega G8660
  • Proteasome-Glo Trypsin-Like Cell-Based Assay kit Promega G8761
  • ID3 ID3
  • BTZ ID3
  • Fig. 11A and Fig. 11B The results show that mechanism studies indicate that indirubin monooxime combined with BTZ can promote apoptosis by inhibiting the activity of proteasomes in multiple myeloma cells.
  • indirubin monooxime and BTZ can synergistically inhibit the levels of intracellular free ubiquitin and ubiquitinated protein in multiple myeloma cell line ARP1.
  • ID3 (Id3) purchased from MCE
  • BTZ Bortezomib
  • RIPA protein lysate purchased from Biyuntian Biotechnology Institute
  • Biorad wet film transfer instrument Biorad
  • PVDF membrane Millipore
  • ECL chemiluminescent mixture Thermo scientific, Pierce TM ECL Western Blotting Substrate 32106
  • the PVDF membrane was placed in an ECL chemiluminescence mixture (1: 1) and incubated at room temperature with shaking for 1 min. It was placed in an ImageQuant LAS-4010 fluorescence / chemiluminescence imaging analyzer and developed according to the instrument operation steps.
  • indirubin monooxime and BTZ can synergistically inhibit the level of multiple myeloma cell line ARP1 proteasome activation complex subunit 4 (PSME4) and ubiquitin dissociase (USP7).
  • PSME4 proteasome activation complex subunit 4
  • USP7 ubiquitin dissociase
  • ID3 (Id3) purchased from MCE
  • BTZ Bortezomib
  • RIPA protein lysate purchased from Biyuntian Biotechnology Institute
  • Biorad wet film transfer instrument Biorad
  • PVDF membrane Millipore
  • ECL chemiluminescent mixture Thermo scientific, Pierce TM ECL Western Blotting Substrate 32106
  • the PVDF membrane was placed in an ECL chemiluminescence mixture (1: 1) and incubated at room temperature with shaking for 1 min. It was placed in an ImageQuant LAS-4010 fluorescence / chemiluminescence imaging analyzer and developed according to the instrument operation steps.
  • Example 11 shows that indirubin monooxime combined with BTZ can promote apoptosis by inhibiting the ubiquitin proteasome pathway of multiple myeloma cells.

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Abstract

本发明为靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，属于医学和制药领域。本发明发现了靛玉红类化合物对硼替佐米耐药的患者有效。靛玉红类化合物与硼替佐米联用药物毒性小，降低了硼替佐米的用量。

Description

靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途 技术领域
本发明涉及靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，属于医疗和药学领域。
背景技术
多发性骨髓瘤(Multiple Myeloma，MM)是一种常见于中老年人群的血液系统恶性肿瘤，约占血液系统恶性肿瘤的10％。近年来随着蛋白酶体抑制剂(硼替佐米)、免疫调节剂(沙利度胺、来那度胺)等新药的应用，以及自体造血干细胞移植的广泛开展，大多数MM患者可获得较好的缓解，其中位总生存期由原来的不足3年延长至5～7年。然而目前临床应用的蛋白酶体抑制剂及免疫调节剂等抗骨髓瘤药物多为国外医药公司专利产品，其价格昂贵，以其为基础的化疗方案使患者显著获益的同时也带来了巨额医疗费用；医疗负担过重严重影响了患者的治疗进程。第一代蛋白酶体抑制剂硼替佐米是目前国内MM治疗的骨架药物之一，可特异性抑制蛋白酶体的活性，抑制与MM细胞增殖相关基因的表达，减少白细胞介素6(IL-6)等MM细胞生长因子的分泌和黏附因子的表达，最终导致肿瘤细胞凋亡。但硼替佐米价格昂贵，一支国外进口的硼替佐米(3.5毫克)的价格7千元左右，即便是国产的硼替佐米(3.5毫克)也要5千元左右，一般一个疗程使用4支，6-8个疗程为一个治疗周期，每个治疗周期仅硼替佐米的药品费用就需要十几万元，给患者造成很大的经济压力。此外由于进口药品在国内上市流程滞后的原因，许多新药目前在国内市场不可及，例如第二、第三代蛋白酶体抑制剂。随着我国人口的快速老龄化，MM发病率呈明显上升趋势。这种依赖国外进口药品的现状急需改变，研发具有自主知识产权的抗MM药物具有重要的现实意义。
此外，随着硼替佐米在临床的广泛应用以及给药时间的延长，几乎所有的患者均会出现对现有治疗药物耐药而复发难治，复发难治患者疾病进展迅速、生存期短、预后极差，复发/难治到无治仍是几乎所有MM的最终结局，因此，MM的治疗仍面临巨大挑战，阐明硼替佐米等抗MM药物的耐药机制并寻求新的药物及干 预策略对提高、改善复发难治MM疗效有重要的理论及现实意义。
发明内容
本发明要解决的技术问题是提供靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，尤其是指在制备对硼替佐米耐药的多发性骨髓瘤的药物中的用途。
本发明要解决的另一个技术问题是，提供一种治疗对硼替佐米耐药的多发性骨髓瘤的药物。
为实现上述目的，本发明采用以下技术方案：
靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途。
所述靛玉红类化合物为靛玉红和/或靛玉红-3’-单肟(Indirubin-3’-oxime，ID3，Id3)。
靛玉红(Indirubin)是我国从中草药青黛中发现的一种对慢性粒细胞白血病(慢粒)有治疗作用的新型双吲哚类抗肿瘤药物，为我国首创的新型结构的抗白血病药物。靛玉红的CAS号为479-41-4，结构式如下：
Figure PCTCN2019084873-appb-000001
所述靛玉红-3’-单肟(Indirubin-3’-oxime，ID3，Id3)，结构式如下：
Figure PCTCN2019084873-appb-000002
靛玉红-3’-单肟，CAS号：160807-49-8。ID3(Id3)及其衍生物是一类双吲哚杂环平面结构的化合物。国外学者近年来对ID3(Id3)及其衍生物抗癌机理的研究表明，ID3(Id3)及其衍生物对细胞内广泛存在的细胞周期依赖性蛋白激酶 (CDK)具有抑制作用，导致癌细胞的细胞周期停滞，从而抑制肿瘤细胞增殖。
所述多发性骨髓瘤是指普通的多发性骨髓瘤和/或对硼替佐米产生耐药性的多发性骨髓瘤。
所述治疗是指抑制多发性骨髓瘤瘤体积生长和/或抑制瘤体重量的生长。
本发明首次发现：
1.靛玉红类化合物ID3(Id3)单药或小剂量联合硼替佐米(BTZ)可显著抑制MM细胞增殖，促进MM细胞凋亡。在降低BTZ剂量的同时，可达到与BTZ相当的疗效，并显著降低了患者用药的经济成本。
2.靛玉红类化合物ID3(Id3)可通过持续诱导内质网应激反应，削弱未折叠蛋白应答反应(UPR)克服硼替佐米耐药。并可显著抑制BTZ耐药的MM细胞增殖，促进其凋亡。
一种治疗多发性骨髓瘤的药物组合物，活性成分为靛玉红类化合物和硼替佐米。
所述靛玉红类化合物为靛玉红(Indirubin，CAS号479-41-4)和/或靛玉红-3’-单肟(Indirubin-3’-oxime，ID3，Id3，CAS号：160807-49-8)。
所述多发性骨髓瘤是指普通的多发性骨髓瘤和/或对硼替佐米产生耐药性的多发性骨髓瘤。
所述治疗是指抑制多发性骨髓瘤瘤体积生长和/或抑制瘤体重量的生长。
本发明药物组合物具有良好的抑制骨髓瘤生长的效果，可以解决多发性骨髓瘤对现有药物具有耐药性的问题。
本发明药物组合物可以以任何途径给药，例如以液体或固体的形式采用口服、经鼻、非肠胃、静脉、皮内注射、皮下注射或者局部给药。
为了非肠胃给药的目的，可以将活性成分置于溶液或混悬液中。溶液和混悬液还可以包含下面的成分：用于注射的无菌稀释剂，例如，注射用水：脂质体颗粒混悬物，其中在颗粒的中心包含稳定的活性药，该颗粒具有预定的pH值和受保护的内环境；脂质体颗粒混悬物，其中活性药外挂在颗粒的外表或是双膜的任何一膜；盐水溶液、不挥发油、聚乙二醇、甘油、丙二醇或者其他合成溶剂；抗菌剂，例如苄醇；抗氧化剂，例如抗坏血酸或者亚硫酸氢钠；螯合剂，例如乙二 胺四乙缓冲剂，例如乙酸(盐)、柠檬酸(盐)以及用于调节张力的物质，例如氯化钠或者葡萄糖。非肠胃给药的制剂可以装在由玻璃或塑料制成的安瓿、一次性注射器或者多次剂量的小瓶中。
本发明的优点是：本发明首次发现了靛玉红类化合物ID3(Id3)单药对硼替佐米耐药的患者有效。ID3(Id3)与硼替佐米联用药物毒性小，显著降低了硼替佐米的用量，可显著降低患者的治疗费用。
附图说明
图1A为ANBL6细胞经2.5μM、5μM、10μM ID3(Id3)联合2.5nM BTZ处理24h后，两药联用对MM细胞增殖的抑制率结果。
图1B为应用CompuSyn软件得出的图1A中两种药物不同浓度组合的协同指数(CI))图。
图1C为U266细胞经1.25μM、2.5μM、5μM ID3(Id3)联合2.5nM或10nM BTZ处理48h后，两药联用对MM细胞增殖的抑制率结果。
图1D为应用CompuSyn软件得出的图1C中两种药物不同浓度组合的协同指数(CI))图。
图1E为ARP1细胞经1.25μM、2.5μM、5μM ID3(Id3)联合5nM BTZ处理24h后，两药联用对MM细胞增殖的抑制率结果。
图1F为应用CompuSyn软件得出的图1E中两种药物不同浓度组合的协同指数(CI))图。
图2A为ID3(Id3)联合BTZ可协同促进MM细胞(ANBL6)凋亡试验结果。
图2B为ID3(Id3)联合BTZ可协同促进MM细胞(U266)凋亡试验结果。
图2C为ID3(Id3)联合BTZ可协同促进MM细胞(ARP1)凋亡试验结果。
图3A为ID3(Id3)与BTZ联用可显著抑制小鼠体内肿瘤细胞的生长试验结果。
图3B为低剂量ID3(Id3)与BTZ联合应用对小鼠体内肿瘤生长的抑制作用与高剂量ID3(Id3)和高剂量BTZ单药应用及对照组的比较。
图3C为低剂量ID3(Id3)与BTZ联合应用对小鼠体内肿瘤生长的抑制作用与低剂量ID3(Id3)和低剂量BTZ单药应用及对照组的比较。
图4为ID3(Id3)联合BTZ可协同抑制内质网应激反应试验结果。
图5为ANBL6和ANBL6-BR两种MM细胞系对BTZ敏感性不同的试验结果。
图6为ID3(Id3)可通过减少ANBL6-BR细胞的S期比例促进MM细胞凋亡试验结果。
图7为ANBL6和ANBL6-BR对ID3(Id3)的敏感性试验结果。
图8A为ID3(Id3)可逆转MM细胞BTZ耐药试验结果。
图8B为应用CompuSyn软件得出的图8A中两种药物不同浓度组合的协同指数(CI)图；图中居中的横线代表CI值＝1，线下的点代表两药联用CI＜1。
图9为ID3(Id3)联合BTZ可协同促进BTZ耐药的MM细胞(ANBL6-BR)凋亡试验结果图。
图10为ID3(Id3)可抑制BTZ耐药的ANBL6-BR细胞内质网应激反应结果图。
图11A为ID3(Id3)与BTZ联用可协同抑制多发性骨髓瘤细胞系(ANBL6，ANBL6BR，ARP1，U266)糜蛋白酶样蛋白酶体活性的实验结果图。
图11B为ID3(Id3)与BTZ联用可协同抑制多发性骨髓瘤细胞系(ANBL6，ANBL6BR，ARP1，U266)胰蛋白酶样蛋白酶体活性的实验结果图。
图12为ID3(Id3)与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1胞内游离泛素及泛素化蛋白的水平的实验结果图。
图13为ID3(Id3)与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1蛋白酶体活化复合物亚单位4(PSME4)和泛素解离酶(USP7)的水平的实验结果图。
具体实施方式
实施例1：ID3(Id3)联合BTZ可协同抑制MM细胞增殖
一、材料：
ANBL6细胞株：美国哈佛大学医学院
U266细胞株：购于ATCC
ARP1细胞株：美国爱荷华大学
CCK8试剂盒：日本同仁化学研究所(Dojindo)CK04CCK-8 WST-8
靛玉红-3’-单肟(ID3，Id3)：购于Med Chem express(MCE)公司
硼替佐米(BTZ)：西安杨森制药有限公司
二、方法：MM细胞增殖实验采用CCK8法。
CCK8法是一种运用比色法测量细胞数量的测定方法。CCK8试剂盒中含有WST-8【2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2，4-二磺酸苯)-2H-四唑单钠盐】。它在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-Methoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物(Formazan dye)。生成的甲瓒物的数量与活细胞的数量成正比。因此可利用这一特性直接进行细胞增殖和毒性分析。
96孔板中接入MM细胞系2×10 4/100ul/孔的ANBL6、U266和ARP1。设置对照组和加样组。对照组不加药物，加样组加入含有不同浓度ID3(Id3)或/和BTZ的1640培养基(HyClone RPMI-1640)。ANBL6、U266、ARP1细胞系分别经1.25μM、2.5μM、5μM、10μM ID3(Id3)单药或(和)2.5nM、5nM、10nM BTZ处理24h或48h，每个浓度重复三个孔，CCK8检测细胞增殖，并应用CompuSyn软件计算两药协同指数CI(Combination Index)，(CI＜1为两药协同)。不同浓度药物组合，CI值不同，协同指数越小，代表两药协同作用越强。
三、结果与评价：
ANBL6、U266、ARP1细胞系中ID3(Id3)和BTZ联用，CI＜1的浓度组合结果如下：
ANBL6细胞经2.5μM、5μM、10μM ID3(Id3)联合2.5nM BTZ处理24h后，两药联用对MM细胞增殖的抑制率明显高于两药单用(CI＜1)(图1A)。
U266细胞经1.25μM、2.5μM、5μM ID3(Id3)联合2.5nM、10nM BTZ处理48h后，两药联用对MM细胞增殖的抑制率明显高于两药单用(CI＜1)(图1C)。
ARP1细胞经1.25μM、2.5μM、5μM ID3(Id3)联合5nM BTZ处理24h后，两药联用对MM细胞增殖的抑制率明显高于两药单用(CI＜1)。(图1E)。
与ID3(Id3)和BTZ单用组相比，两药联用可明显抑制MM细胞的增殖。
图1B、图1D和图1F是应用CompuSyn软件得出的两种药物不同浓度组合的协同指数(CI))图。图中居中的横线代表CI值＝1，线下的点代表两药联用CI＜1。
实施例2：ID3(Id3)联合BTZ可协同促进MM细胞凋亡。
一、材料
ANBL6细胞株：同实施例1
U266细胞株：同实施例1
ARP1细胞株：同实施例1
ID3(Id3)：同实施例1
流式细胞仪：BD公司Canto II，7AAD/PE Annexin V Apoptosis Detection Kit：BD公司559763
二、方法
1.目的和原理：细胞在晚期凋亡或死亡后细胞膜结构破坏，此时Annexin V染色呈阳性。而在细胞早期凋亡时，细胞膜完整性尚未破坏，此时非浸透性荧光染料如7-AAD或PI无法进入细胞，染色呈阴性。因此结合Annexin V和7-AAD/PI染色，通过流式细胞仪检测用药前后AnnexinV/7AAD细胞比例的变化，来评价药物对MM细胞促凋亡的作用。
2.方法：6孔板中接入MM细胞系2×10 5/1ml/孔，加入不同浓度ID3(Id3)或(和)BTZ，于5％CO 2，37度培养箱中，药物作用24h或48h后，加入PE Annexin V Apoptosis Detection Kit对细胞进行孵育。流式细胞仪检测AnnexinV/7AAD细胞比例的变化。
三、结果与评价：
图2为试验结果。
MM细胞系ANBL6、U266和ARP1应用CI指数最小的ID3(Id3)和BTZ浓度组合处理24h或48h后，采用流式细胞术检测凋亡(AnnexinV/7AAD)MM细胞比例的变化。
图2A显示两药联用组(5μM ID3(Id3)，2.5nM BTZ)处理ANBL6细胞系24h后，AnnexinV阳性细胞的比例明显高于两药分别单用。其中联用组AnnexinV阳性细胞的比例为22.6％；而5μM ID3(Id3)、2.5nM BTZ，对照组分别为17.9％、16.2％和6.4％。
图2B显示1.25μM ID3(Id3)和2.5nM BTZ联合处理U266细胞系48h后， AnnexinV阳性细胞的比例为29.6％；而1.25μM ID3(Id3)，2.5nM BTZ、对照组分别为18.1％、23.8％和10.7％。
图2C显示两药联用组(5μM ID3(Id3)，5nM BTZ)处理ARP1细胞系24h后，AnnexinV阳性细胞的比例亦明显高于两药分别单用。其中联用组AnnexinV阳性细胞的比例为34.7％；而5μM ID3(Id3)、5nM BTZ、对照组分别为28.1％、25.1％和7.6％。
实施例3：体内动物模型证实ID3(Id3)与BTZ联用的抗MM活性
一、材料：
NOD-SCID小鼠(购于北京华阜康生物科技股份有限公司)
ID3(Id3)：同实施例1
硼替佐米(BTZ)：购于西安杨森制药有限公司
二、方法
1.目的和原理：使用体外培养的肿瘤细胞和免疫缺陷小鼠建立实验动物肿瘤实验模型，观察药物对肿瘤细胞的杀伤作用或增殖抑制作用。
2.方法：
(1)制备动物成瘤模型：将体外培养生长良好的ARP1细胞经皮下注射于5-6周龄，20g左右雌性NOD-SCID小鼠左下腹皮下，制备异种移植MM小鼠模型，每只注射10 6/100ul。注射10天以后，小鼠皮下肿瘤组织可见。
(2)分组：将动物随机分成不同的治疗组，并根据文献确定BTZ和ID3(Id3)的给药剂量及方式(对照组，硼替佐米1mg/kg组，ID3(Id3)6mg/kg组，硼替佐米0.5mg/kg组，ID3(Id3)1.25mg/kg组和硼替佐米(0.5mg/kg)ID3(Id3)(1.25mg/kg)联合用药组，每组5只小鼠。
(3)给药：
对照组：每周两次皮下注射PBS。
硼替佐米组：每周两次皮下注射不同浓度BTZ。
ID3(Id3)组：隔天一次腹腔注射不同浓度的ID3(Id3)。
治疗后，每天观察小鼠的一般情况，每3-4天用游标卡尺测量肿瘤直径。肿瘤体积计算公式如下：V＝0.5×a×b 2(a和b分别为肿瘤的长径和短径)，绘制肿瘤生长曲线。
三、结果与评价：
图3A为ID3(Id3)与BTZ联用可显著抑制小鼠体内肿瘤细胞的生长实验结果。
图3B为低剂量ID3(Id3)与BTZ联合应用对小鼠体内肿瘤生长的抑制作用与高剂量ID3(Id3)和高剂量BTZ单药应用及对照组的比较，说明ID3(Id3)与BTZ联用，可以显著降低硼替佐米的用量。
图3C为低剂量ID3(Id3)与BTZ联合应用对小鼠体内肿瘤生长的抑制作用与低剂量ID3(Id3)和低剂量BTZ单药应用及对照组的比较
ID3(Id3)(6mg/kg)可显著抑制小鼠体内肿瘤细胞的生长，与对照组相比有显著性差异(p＜0.001)，与硼替佐米1mg/kg组没有明显差异。硼替佐米(0.5mg/kg)组和ID3(Id3)(1.25mg/kg组)，与对照组相比没有显著性差异。硼替佐米(0.5mg/kg)ID3(Id3)(1.25mg/kg)联合用药组的肿瘤体积与对照组和硼替佐米(1mg/kg组)相比均有显著性差异，p值分别为p＜0.001，p＜0.05。
实施例4：ID3(Id3)与BTZ联用可通过抑制内质网应激反应，活化JNK凋亡途径，导致MM细胞凋亡
一、材料
ARP1细胞株：同实施例1
ID3(Id3)：同实施例1
硼替佐米(BTZ)：购于西安杨森制药有限公司
RIPA蛋白裂解液：购于碧云天生物技术研究所
Pierce TM BCA Protein Assay Kit：Thermo scientific公司23225
10％Mini-PROTEIN TGX预制胶：Biorad公司4561033
Biorad湿转膜仪：Biorad公司
PVDF膜：密理博公司(Millipore)
anti-CHOP：Cell Signaling 5554s
anti-p-iRE1a：Abcam ab124945
anti-iRE1a：Cell Signaling 3294s
anti-XBP-1s：Cell Signaling 12782s
anti-p-SAPK/JNK：Cell Signaling 9255s
anti-GAPDH：Cell Signaling 5174s
二抗：Anti-rabbit IgG，HRP-linked Antibody Cell Signaling 7074
ECL化学发光混合液：Thermo scientific，Pierce TM ECL Western Blotting Substrate 32106
ImageQuant LAS-4010荧光/化学发光成像分析仪：通用电气医疗集团
二、方法：
1.目的和原理：
免疫印迹法(western blot)通过电泳区分不同的组分，并转移至固相支持物，通过抗体作为探针，对靶蛋白进行检测。
2.方法：
6孔板中接入MM细胞系ARP1，加入不同浓度ID3(Id3)或(和)BTZ，同时设空白对照组，于5％CO 2，37度培养箱中，药物作用24h后，收集细胞，制备蛋白裂解液(10 6/100ul RIPA)，应用Pierce TM BCA Protein Assay Kit，按说明书操作对蛋白进行定量。应用10％Mini-PROTEIN TGX预制胶，电压80V进行蛋白电泳至溴酚蓝到达凝胶底部为止。应用Biorad湿转膜仪，电流250mA，60min将蛋白转移至PVDF膜。5％(w/v)脱脂奶粉，溶于PH 7.4的TBST中，室温60min孵育PVDF膜。随后进行抗体与靶蛋白的结合反应。
①一抗与PVDF膜的共温育
a.在10cm培养皿中加入10ml第一抗体(anti-CHOP；anti-p-iRE1a；
anti-iRE1a；anti-XBP-1s；anti-p-JNK；anti-GAPDH，抗体均为1∶1000稀释)，水平摇床上4℃过夜；
b.用TBST漂洗3，每次10min。
②二抗与PVDF膜温育
a.经TBST最后一次洗涤后，把膜转移至一个新的10cm培养皿，加入1∶ 1000稀释辣根过氧化物酶标记的二抗10ml，37℃摇床平缓摇动，温育1h；
b.用TBS洗涤3次，每次5min。
将PVDF膜置于ECL化学发光混合液(1∶1)中于室温下振荡温育1min，置于ImageQuant LAS-4010荧光/化学发光成像分析仪中按仪器操作步骤显影。三、结果与评价：
图4为ID3(Id3)联合BTZ可协同抑制内质网应激反应结果。
2.5nM BTZ与2.5μM ID3(Id3)联用可活化CHOP的表达，促进JNK的磷酸化，并显著抑制iRE1a的表达及其磷酸化水平，同时上调剪切型XBP-1的表达。
实施例5：验证ANBL6和ANBL6-BR对BTZ的敏感性
一、材料
BTZ敏感的MM细胞系ANBL6和BTZ耐药的MM细胞系ANBL6-BR：美国哈佛大学
硼替佐米(BTZ)：购于西安杨森制药有限公司
CCK8试剂盒：日本同仁化学研究所(Dojindo)CK04CCK-8WST-8
二、方法
分别单用BTZ系列浓度(1.25nM、2.5nM、5nM、10nM)处理细胞ANBL6和ANBL6-BR，72h后(同时设空白不加药对照组)，CCK8法检测细胞增殖。
三、结果与评价：
图5显示，ANBL6和ANBL6-BR两种MM细胞系对BTZ敏感性不同。
实施例6：ID3(Id3)可通过减少BTZ耐药的MM细胞系ANBL6-BR的S期比例促进MM细胞凋亡。
一、材料
BTZ耐药的MM细胞系ANBL6-BR：美国哈佛大学
PI/RNase Staining Buffer：碧云天生物技术研究所
ID3(Id3)：同实施例1
硼替佐米(BTZ)：购于西安杨森制药有限公司
二、方法
1.目的和原理：PI可以与细胞内DNA和RNA结合，采用RNA抑制剂将RNA消化后，通过流式细胞术检测到的与DNA结合的PI的荧光强度直接反映了细胞内DNA含量的多少。细胞周期各时相的DNA含量不同，正常细胞的G1/G0期具有二倍体细胞的DNA含量(2N)，而G2/M期具有四倍体细胞的DNA含量(4N)，而S期的DNA含量介于二倍体和四倍体之间。因此，通过流式细胞术PI染色法对细胞内DNA含量进行检测，可以区分G1/G0期、S期以及G2/M期细胞。
2.方法：6孔板中接入MM细胞系，加入不同浓度ID3(Id3)，于5％CO 2，37度培养箱中，药物作用24h后，加入PI/RNase Staining Buffer，用流式细胞仪检测，并计算得到药物作用前后细胞各个时期的分布的变化。
三、结果与评价：
与对照组相比，加入ID3(Id3)的实验组，随ID3(Id3)浓度增加，ANBL6-BR的S期逐渐减少，G2/M期增多，说明ID3(Id3)可通过调节细胞周期促进硼替佐米耐药的MM细胞凋亡。图6显示，ID3(Id3)可通过减少BTZ耐药的MM细胞系ANBL6-BR的S期比例促进MM细胞凋亡。
实施例7：ID3(Id3)对BTZ耐药细胞系ANBL6-BR的杀伤作用呈浓度依赖一、材料
BTZ敏感的MM细胞系ANBL6和BTZ耐药的MM细胞系ANBL6-BR：美国哈佛大学
ID3(Id3)：同实施例1
硼替佐米(BTZ)：西安杨森制药有限公司
7AAD/PE Annexin V Apoptosis Detection Kit：BD公司559763
二、方法
1.目的和原理：细胞在晚期凋亡或死亡后细胞膜结构破坏，此时Annexin V染色呈阳性。而在细胞早期凋亡时，细胞膜完整性尚未破坏，此时非浸透性荧光染料如7-AAD或PI无法进入细胞，染色呈阴性。因此结合Annexin V和7-AAD/PI 染色，通过流式细胞仪检测用药前后AnnexinV/7AAD细胞比例的变化，来评价药物对MM细胞促凋亡的作用。
2.方法：6孔板中接入MM细胞系，加入不同浓度ID3(Id3)，于5％CO 2，37度培养箱中，药物作用24h后，加入PE Annexin V Apoptosis Detection Kit对细胞进行孵育。流式细胞仪检测AnnexinV/7AAD细胞比例的变化。
三、结果和评价：
图7显示，两种细胞系对ID3(Id3)的敏感性有显著差异，且ID3(Id3)对BTZ耐药细胞系ANBL6-BR的杀伤作用呈浓度依赖。
实施例8：ID3(Id3)联合BTZ可协同抑制BTZ耐药的MM细胞增殖。
一、材料
BTZ耐药的MM细胞系ANBL6-BR：美国哈佛大学
CCK8检测试剂盒：日本同仁化学研究所(Dojindo)CK04CCK-8 WST-8
ID3(Id3)：同实施例1
硼替佐米(BTZ)：购于西安杨森制药有限公司
二、方法
1.目的和原理：应用CCK8法检测对照组和用药组的细胞增殖活性。
2.方法：96孔板中接入BTZ耐药的MM细胞系ANBL6-BR。设置对照组和加样组。对照组不加药物，加样组加入含有不同浓度ID3(Id3)或/和BTZ的培养基培养，每个浓度重复三个孔，作用24h或48h后，CCK8检测细胞增殖，并计算两药协同指数CI(CI＜1为两药协同)。不同浓度药物组合，CI值不同，协同指数越小，代表两药协同作用越强。
三、结果与评价：
图8(图8A和图8B)显示，ANBL6-BR细胞经1.25μM、2.5μM、5μM ID3(Id3)联合10nM BTZ处理24h后，两药联用对MM细胞增殖的抑制率明显高于两药单用(CI＜1)，说明ID3(Id3)能克服硼替佐米的耐药。
实施例9：ID3(Id3)联合BTZ可协同促进BTZ耐药的MM细胞(ANBL6-BR) 凋亡
一、材料
BTZ耐药的MM细胞系ANBL6-BR：美国哈佛大学
ID3(Id3)：同实施例1
流式细胞仪：BD公司Canto II
7AAD/PE Annexin V Apoptosis Detection Kit：BD公司559763
二、方法
1.目的和原理：细胞在晚期凋亡或死亡后细胞膜结构破坏，此时Annexin V染色呈阳性。而在细胞早期凋亡时，细胞膜完整性尚未破坏，此时非浸透性荧光染料如7-AAD或PI无法进入细胞，染色呈阴性。因此结合Annexin V和7-AAD/PI染色，通过流式细胞仪检测用药前后AnnexinV/7AAD细胞比例的变化，来评价药物对MM细胞促凋亡的作用。
2.方法：6孔板中接入MM细胞系2×10 5/1ml/孔，加入不同浓度ID3(Id3)或(和)BTZ，于5％CO 2，37度培养箱中，药物作用24h后，加入PE Annexin V Apoptosis Detection Kit对细胞进行孵育。流式细胞仪检测AnnexinV/7AAD细胞比例的变化。
三、结果与评价：
图9为试验结果。
MM细胞系ANBL6-BR应用CI指数最小的ID3(Id3)和BTZ浓度组合处理24h后，采用流式细胞术检测凋亡(AnnexinV/7AAD)MM细胞比例的变化。
图9显示两药联用组(1.25μM ID3(Id3)，10nM BTZ)处理ANBL6-BR细胞系24h后，AnnexinV阳性细胞的比例明显高于两药分别单用。其中联用组AnnexinV阳性细胞的比例为16.6％；而1.25μM ID3(Id3)、10nM BTZ，对照组分别为7.4％、7.8％和4.8％。
实施例10：ID3(Id3)可通过活化P53，抑制检查点1(checkpoint 1)的磷酸化，抑制内质网应激反应，活化JNK介导的细胞凋亡途径等多条信号通路，促进BTZ耐药的MM细胞凋亡，并克服硼替佐米耐药。
一、材料
ANBL6-BR细胞系(BTZ耐药)：美国哈佛大学
ANBL6细胞系(BTZ敏感)：美国哈佛大学
ID3(Id3)：同实施例1
硼替佐米(BTZ)：购于西安杨森制药有限公司
RIPA蛋白裂解液：购于碧云天生物技术研究所
PierceTM BCA Protein Assay Kit：Thermo scientific公司23225
10％Mini-PROTEIN TGX预制胶：Biorad公司4561033
Biorad湿转膜仪：Biorad公司
PVDF膜：密理博公司(Millipore)
anti-P53：Cell Signaling 2527s
anti-p-iRE1a：Abcam ab124945
anti-iRE1a：Cell Signaling 3294s
anti-p-Chk1：Cell Signaling 12302s
anti-BCL2：Cell Signaling 2872s
anti-p-SAPK/JNK：Cell Signaling 9255s
anti-GAPDH：Cell Signaling 5174s
二抗：Anti-rabbit IgG，HRP-linked Antibody Cell Signaling 7074
ECL化学发光混合液：Thermo scientific，Pierce TM ECL Western B1otting Substrate 32106
ImageQuant LAS-4010荧光/化学发光成像分析仪：通用电气医疗集团
二、方法
1.目的和原理：免疫印迹法(Western blot)通过电泳区分不同的组分，并转移至固相支持物，通过抗体作为探针，对靶蛋白进行检测。
2.方法：六孔板中接入MM细胞系ANBL6-BR及其对照细胞系ANBL6。分别设置对照组和加样组。对照组不加药物，加样组在培养基加入10μM ID3(Id3)。药物作用24h后，收集细胞，制备蛋白裂解液，应用Western blot法检测目的蛋白的表达情况。
三、结果与评价：
图10显示，与硼替佐米敏感的细胞系相比，硼替佐米耐药的细胞系在ID3(Id3)处理24h以后，内质网应激相关的JNK凋亡途径被活化，内质网应激反应被抑制。BCL2及p-chk1蛋白表达明显受抑，P53蛋白表达增加。表明ID3(Id3)可通过作用多条信号通路，对硼替佐米耐药的细胞进行杀伤。
实施例11：机制研究
一、靛玉红单肟与BTZ联用可协同抑制多发性骨髓瘤细胞系(ANBL6，ANBL6BR，ARP1，U266)糜蛋白酶样和胰蛋白酶样蛋白酶体的活性。
1.材料：
ANBL6，ANBL6BR，ARP1，U266细胞株
ID3(Id3)：购于MCE公司
硼替佐米(BTZ)：购于西安杨森制药有限公司
Proteasome-Glo TM Chymotrypsin-Like Cell-Based Assay kit：Promega公司G8660
Proteasome-Glo Trypsin-Like Cell-Based Assay kit：Promega公司G8761
2.方法：
96孔板中接入MM细胞系ANBL6，ANBL6BR，ARP1，U266，加入不同浓度ID3(Id3)或(和)BTZ，同时设空白对照组，于5％CO 2，37度培养箱中，药物作用24h后，按说明书操作在读板机上检测蛋白酶体活性。
3.结果：
结果见图11A和图11B所示，结果显示：机制研究表明靛玉红单肟与BTZ联用可通过抑制多发性骨髓瘤细胞蛋白酶体的活性促进细胞凋亡。
二、靛玉红单肟与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1胞内游离泛素及泛素化蛋白的水平。
1.材料：
ARP1细胞株
ID3(Id3)：购于MCE公司
硼替佐米(BTZ)：购于西安杨森制药有限公司
RIPA蛋白裂解液：购于碧云天生物技术研究所
Pierce TM BCA Protein Assay Kit：Thermo scientific公司23225
10％Mini-PROTEIN TGX预制胶：Biorad公司4561033
Biorad湿转膜仪：Biorad公司
PVDF膜：密理博公司(Millipore)
anti-Ubiquitin Antibody：Cell Signaling 3933s
anti-GAPDH：Cell Signaling 5174s
二抗：Anti-rabbit IgG，HRP-linked Antibody Cell Signaling 7074
ECL化学发光混合液：Thermo scientific，Pierce TM ECL Western Blotting Substrate 32106
ImageQuant LAS-4010荧光/化学发光成像分析仪：通用电气医疗集团
2.方法：
6孔板中接入MM细胞系ARP1，加入不同浓度ID3(Id3)或(和)BTZ，同时设空白对照组，于5％CO 2，37度培养箱中，药物作用24h后，收集细胞，制备蛋白裂解液(10 6/100ul RIPA)，应用Pierce TM BCA Protein Assay Kit，按说明书操作对蛋白进行定量。应用10％Mini-PROTEIN TGX预制胶，电压80V进行蛋白电泳至溴酚蓝到达凝胶底部为止。应用Biorad湿转膜仪，电流250mA，60min将蛋白转移至PVDF膜。5％(w/v)脱脂奶粉，溶于PH 7.4的TBST中，室温60min孵育PVDF膜。随后进行抗体与靶蛋白的结合反应。
①一抗与PVDF膜的共温育
在10cm培养皿中加入10ml第一抗体(anti-Ubiquitin Antibody；anti-GAPDH，抗体均为1∶1000稀释)，水平摇床上4℃过夜；用TBST漂洗3，每次10min。
②二抗与PVDF膜温育
a.经TBST最后一次洗涤后，把膜转移至一个新的10cm培养皿，加入1∶1000稀释辣根过氧化物酶标记的二抗10ml，37℃摇床平缓摇动，温育1h；
b.用TBS洗涤3次，每次5min。
将PVDF膜置于ECL化学发光混合液(1∶1)中于室温下振荡温育1min，置于ImageQuant LAS-4010荧光/化学发光成像分析仪中按仪器操作步骤显影。
3.结果：
结果见图12所示。结果显示：靛玉红单肟与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1胞内游离泛素及泛素化蛋白的水平。
三、靛玉红单肟与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1蛋白酶体活化复合物亚单位4(PSME4)和泛素解离酶(USP7)的水平。
1.材料：
ARP1细胞株
ID3(Id3)：购于MCE公司
硼替佐米(BTZ)：购于西安杨森制药有限公司
RIPA蛋白裂解液：购于碧云天生物技术研究所
Pierce TM BCA Protein Assay Kit：Thermo scientific公司23225
10％Mini-PROTEIN TGX预制胶：Biorad公司4561033
Biorad湿转膜仪：Biorad公司
PVDF膜：密理博公司(Millipore)
anti-USP7：Proteintech 26948-1-AP
anti-PSME4：Proteintech 18799-1-AP
anti-GAPDH：Cell Signaling 5174s
二抗：Anti-rabbit IgG，HRP-linked Antibody Cell Signaling 7074
ECL化学发光混合液：Thermo scientific，Pierce TM ECL Western Blotting Substrate 32106
ImageQuant LAS-4010荧光/化学发光成像分析仪：通用电气医疗集团
2.方法：
6孔板中接入MM细胞系ARP1，加入不同浓度ID3(Id3)或(和)BTZ，同时设空白对照组，于5％CO 2，37度培养箱中，药物作用24h后，收集细胞，制备蛋白裂 解液(10 6/100ul RIPA)，应用Pierce TM BCA Protein Assay Kit，按说明书操作对蛋白进行定量。应用10％Mini-PROTEIN TGX预制胶，电压80V进行蛋白电泳至溴酚蓝到达凝胶底部为止。应用Biorad湿转膜仪，电流250mA，60min将蛋白转移至PVDF膜。5％(w/v)脱脂奶粉，溶于PH 7.4的TBST中，室温60min孵育PVDF膜。随后进行抗体与靶蛋白的结合反应。
①一抗与PVDF膜的共温育
a.在10cm培养皿中加入10ml第一抗体(anti-USP7；anti-PSME4；anti-GAPDH，抗体均为1∶1000稀释)，水平摇床上4℃过夜；
b.用TBST漂洗3，每次10min。
②二抗与PVDF膜温育
a.经TBST最后一次洗涤后，把膜转移至一个新的10cm培养皿，加入1∶1000稀释辣根过氧化物酶标记的二抗10ml，37℃摇床平缓摇动，温育1h；
b.用TBS洗涤3次，每次5min。
将PVDF膜置于ECL化学发光混合液(1∶1)中于室温下振荡温育1min，置于ImageQuant LAS-4010荧光/化学发光成像分析仪中按仪器操作步骤显影。
3.结果：
结果见图13所示。结果显示：靛玉红单肟与BTZ联用可协同抑制多发性骨髓瘤细胞系ARP1蛋白酶体活化复合物亚单位4(PSME4)和泛素解离酶(USP7)的蛋白水平。
实施例11的机制研究表明，靛玉红单肟与BTZ联用可通过抑制多发性骨髓瘤细胞泛素蛋白酶体途径促进细胞凋亡。

Claims (10)

  1. 靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途。
  2. 根据权利要求1所述的靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，其特征在于：所述靛玉红类化合物为靛玉红和/或靛玉红-3’-单肟(Indirubin-3’-oxime，ID3，Id3)。
  3. 根据权利要求1或2所述的靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，其特征在于：所述多发性骨髓瘤是指普通的多发性骨髓瘤和/或对硼替佐米产生耐药性的多发性骨髓瘤。
  4. 根据权利要求3所述的靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途，其特征在于：所述治疗是指抑制多发性骨髓瘤瘤体积生长和/或抑制瘤体重量的生长。
  5. 一种治疗多发性骨髓瘤的药物组合物，其特征在于：活性成分为靛玉红类化合物和硼替佐米。
  6. 根据权利要求5所述的药物组合物，其特征在于：所述靛玉红类化合物为靛玉红和/或靛玉红-3’-单肟(Indirubin-3’-oxime，ID3，Id3)。
  7. 根据权利要求5所述的药物组合物，其特征在于：所述多发性骨髓瘤是指普通的多发性骨髓瘤和/或对硼替佐米产生耐药性的多发性骨髓瘤。
  8. 根据权利要求5-7中任何一项所述的药物组合物，其特征在于：所述药物组合物为口服、经鼻、非肠胃、静脉、皮内注射、皮下注射或者局部给药剂型。
  9. 根据权利要求8所述的药物组合物，其特征在于：所述药物组合物的活性成分置于溶液或混悬液中。
  10. 根据权利要求9所述的药物组合物，其特征在于所述溶液或混悬液还包含下述成分中的一种或多种：无菌稀释剂、脂质体颗粒混悬物、盐水溶液、不挥发油、抗菌剂、抗氧化剂、螯合剂、缓冲剂、调节张力的物质。
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