WO2019034069A1 - 一种抗肿瘤联合用药物及其在制备抗癌药物中的用途 - Google Patents

一种抗肿瘤联合用药物及其在制备抗癌药物中的用途 Download PDF

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WO2019034069A1
WO2019034069A1 PCT/CN2018/100603 CN2018100603W WO2019034069A1 WO 2019034069 A1 WO2019034069 A1 WO 2019034069A1 CN 2018100603 W CN2018100603 W CN 2018100603W WO 2019034069 A1 WO2019034069 A1 WO 2019034069A1
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drug
egfr
chlorogenic acid
preparation
tumor
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French (fr)
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张洁
黄望
杨华蓉
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四川九章生物科技有限公司
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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to an anti-tumor combination drug and the use thereof in preparing an anticancer drug.
  • Chlorogenic acid is a carboxyphenolic acid composed of caffeic acid and Quinicic acid. Chlorogenic acid has a wide range of pharmacological effects, including anti-oxidation, anti-bacterial and anti-inflammatory, inhibiting tumor, protecting liver and gallbladder, and promoting blood pressure and blood pressure.
  • EGFR epidermal growth factor receptor
  • erbB-1 epidermal growth factor receptor
  • HER2/neu erbB-2
  • HER3 erbB-3
  • HER4 erbB-4
  • EGFR is a receptor-type tyrosine protein kinase (RTK), which is overexpressed or abnormally expressed in various tumors such as non-small cell lung cancer, breast cancer, colorectal cancer, head and neck cancer, gastric cancer, ovarian cancer, and pancreatic cancer. The occurrence and development of tumors are closely related.
  • RTK receptor-type tyrosine protein kinase
  • EGFR activation can be divided into three steps: (1) EGFR binding to the ligand can lead to the formation of homodimers to the receptor, and can also form heterodimers with other EGFR families; (2) the dimerization Cross-linking phosphorylation occurs, that is, phosphorylation of specific tyrosine residues on one receptor and another receptor activates the TK subregion of the intracellular region, thereby inducing the next level of signaling. Eventually lead to cell cycle progression, decreased ability to apoptosis, and the emergence of metastatic phenotype.
  • the EGFR pathway is thought to play an important role in the evolution and progression of epithelial malignancies, and thus can be used as a potential target for systemic therapy, while inhibition of the epidermal growth factor receptor tyrosine kinase has become a research hotspot for the treatment of tumors.
  • the current common treatments are:
  • EGFR tyrosine kinase inhibitors can be divided into two broad categories: one is a non-specific tyrosine kinase inhibitor that inhibits all tyrosine kinases; the other is currently used more selective EGFR tyrosine.
  • Kinase inhibitors such as gefitinib (Iressa), erlotinib (Troquet) and ectinib (Kemena), which are already marketed in China.
  • Iressa gefitinib
  • Troquet erlotinib
  • emena ectinib
  • Monoclonal antibodies Binding to EGFR competes with and blocks the binding of ligands such as EGF and TGF ⁇ to inhibit tumor growth.
  • ligands such as EGF and TGF ⁇
  • small molecule drugs targeting EGFR there have been many small molecule drugs targeting EGFR.
  • the monoclonal antibody represented by nimotuzumab, panitumumab and cetuximab has been successfully applied to the experimental treatment of clinical tumors.
  • this class also has the problem of insufficient targeting and significant side effects.
  • the technical problem to be solved by the present invention is to provide an anti-tumor combination drug and use thereof in preparing an anticancer drug, and the invention also provides a chlorogenic acid in the preparation of anti-cancer
  • the technical scheme of the invention can solve the defects that the drug targeting EGFR is poorly selected, produces various toxic side effects, produces drug resistance, improves the targeting of drugs targeting EGFR, reduces toxic side effects, and reverses Its resistance, improve patient compliance
  • the technical solution of the present invention is an anti-tumor combination drug, which comprises chlorogenic acid, an antitumor drug and a pharmaceutically acceptable adjuvant.
  • the anti-tumor drug is a drug targeting EGFR.
  • the anti-tumor combination drug comprises: a first preparation formed by chlorogenic acid and a pharmaceutically acceptable adjuvant, and a second preparation formed by the EGFR-targeted drug and a pharmaceutically acceptable adjuvant. .
  • the first formulation is 10-40 parts by weight
  • the second formulation is 2-40 parts by weight.
  • the EGFR-targeted drug is one or both of an EGFR tyrosine kinase inhibitor and an anti-EGFR antibody.
  • the EGFR tyrosine kinase inhibitor is one or more of gefitinib, erlotinib, ectinib, afatinib and lapatinib.
  • the anti-EGFR antibody is one or more of cetuximab, trastuzumab, nimotuzumab and panitumumab.
  • the dosage form of the first preparation is an injection or an oral preparation
  • the dosage form of the second preparation is an injection or an oral preparation.
  • the invention also provides a use of the anti-tumor combination medicament of the invention for preparing an anticancer medicament.
  • the cancer is non-small cell carcinoma, breast cancer, malignant glioma or nasopharyngeal carcinoma.
  • the invention also provides a use of chlorogenic acid for preparing an anticancer drug toxic side effect inhibitor.
  • the anticancer drug is a drug targeting EGFR.
  • the main problems of molecular targeted drugs of solid tumors are: more toxic and side effects, unsatisfactory effect of drug alone, and prone to drug-resistant mutations. How to better reduce toxic and side effects, optimize the combination of drug regimens, and overcome drug resistance have become urgent problems to be solved by molecular targeted drugs.
  • the present invention discloses an anti-tumor combination drug and the use thereof in preparing an anticancer drug, and the combination drug of the present invention comprises chlorogenic acid, an antitumor drug and a pharmaceutically acceptable adjuvant, Studies have shown that the anti-tumor combination drug provided by the present invention can enhance the targeting of EGFR-targeted drugs in non-small cell carcinoma, breast cancer, malignant glioma, nasopharyngeal carcinoma, and reduce EGFR Target drug toxic side effects and reverse the drug resistance of EGFR-targeted drugs.
  • chlorogenic acid has anti-mutagenic and anti-cancer effects.
  • G6PT glycose-6-phosphotransferase
  • chlorogenic acid can initiate programmed cell death of neutrophils and promyelocytic HL-60 and inhibit the secretion of metallothionein (MMP) from human Hep3B hepatoma cells. , inhibits the migration of gliomas.
  • MMP metallothionein
  • the sensitization effect of chlorogenic acid on EGFR-targeted drugs may be related to the above mechanism.
  • the chlorogenic acid molecule contains a certain amount of active hydroxyl groups, which can form hydrogen radicals with anti-oxidation effect, can eliminate the activity of free radicals such as hydroxyl radicals and superoxide anions, and protect tissues from oxidation damage.
  • chlorogenic acid had a greater effect on the levels of NF- ⁇ and TNF- ⁇ in supernatants of intraepithelial lymphocytes (IEL) and lymphocytes of intestinal lamina intestinal (LPL).
  • IEL intraepithelial lymphocytes
  • LPL intestinal lamina intestinal lamina limbal
  • chlorogenic acid can induce the production of IFN- ⁇ and IFN- ⁇ by human lymphocytes and human peripheral blood leukocytes, which may also be related to the toxic side effects of chlorogenic acid in this application to reduce EGFR-targeted drugs. .
  • EGFR-TKI epidermal growth factor receptor-tyrosine kinase inhibitor
  • clinical classification is divided into primary drug resistance and secondary drug resistance.
  • Primary resistance ie, the patient did not respond to the first use of TKI, and did not receive significant benefits in terms of symptom improvement, lesion control, and survival time.
  • Approximately 25% of EGFR-mutated tumor patients are ineffective for TKI therapy, which may be associated with primary resistance to TKI.
  • the main reasons include: 1 there are other EGFR mutations that occur simultaneously with drug-sensitive mutations, 2 other gene mutations affecting the downstream signal of EGFR, the presence of 3RAS gene mutations, and 4 patients' own factors such as low immune function and rapid loss of metabolism Live, and the decline in absorptive capacity.
  • EGFR-TKI acquired resistance is related to the two mechanisms of secondary mutation theory and Met gene amplification.
  • Other drug resistance mechanisms may be due to the patient's own influencing factors (such as smoking or not, gender, race and pathological type, etc.) ) caused by different.
  • chlorogenic acid reverses the drug resistance of EGFR tyrosine kinase inhibitors, and its mechanism may be related to various causes of drug resistance mentioned above, and the specific mechanism needs further study.
  • Figure 1 shows the inhibition of proliferation of PC9 cell line by chlorogenic acid
  • Figure 2 shows the inhibition of proliferation of PC9/ZD cell lines by chlorogenic acid.
  • EGFR is a member of the tyrosine kinase type I receptor subfamily, and other members of the family include HER2/neu, HER3 and HER4. Their receptors are composed of three parts: an extracellular ligand binding region, a transmembrane region composed of a single strand, and an intracellular tyrosine kinase region.
  • EGFR is a constitutive expression component of many normal epithelial tissues such as skin and hair follicles, and overexpression of EGFR is found in many human tumors.
  • Activated EGFR is primarily involved in the following signaling pathways: the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, initiates DNA replication, causes cell proliferation and differentiation, and mediates CLE regulation of the cell cycle; PI3K- Akt pathway, inhibits apoptosis; activates downstream VEGF, promotes microvascular network formation; epithelial growth factor receptor-signaling and transcription activator 3 (EGFR-STAT3) pathway, activates STAT3 in many tumors, regulates multiple genes The activity is thus involved in tumorigenesis, development and apoptosis.
  • EGFR signal transduction pathway plays an important role in tumor cell proliferation, injury repair, invasion and neovascularization. In recent years, targeting EGFR drugs has become a hot spot in cancer therapy.
  • Tumor molecular targeting drugs against EGFR are mainly divided into two categories according to their nature: one is monoclonal antibody, and cetuximab (cetuximab, erbitux) and panitumumab (panitumumab) are currently available in China. , vectibix), nimotuzumab (nimotuzumab, Taixinsheng), etc.; the other is small molecule inhibitors (Table 2), gefitinib, iressa, erlotinib , tarceva) and lapatinib (lapatinib, tykerb) and so on.
  • small molecule inhibitors are different from that of monoclonal antibodies, mainly by competitively binding to the phosphorylation site of EGFR intracellular tyrosine kinase, blocking its interaction with ATP, and then inhibiting tyrosine phosphorylation of EGFR. And a series of downstream signal transduction, so small molecule inhibitors are not as specific as monoclonal antibodies, which will produce a series of toxic side effects. Common adverse reactions include rash, diarrhea, liver damage, and rare adverse reactions including pulmonary interstitial. Lesion.
  • anti-EGFR monoclonal antibodies bind to the extracellular receptor binding domain of EGFR, thereby preventing the binding of natural ligands to receptors in the organism, thereby preventing activation of the receptor and signaling of downstream signaling pathways.
  • the resulting biological effects are manifested by cell cycle inhibition, promotion of apoptosis, inhibition of tumor angiogenesis, and the like.
  • monoclonal antibodies significantly increase the risk of FAEs (risk of death and adverse events) in targeted therapy in patients with solid malignancies. Toxicity is mainly manifested in the skin and mucous membrane system, cardiovascular system, hematopoietic system and gastrointestinal system.
  • the epidermal growth factor receptor (EGFR) mutation in lung cancer occurs mainly in the intracellular coding domain (exons 18-21), including the deletion mutation of exon 19 (delE746-A750) and the explicit 21 point mutation (L858R), which accounts for more than 90% of all EGFR kinase mutations, and is associated with sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI); There are also exon 18 point mutations (G719S) and exon 20 insertion mutations.
  • the former belongs to the sensitive mutation of EGFR-TKI and the latter is related to the resistance of EGFR-TKI, the incidence rate is about 5%.
  • EGFR-mutated NSCLC patients are more sensitive to the treatment of TKI.
  • EGFR-TKIs inhibit the autophosphorylation of tyrosine in EGFR and the activation of tyrosine kinase by competitively binding to the extracellular ligand binding site with ATP or substrate, inhibiting EGFR homology or heterologous to ERBB3 The formation of dimers, thereby inhibiting EGFR activation, preventing downstream signal transduction, inhibiting cell cycle progression, accelerating apoptosis, inhibiting angiogenesis and metastasis.
  • drug resistance mechanisms mainly include primary resistance and acquired resistance.
  • Primary drug resistance refers to the first use of EGFR-TKI to produce drug resistance, and approximately 60% of patients with NSCLC are resistant to TKI. Among them, nearly 30% of EGFR gene activating mutations were resistant to primary TKI. In addition, although gefitinib and erlotinib can be effective in patients with EGFR-mutant NSCLC, most patients develop acquired resistance within 6 months to 12 months of treatment.
  • the invention discloses an anti-tumor combination drug, which comprises chlorogenic acid, an antitumor drug and a pharmaceutically acceptable adjuvant; wherein the antitumor drug is preferably a drug targeting EGFR; More specifically, the anti-tumor combination drug comprises: a first preparation formed by chlorogenic acid and a pharmaceutically acceptable adjuvant, and a second formed by a drug targeting EGFR and a pharmaceutically acceptable adjuvant a formulation; wherein the present invention has no special requirements for the pharmaceutically acceptable adjuvant, and a person skilled in the art can select a suitable adjuvant according to actual needs; and the anti-tumor combination drug is in parts by weight.
  • the first formulation is preferably 10-40 parts, more preferably 10-30 parts, most preferably 10-20 parts
  • the second formulation is preferably 2-40 parts, more preferably 5-30 parts, most preferably 10 -20 servings.
  • the EGFR-targeted drug is preferably one or both of an EGFR tyrosine kinase inhibitor and an anti-EGFR antibody, preferably gefitinib, erlotinib One or more of ectinib, afatinib and lapatinib
  • the anti-EGFR antibody is preferably cetuximab, trastuzumab, nimotuzumab and panis One or several of the monoclonal antibodies.
  • the present invention has no special requirements for the dosage form of the combination drug, and a dosage form known in the art which can be used for an anticancer drug can be used;
  • the invention also discloses the use of chlorogenic acid for preparing an anticancer drug toxic side effector, wherein the anticancer drug is a drug targeting EGFR.
  • chlorogenic acid can reduce erlotinib, ectinib, gefitinib, afatinib, trastuzumab, lapatinib, nimotuzumab, cetuximab
  • the toxicity against human normal nasal epithelial cells, human normal lung epithelial cells and human normal breast cells improves the targeting of such drugs to a certain extent, and can reduce the side effects of drugs targeting EGFR.
  • chlorogenic acid and erlotinib, ectinib, gefitinib, afatinib, trastuzumab, lapatinib, nimotuzumab, cetuximab When combined with tumor cells, it produces a synergistic synergistic effect and achieves a sensitizing effect on tumor cells.
  • mice Animal experiments also showed that chlorogenic acid combined with afatinib, erlotinib, panitumumab, and nimotuzumab, G422 glioma BABLC mice and Lewis lung cancer C57BL/6 small
  • the mouse not only achieved synergistic effects, but also reduced the side effects of afatinib, erlotinib, panitumumab and nimotuzumab.
  • the experimental results of the present invention also showed that the combination of chlorogenic acid and gefitinib in human lung tumor cells significantly reversed the resistance of gefitinib.
  • the technical solution of the present invention can improve the targeting of EGFR-targeted drugs in non-small cell carcinoma, breast cancer, malignant glioma, nasopharyngeal carcinoma, reduce its toxic side effects, and reverse Drug resistance of drugs targeting EGFR.
  • Test drugs chlorogenic acid, erlotinib, gefitinib, ectinib, afatinib, trastuzumab, lapatinib, nimotuzumab, cetuximab .
  • Test cell strain human lung cancer cell line A549, human breast cancer cell line MDA-MB-453, human nasopharyngeal carcinoma cell line CNE-1, human normal lung epithelial cell BEAS-2B, human normal breast cell MCF10A, human normal nasal cavity Epithelial cells RPMI2650.
  • a bottle of logarithmic growth phase cells was taken, washed twice with PBS, digested with 0.25% trypsin, and counted in a single cell suspension with RPMI-1640 medium containing 10% calf serum.
  • the cells were inoculated in a 96-well culture plate under aseptic conditions, and the number of cells inoculated was 5 ⁇ 10 3 /well.
  • the cells were incubated for 24 h at 37 ° C in a 5% CO 2 incubator and the medium was changed.
  • the cells were divided into a negative control group, a EGFR-targeted drug alone group, a chlorogenic acid group, and a combination drug group, and the blank control group was not seeded.
  • the total amount of the culture medium and the drug per well was 200 ⁇ l, and 3 wells were set for each concentration, and an equal volume of the culture solution was used instead of the drug as a blank control.
  • Inhibition rate (1-OD570 (experimental group - blank group) / OD570 (control group - blank group)) * 100%.
  • the IC50 value at the time of use indicates that chlorogenic acid can reduce the toxicity of erlotinib, ectinib, gefitinib and afatinib on human normal lung epithelial cells BEAS-2B, and to some extent improve these drugs.
  • Targeting In the MCF10A inhibition test of human normal breast cells, the IC50 values of chlorogenic acid (20 ⁇ g/ml) combined with trastuzumab and lapatinib were higher than IC50 values when used alone, indicating that chlorogenic acid can be used. Reduce the toxicity of trastuzumab and lapatinib on human normal breast cell MCF10A, and improve the targeting of the two drugs to some extent.
  • the IC50 values of chlorogenic acid (20 ⁇ g/ml) combined with trastuzumab, nimotuzumab and cetuximab were higher than those used alone.
  • the IC50 value indicates that chlorogenic acid can reduce the toxicity of trastuzumab, nimotuzumab and cetuximab to human normal nasal epithelial cells RPMI2650, and improve the targeting of the third monoclonal antibody to some extent.
  • the above experimental data indicate that chlorogenic acid can reduce the toxic side effects of drugs targeting EGFR and improve its targeting to some extent.
  • the Q value is between 1.15 and 20, the combined effect of the two drugs is synergistic (++); the Q value of 4uM lapatinib combined with 10-50 ⁇ g/ml chlorogenic acid is 1.15-20, and the combined effect of the two drugs is synergistic ( ++).
  • the Q value is between 1.15 and 20, and the combined effect of the two drugs is synergistic (++); the Q value of 100 uM nimotuzumab combined with 10-50 ⁇ g/ml chlorogenic acid is 1.15-20, and the combined effect of the two drugs is synergistic ( ++); The Q value of 55uM cetuximab combined with 10-50 ⁇ g/ml chlorogenic acid is 1.15-20, and the combined effect of the two drugs is synergistic (++).
  • Example 2 Chlorogenic acid reverses resistance of human lung adenocarcinoma cell line PC9 to gefitinib in vitro
  • Test drug chlorogenic acid; gefitinib
  • Test cell strain PC9 cells, PC9/ZD is self-screening gefitinib cell line
  • PC9 cells Human lung adenocarcinoma cell line PC9 cells were exposed to gefitinib (200 nmol/L) for 3 months, and cell clones resistant to gefitinib were selected to become PC9/ZD cell lines. These two cells have the ability to be stably resistant to gefitinib, respectively.
  • PC9 cells and PC9/ZD cells were grown in a 25 cm 2 flask, cultured in DMEM medium containing 10% fetal bovine serum, and incubated in a 5% CO 2 incubator at 37 ° C. The cells were observed every day. When the cell growth covered 70%-80% of the bottom of the bottle, the cells were passaged 1:3, and the average 3-4d was passaged once to ensure the cell viability.
  • PC9 cells and drug-resistant cell lines in logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA, adjusted to a cell concentration of 5 ⁇ 10 4 /ml, and seeded in 96-well plates at 100 ⁇ l/5000 cells per well. After incubating for 24 hours at 5% CO 2 and 37 ° C, the original culture solution was aspirated, and 100 ⁇ l per well of DMEM medium containing different concentrations of gefitinib was added to make the final concentrations 0 , 0.1, 0.2, 0.5, 1, 5 respectively.
  • the culture and treatment methods of PC9 cells and drug-resistant cells are the same as above.
  • the experiments were divided into three groups: blank group, control group and experimental group.
  • the blank group was only added to the DMEM medium, and no cells were inoculated;
  • the control group was added with the medium and the cells were inoculated;
  • the experimental group was further added with different concentrations of chlorogenic acid working solution to make the final concentration of 1, 2, 4, respectively.
  • 8, 16, 32, 64, 128 ⁇ g / mL placed in the incubator for 48h, add 5mg / ml MTT20 ⁇ l per well, continue to incubate for 4h, aspirate the supernatant, add 150 ⁇ l of DMSO per well, let stand for 30min, until the crystal is completely dissolved.
  • PC9/ZD group PC9/ZD+chlorogenic acid 8 ⁇ g/ml group
  • PC9/ZD+chlorogenic acid 16 ⁇ g/ml group experimental group added gefitinib to the final concentration of 0.1, 0.2, 0.5, 1 5, 10 ⁇ mol/L, 3 wells per concentration, determine the OD value of each well, observe the non-cytotoxic chlorogenic acid (5 ⁇ g/ml, 10 ⁇ g/ml) combined with gefitinib in PC9/ZD
  • the IC50 of gefitinib against human lung adenocarcinoma PC9/ZD was calculated by the change in the killing effect of fetinib on PC9/ZD cells.
  • Reversal multiple IC50 value before reversal / IC50 value after reversal.
  • the IC50 of PC9 and drug-resistant cells PC9/ZD were 0.21 ⁇ mol/L and 5.45 ⁇ mol/L, respectively. According to the formula, the drug resistance multiple was calculated to be 25.9.
  • Chlorogenic acid inhibited proliferation of human lung adenocarcinoma sensitive cell PC9 and drug resistant cell PC9/ZD with IC50 of 190ug/ml.
  • concentration of chlorogenic acid was less than 16 ⁇ g/ml, the inhibition rate of human lung adenocarcinoma PC9 and drug-resistant cells PC9/ZD was ⁇ 10%, and there was no cytotoxicity.
  • Fig. 1 and Fig. 2 The results are shown in Fig. 1 and Fig. 2 .
  • the IC50 of gefitinib against PC9/ZD was 3.25, and the reversal multiple was 1.67.
  • the IC50 is 2.03 and the reversal factor is 2.68.
  • Example 2 demonstrates that chlorogenic acid combined with gefitinib can reverse the resistance of gefitinib to human lung adenocarcinoma cell line PC9/ZD.
  • Test drugs chlorogenic acid; afatinib, erlotinib, panitumumab, nimotuzumab
  • Test cell strain G422 cells, Lewis mouse lung cancer cell lines
  • Test animals BABLc mice, C57BL/6 mice
  • the logarithmic growth phase cells were collected, centrifuged at 1000 rpm for 5 min, and the cells were washed twice with PBS. After counting, the cell concentration was adjusted to 1 x 10 7 /ml with serum-free medium, and sent to the animal room under aseptic conditions. Under the conditions of aseptic experiment, 1x10 7 /ml cells were injected subcutaneously into the right axilla of the mice, 0.1ml each, and the skin was partially exposed. After 1 week, large nodules appeared in the left axilla of the nude mice, indicating transplantation. The model was successfully established and was divided into 6 groups according to the random grouping method.
  • BABLc mouse G422 glioma xenografts were divided into: 1, saline group (NS group); 2, nimotuzumab group; 3, chlorogenic acid group; 4, panitumumab group; 5, green Group of ortho-acid + nimotuzumab; 6, chlorogenic acid + panitumumab group, 5 in each group.
  • mice Lewis lung cancer were divided into: 7, saline group (NS group); 8, afatinib group; 9, chlorogenic acid group; 10, erlotinib group; 11, chlorogenic acid + A Fatinib group; 12, chlorogenic acid + erlotinib group, 5 in each group
  • the treatment was started after the average diameter of the tumor reached 100 mm 3 , and the intraperitoneal injection was performed once a day, and the physiological saline control group was given 0.2 ml of sterile physiological saline.
  • the body weight of the animal was measured every day after the administration; the tumor was stopped when the tumor volume was about 0.5 cm 3 in the negative group, and the WBC, HBC and HGB contents were determined by eyeball removal, and the mice were sacrificed by cervical dislocation and weighed, and the tumor was removed and weighed. .
  • Tumor inhibition rate (control group tumor weight - experimental group tumor weight) / control group tumor weight x 100%.
  • the inhibition rate, the denominator (Ea+Eb-Ea ⁇ Eb) is the expected combination effect, and Q is the ratio of the two.
  • the Q value is between 0.85 and 1.15
  • the combined effect of the two drugs is additive (+)
  • the Q value is synergistic (++) when the value is 1.15-20
  • the Q value is >20 is the obvious synergy (+++)
  • the Q value is 0.05.
  • Antagonism at ⁇ 0.85, and significant inhibition of Q value ⁇ 0.05.
  • the experimental data were processed using SPSS 17.0 statistical software, and all data were expressed as mean ⁇ SD. One-way analysis of variance was used for statistical analysis between groups. P ⁇ 0.05 was considered to be different, and P ⁇ 0.01 was considered to be significant.
  • chlorogenic acid 10 mg/kg, nimotuzumab 20 mg/kg, and panitumumab 20 mg/kg had a weak inhibitory effect on G422 glioma xenografts, compared with saline group. There was no significant difference. However, chlorogenic acid and nimotuzumab, as well as chlorogenic acid combined with panitumumab have a good tumor inhibition rate, and there is a significant difference compared with single use. The combined Q value calculation showed that chlorogenic acid combined with panitumumab and nimotuzumab had a synergistic effect.
  • chlorogenic acid and afatinib, and chlorogenic acid combined with erlotinib have a better tumor inhibition rate, and there is a significant difference compared with single use.
  • the Q value calculation of the combined drug showed that chlorogenic acid had a synergistic effect with afatinib and erlotinib.
  • chlorogenic acid (10 mg/kg) significantly improved the reduction of WBC, RBC and HGB in blood routines of G422 glioma mice after using nimotuzumab and panitumumab; in addition, Lewis The reduction of WBC, RBC, and HGB in blood routines in lung cancer mice after using afatinib and erlotinib was also significantly improved, and the increase in white blood cell counts indicated that chlorogenic acid can alleviate myelosuppression induced by VEGF pathway inhibitors.
  • the increase of platelet count can reduce the occurrence of hemorrhagic adverse reactions, and the increase of hemoglobin can prevent the occurrence of anemia and anemia-related adverse reactions, indicating that chlorogenic acid has reduced the side effects of EGFR-targeted drugs in many aspects. .

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Abstract

一种抗肿瘤联合用药物及其在制备抗癌药物中的用途，所述联合用药物包括绿原酸、抗肿瘤药物和药学上可接受的辅剂。还包括绿原酸在制备抗癌药物毒副作用抑制剂中的用途，所述抗癌药物为以EGFR为靶点的药物。该抗肿瘤联合用药物可提高以EGFR为靶点的药物在非小细胞癌，乳腺癌，恶性神经胶质瘤、鼻咽癌中的靶向性，且降低了以EGFR为靶点的药物毒副作用，并逆转以EGFR为靶点的药物的耐药性。

Description

一种抗肿瘤联合用药物及其在制备抗癌药物中的用途
本申请要求于2017年8月18日提交中国专利局、申请号为201710714132.4、发明名称为“一种抗肿瘤联合用药物及其在制备抗癌药物中的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药领域，尤其涉及一种抗肿瘤联合用药物及其在制备抗癌药物中的用途。
背景技术
绿原酸是由咖啡酸(Caffeic acid)与奎尼酸(Quinicacid)组成的羧酚酸。绿原酸具有广泛的药理作用，包括抗氧化、抗菌消炎、抑制肿瘤、保肝利胆、活血降压等生物活性。
EGFR(表皮生长因子受体)(erbB-1/HER1)是HER家族中的一个成员，另外三个成员是：erbB-2(HER2/neu)、erbB-3(HER3)和erbB-4(HER4)。EGFR属于受体型酪氨酸蛋白激酶(RTK)，在非小细胞肺癌，乳腺癌，大肠癌，头颈癌，胃癌，卵巢癌，和胰腺癌等多种肿瘤中有过表达或异常表达，与肿瘤的发生、发展有密切关系。EGFR活化可分为3个步骤：(1)EGFR与配体结合后可导致受体形成同源二聚体，也可与其他EGFR家族形成异源二聚体；(2)二聚化的受体发生交联磷酸化，即一个受体和另外一个受体上特定酪氨酸残基磷酸化，激活胞内区的TK亚区，从而激发下一级信号传导。最终导致细胞周期演进，凋亡能力下降以及出现转移表型等。
EGFR通路被认为在上皮恶性肿瘤演化及进展过程中发挥了重要作用，因而可作为全身性治疗的一个潜在靶点、而抑制表皮生长因子受体酪氨酸激酶则成为治疗肿瘤的一个研究热点。目前常见的治疗药物为：
1、酪氨酸激酶抑制剂。EGFR酪氨酸激酶抑制剂可分为两大类：一类为非特异性酪氨酸激酶抑制剂，能抑制所有的酪氨酸激酶；另一类为目 前使用较多的选择性EGFR酪氨酸激酶抑制剂，如已在我国上市的吉非替尼(易瑞沙)、厄洛替尼(特罗凯)和埃克替尼(凯美纳)等。但目前研究发现，几乎所有病人在接受此类药物治疗的10个月内转变成耐药性肿瘤病人，最终无药可治失去生命，并且，该类药物因为靶向性不足，也具有毒副作用显著的问题。
2、单克隆抗体。与EGFR结合，竞争和阻断EGF、TGFα等配体的结合，达到抑制肿瘤生长之目的。近年来出现许多靶向EGFR的小分子药物，其中以尼妥珠单抗、帕尼单抗、西妥昔单抗等为代表的单抗类药物部分已经成功应用于临床肿瘤的试验性治疗，但该类同样具有具有靶向性不足，毒副作用显著的问题。
发明内容
针对上述现有技术中存在的缺陷，本发明所要解决的技术问题是提供一种抗肿瘤联合用药物及其在制备抗癌药物中的用途，本发明还提供了一种绿原酸在制备抗癌药物毒副作用抑制剂中的用途，所述抗癌药物为以EGFR为靶点的药物。本发明的技术方案可以解决以EGFR为靶点的药物选择性较差，产生各种毒副作用，产生耐药性的缺陷，提高以EGFR为靶点的药物的靶向性，降低毒副作用，逆转其耐药性，提高患者依从性
为解决以上技术问题，本发明技术方案为一种抗肿瘤联合用药物，其特征在于，包括绿原酸、抗肿瘤药物和药学上可接受的辅剂。
优选的，所述抗肿瘤药物为以EGFR为靶点的药物。
优选的，所述抗肿瘤联合用药物包括：绿原酸和药学上可接受的辅剂形成的第一制剂，及以EGFR为靶点的药物和药学上可接受的辅剂形成的第二制剂。
优选的，所述第一制剂10-40重量份，所述第二制剂2-40重量份。
优选的，所述以EGFR为靶点的药物为EGFR酪氨酸激酶抑制剂和抗EGFR抗体中的一种或两种。
优选的，所述EGFR酪氨酸激酶抑制剂为吉非替尼、厄洛替尼、埃克替尼、阿法替尼和拉帕替尼中的一种或几种。
优选的，所述抗EGFR抗体为西妥昔单抗、曲妥珠单抗、尼妥珠单抗和帕尼单抗中的一种或几种。
优选的，所述第一制剂的剂型为注射剂或口服制剂；
所述第二制剂的剂型为注射剂或口服制剂。
本发明还提供了一种本发明所述的抗肿瘤联合用药物在制备抗癌药物中的用途。
优选的，所述癌症为非小细胞癌，乳腺癌，恶性神经胶质瘤或鼻咽癌。
本发明还提供了一种绿原酸在制备抗癌药物毒副作用抑制剂中的用途。
优选的，所述抗癌药物为以EGFR为靶点的药物。从EGFR的分子靶向药物临床治疗效果来看，目前实体瘤的分子靶向药物存在的主要问题有：毒副作用较多，单独用药效果不理想，易发生耐药突变等。如何更好的减少毒副作用，优化联合用药方案，克服耐药等成为分子靶向药物急需解决的问题。
基于此，本发明公开了一种抗肿瘤联合用药物及其在制备抗癌药物中的用途，本发明所述联合用药物包括绿原酸、抗肿瘤药物和药学上可接受的辅剂，通过研究表明，本发明提供的抗肿瘤联合用药物可提高以EGFR为靶点的药物在非小细胞癌，乳腺癌，恶性神经胶质瘤、鼻咽癌中的靶向性，且降低了以EGFR为靶点的药物毒副作用，并逆转以EGFR为靶点的药物的耐药性。
目前，大量的实验均证明绿原酸具有抗诱变及抗癌的作用。绿原酸作为G6PT(葡萄糖-6-磷酸转移酶)的抑制剂，可以启动中性粒细胞及前髓细胞HL-60的程序性细胞死亡，抑制人Hep3B肝癌细胞的金属硫蛋白(MMP)分泌，抑制神经胶质瘤的迁移。绿原酸对以EGFR为靶点的药物的增敏作用可能与上述机理有关。其次绿原酸分子中含有一定量的活性羟基，能形成具有抗氧化作用的氢自由基，可以消除羟基自由基和超氧阴离子等自由基的活性，保护组织免受氧化作用的损伤。此外，绿原酸对上皮内淋巴细胞(IEL)上清液以及肠道固有层淋巴细胞(LPL)上清液中NF-γ和TNF-α水平影响均较大。体外研究显示绿原酸能诱导人淋巴细胞及人外周血白细胞生成IFN-γ和IFN-α，这些作用也可能与绿原酸在本申请中能降低以EGFR为靶点的药物的毒副作用有关。
关于患者对EGFR-TKI(表皮生长因子受体-酪氨酸激酶抑制剂)的耐药，临床分为原发性耐药及继发性耐药。原发性耐药即患者对首次使用TKI治疗无反应，在症状改善、病灶控制和生存时间等方面未获得明显益处。EGFR突变的肿瘤患者中约25％对TKI治疗无效，这可能与TKI原发性耐药相关。其原因主要包括：①存在与药敏突变同时发生的其他EGFR突变，②存在影响EGFR下游信号的其他基因突变，③RAS基因突变的存在，④患者自身的因素，如免疫功能低下，新陈代谢的快速失活，以及吸收能力的下降等。EGFR-TKI获得性耐药与二次突变学说和Met基因扩增这2种机制有关，其他的耐药机制可能是由于患者自身的影响因素(如吸烟与否、性别、人种及病理类型等)不同造成的。绿原酸在本发明中，逆转了EGFR络氨酸激酶抑制剂的耐药性，其机理可能与上述产生耐药性的各类原因相关，具体机制有待进一步的研究。
附图说明
图1为绿原酸对PC9细胞株的增殖抑制；
图2为绿原酸对PC9/ZD细胞株的增殖抑制。
具体实施方式
为了使本领域的技术人员更好地理解发明的技术方案，下面结合具体 实施方式对本发明作进一步的详细说明。
EGFR是酪氨酸激酶I型受体亚家族的一个成员，该家族的其他成员包括HER2/neu、HER3和HER4。它们的受体均由三部分组成：胞外的配体结合区、由单链构成的跨膜区以及胞内的酪氨酸激酶区。EGFR是许多正常上皮组织(如皮肤和毛囊)的组成性表达成分，在很多人类肿瘤中发现有EGFR的过量表达。活化的EGFR主要与以下信号传导通路有关：丝裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)途径，启动DNA复制，引起细胞增殖与分化，并介导CLE调节细胞周期；PI3K-Akt途径，抑制细胞凋亡；活化下游VEGF，促进微血管网生成；上皮生长因子受体-信号转导与转录激活因子3(EGFR-STAT3)途径，使STAT3在许多肿瘤中活化，调节多种基因的活性，从而参与肿瘤的发生、发展和细胞凋亡。EGFR信号转导途径在肿瘤细胞的增殖、损伤修复、侵袭及新生血管形成等方面起重要作用，近年来靶向EGFR药物已成为肿瘤治疗的热点。
针对EGFR的肿瘤分子靶向药物，按其性质主要分为两大类：一类是单克隆抗体，目前在我国已上市的有西妥昔单抗(cetuximab、erbitux)、帕尼单抗(panitumumab、vectibix)、尼妥珠单抗(nimotuzumab、泰欣生)等；另一类是小分子抑制剂(表2)，已上市的有吉非替尼(gefitinib、iressa)、厄洛替尼(erlotinib、tarceva)和拉帕非尼(lapatinib、tykerb)等。小分子抑制剂的作用机制同单克隆抗体不同，主要通过竞争性结合EGFR胞内段酪氨酸激酶的磷酸化位点，阻断其与ATP的相互作用，继而抑制EGFR的酪氨酸磷酸化及下游一系列的信号传导，因此小分子抑制剂在 特异性方面不如单克隆抗体，会产生一系列毒副作用，其中常见不良反应包括皮疹、腹泻、肝功能损害，少见的不良反应包括肺间质病变。
抗EGFR单克隆抗体的作用机理是，它们可以结合于EGFR的胞外受体结合域，从而阻止生物体内自然配体与受体的结合，进而阻止受体的激活和下游信号通路的信号传递。因此产生的生物学效应表现为细胞周期抑制、促进细胞凋亡、抑制肿瘤血管生成等。然而目前已有系统评价方面的文献表明，此类单抗类药物，在实体恶性肿瘤患者的靶向治疗中显著增加了FAEs(死亡风险与不良事件)的发生风险。毒性主要表现在皮肤黏膜系统、心血管系统、造血系统及胃肠道系统等方面。
肺癌中表皮生长因子受体(epidermal growth factor receptor，EGFR)突变主要发生在胞内段编码结构域(外显子18-21)，包括外显子19的缺失突变(delE746-A750)和外显子21点突变(L858R)，两者占所有EGFR激酶突变的90％以上，与对表皮生长因子受体-酪氨酸激酶抑制剂(EGFR tyrosine kinase inhibitors，EGFR-TKI)的敏感性有关；此外，还有外显子18点突变(G719S)以及外显子20插入突变，前者属于EGFR-TKI的敏感突变而后者与EGFR-TKI的耐药有关，发生率均在5％左右。通常情况下，EGFR突变的NSCLC患者对TKI的治疗较敏感。EGFR-TKIs通过与ATP或底物竞争性结合胞外的配体结合位点，阻断EGFR分子内酪氨酸的自身磷酸化及酪氨酸激酶的活化，抑制EGFR同源或与ERBB3异源二聚体的形成，从而抑制EGFR激活，阻止下游信号转导，抑制细胞周期进程、加速细胞凋亡、抑制血管生成和转移。但是在临床工作中，许多患者对EGFR-TKI的治疗并不敏感，或者是在治疗一段时间后产生耐药。其耐药 机制主要包括原发性耐药与获得性耐药。原发性耐药是指首次使用EGFR-TKI即产生耐药，约60％NSCLC患者的耐药为TKI原发性耐药。其中，EGFR基因激活突变者有近30％对TKI原发耐药。此外，虽然在EGFR突变的NSCLC患者中吉非替尼和厄洛替尼可以起到很好的疗效，但大部分患者在治疗6个月-12个月内会发生获得性耐药。
为了解决上述EGFR小分析络氨酸激酶抑制剂的耐药性及毒副作用，以及抗EGFR单克隆抗体靶向性不足，产生毒副作用的缺陷，结合绿原酸在人体多个系统表现出的有益效果，本申请创造性的将二者结合起来，产生了意料不到的技术效果。本发明公开了一种抗肿瘤联合用药物，其特征在于，包括绿原酸、抗肿瘤药物和药学上可接受的辅剂；其中，所述抗肿瘤药物优选为以EGFR为靶点的药物；更具体的，所述抗肿瘤联合用药物包括：绿原酸和药学上可接受的辅剂形成的第一制剂，及以EGFR为靶点的药物和药学上可接受的辅剂形成的第二制剂；其中，本发明对所述药学上可接受的辅剂并没有特殊要求，本领域技术人员可以根据实际需要选择合适的辅剂；，所述抗肿瘤联合用药物中，按重量份数计，所述第一制剂优选为10-40份，更优选10-30份，最优选10-20份，所述第二制剂优选为2-40份，更优选5-30份，最优选的10-20份。所述以EGFR为靶点的药物优选为EGFR酪氨酸激酶抑制剂和抗EGFR抗体中的一种或两种，所述EGFR酪氨酸激酶抑制剂优选为吉非替尼、厄洛替尼、埃克替尼、阿法替尼和拉帕替尼中的一种或几种；所述抗EGFR抗体优选为西妥昔单抗、曲妥珠单抗、尼妥珠单抗和帕尼单抗中的一种或几种。此外，本发明对联合用药物的剂型没有特殊要求，本领域公知的可用于抗癌药物的剂型均可；
本发明还公开了绿原酸在制备抗癌药物毒副作用抑制剂中的用途，所述抗癌药物为以EGFR为靶点的药物。
实验结果表明，绿原酸可降低厄洛替尼、埃克替尼、吉非替尼、阿法替尼、曲妥珠单抗、拉帕替尼、尼妥珠单抗、西妥昔单抗对人正常鼻腔上皮细胞、人正常肺上皮细胞及人正常乳腺细胞的毒性，一定程度上提高了此类药物的靶向性，可降低以EGFR为靶点的药物的毒副作用。
且实验中，绿原酸与厄洛替尼、埃克替尼、吉非替尼、阿法替尼、曲妥珠单抗、拉帕替尼、尼妥珠单抗、西妥昔单抗等联合应用于肿瘤细胞时，产生了明显的协同增效作用，达到了对肿瘤细胞的增敏效果。
动物实验结果还表明，绿原酸与阿法替尼、厄洛替尼、帕尼单抗、尼妥珠单抗联合应用时，对G422脑胶质瘤BABLC小鼠及Lewis肺癌C57BL/6小鼠，不仅达到了协同增效作用，同时还降低了阿法替尼、厄洛替尼、帕尼单抗与尼妥珠单抗的毒副作用。
本发明实验结果还表明，绿原酸与吉非替尼联合应用于人肺部肿瘤细胞时，明显逆转了吉非替尼的耐药性。
综上所述，本发明所述技术方案可提高以EGFR为靶点的药物在非小细胞癌，乳腺癌，恶性神经胶质瘤、鼻咽癌的靶向性，降低其毒副作用，并逆转以EGFR为靶点的药物的耐药性。
以上是对发明内容的详细分析，下面为该发明的实施例。
实施例1 单独用药及联合用药对不同正常细胞株的影响
1.1材料与仪器
受试药品：绿原酸、厄洛替尼、吉非替尼、埃克替尼、阿法替尼、曲妥珠单抗、拉帕替尼、尼妥珠单抗、西妥昔单抗。
受试细胞株：人肺癌细胞株A549、人乳腺癌细胞株MDA-MB-453、人鼻咽癌细胞株CNE-1、人正常肺上皮细胞BEAS-2B、人正常乳腺细胞MCF10A、人正常鼻腔上皮细胞RPMI2650。
1.2实验方法
取对数生长期细胞一瓶，PBS洗涤2次，0.25％胰蛋白酶消化，用含10％小牛血清的RPMI-1640培养液配成单细胞悬液计数。无菌条件下接种于96孔培养板，接种细胞数均为5×10 3/孔。细胞于37℃、5％CO 2孵箱中分别孵育24h后更换培养基。
细胞分为，阴性对照组、EGFR为靶向的药物单独用药组、绿原酸组以及联合用药组，空白对照组不接种细胞。每个孔培养液和药物总量为200μl，每个浓度设3个复孔，并以等体积培养液代替药物作为空白对照。继续置5％CO 2饱和湿度37℃温箱中继续培养。48h后，每孔加入浓度为5mg/ml MTT 20μl继续培养4h后，小心吸弃孔内液体，加150μl二甲基亚枫溶解沉淀，充分振荡10min，溶解结晶物。选择570nm波长，在酶联免疫检测仪上测定各孔光吸收值，记录结果，计算抑制率及联合指数。以上实验至少重复3次。
1.3数据处理
(1)抑制率＝(1-OD570(实验组-空白组)/OD570(对照组-空白组))*100％。
(2)联合用药以公式Q＝E(a+b)/(Ea+Eb-Ea×Eb)计算有无协 同作用。其中E(a+b)为两药合用的抑制率，即实测合并效应，Ea和Eb为两药单用时的抑制率，分母(Ea+Eb-Ea×Eb)为期望合并效应，Q为两者比值。Q值在0.85～1.15时，两药合并效应为相加(+)，Q值在1.15～20时为协同(++)，Q值＞20为明显协同(+++)，Q值在0.05～0.85时为拮抗，Q值＜0.05为明显拮抗
1.4实验结果
1.4.1单独用药及联合用药对不同正常细胞株的影响
表1 单独用药对不同正常细胞株的影响
Figure PCTCN2018100603-appb-000001
表2 联合用药对不同正常细胞株的影响
Figure PCTCN2018100603-appb-000002
实验结果显示，在人正常肺上皮细胞BEAS-2B、人正常乳腺细胞MCF10A、人正常鼻腔上皮细胞RPMI2650抑制试验中，绿原酸(20μg/ml)单独应用时，IC50值分别为1238.1ug/ml、1352.6ug/ml、1054.4ug/ml，说明绿原酸本身对正常细胞株无毒性。在人正常肺上皮细胞BEAS-2B抑制试验中，绿原酸(20μg/ml)分别与厄洛替尼、埃克替尼、吉非替尼、阿法替尼联合用药IC50值均高于单独使用时的IC50值，说明绿原酸可降低厄洛替尼、埃克替尼、吉非替尼、阿法替尼对人正常肺上皮细胞BEAS-2B的毒性，一定程度上提高此类药物的靶向性。在人正常乳腺细胞MCF10A 抑制试验中，绿原酸(20μg/ml)分别与曲妥珠单抗、拉帕替尼的联合用药IC50值均高于单独使用时的IC50值，说明绿原酸可降低曲妥珠单抗、拉帕替尼对人正常乳腺细胞MCF10A的毒性，一定程度上提高该两种药物的靶向性。在人正常鼻腔上皮细胞RPMI2650抑制试验中，绿原酸(20μg/ml)分别与曲妥珠单抗、尼妥珠单抗、西妥昔单抗的联合用药IC50值均高于单独使用时的IC50值，说明绿原酸可降低曲妥珠单抗、尼妥珠单抗、西妥昔单抗对人正常鼻腔上皮细胞RPMI2650的毒性，一定程度上提高次三种单抗药物的靶向性。上述实验数据说明绿原酸可降低以EGFR为靶点的药物的毒副作用，一定程度上提高其靶向性。
1.4.2单独用药及联合用药对不同肿瘤细胞株的影响
表3 单独用药对不同肿瘤细胞株的影响
Figure PCTCN2018100603-appb-000003
Figure PCTCN2018100603-appb-000004
表4 EGFR为靶向的药物与绿原酸联合用药对人肺癌细胞株A549抑制作用
Figure PCTCN2018100603-appb-000005
结果显示，在以EGFR为靶点的药物与绿原酸联合用药对人肺癌细胞 株A549抑制作用实验中，11uM厄洛替尼与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；5uM埃克替尼与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；20uM吉非替尼与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；3uM阿法替尼与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)。
表5 以EGFR为靶点的药物与绿原酸联合用药对人乳腺癌细胞株MDA-MB-453抑制作用
Figure PCTCN2018100603-appb-000006
结果显示，在以EGFR为靶点的药物与绿原酸联合用药对人乳腺癌细胞株MDA-MB-453抑制作用试验中，50uM曲妥珠单抗与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；4uM拉帕替尼与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)。
表6 以EGFR为靶点的药物与绿原酸联合用药对人鼻咽癌细胞株CNE-1抑制作用
Figure PCTCN2018100603-appb-000007
结果显示，在以EGFR为靶点的药物与绿原酸联合用药对人鼻咽癌细胞株CNE-1抑制作用试验中，75uM曲妥珠单抗与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；100uM尼妥珠单抗与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)；55uM西妥昔单抗与10-50μg/ml绿原酸联合用药的Q值在1.15～20，两药合并效应为协同(++)。
实施例2 绿原酸体外逆转人肺腺癌细胞PC9对吉非替尼的耐药性
2.1材料与仪器
受试药品：绿原酸；吉非替尼
受试细胞株：PC9细胞，PC9/ZD为自筛耐吉非替尼细胞株
2.2实验方法
2.2.1耐药细胞株的培育
人肺腺癌细胞系PC9细胞，将PC9细胞分别暴露于吉非替尼(200nmol/L)长达3个月，挑选出耐吉非替尼的细胞克隆成为PC9/ZD细胞系。此两种细胞分别具有对吉非替尼的稳定耐药的能力。PC9细胞、PC9/ZD细胞分别贴壁生长于25cm 2培养瓶中，以含10％胎牛血清的DMEM培养基培养，置5％CO 2、37℃培养箱中孵育。每天观察细胞，细胞生长覆盖瓶底70％-80％时按1∶3传代，平均3-4d传代1次，保证细胞活力提供。
2.2.2检测吉非替尼对细胞株与耐药株的IC50，计算耐药倍数
取对数生长期PC9细胞及耐药细胞株，用0.25％胰蛋白酶+0.02％EDTA消化离心，调整细胞浓度为5×10 4个/ml，按每孔100μl/5000个细胞接种于96孔板，5％CO 2、37℃孵育24h后吸去原培养液，加入含不同浓度吉非替尼的DMEM培养液每孔100μl，使其终浓度分别为0、0.1、0.2、0.5、1、5、10μM，另设一不含细胞的空白孔，置于5％CO 2、37℃培养箱孵育48h后每孔加入5mg/ml MTT20μl，继续孵育4h，吸去上液，每孔加入DMSO150μl，静置30min，待孔底褐色结晶完全溶解。用酶标仪检测570nm处各孔吸光值OD值，按公式计算耐药倍数。耐药倍数＝耐药细胞 IC50值/敏感细胞IC50值。
2.2.3 MTT法测定无细胞毒的绿原酸浓度
PC9细胞及耐药细胞培养及处理方法同上。实验分别分成3组：空白组、对照组及实验组。空白组只加入DMEM培养基，无需接种细胞；对照组加培养基并接种细胞；实验组在上述基础上再加入不同浓度的绿原酸工作液，使其最终浓度分别为1、2、4、8、16、32、64、128μg/mL，置于培养箱孵育48h后每孔加入5mg/ml MTT20μl，继续孵育4h，吸去上液，每孔加入DMSO150μl，静置30min，待结晶完全溶解。用酶标仪检测570nm处各孔吸光值OD值，计算肿瘤细胞生长抑制率。抑制率＝(1-OD570(实验组-空白组)/OD570(对照组-空白组))×100％，取抑制率10％以下浓度的绿原酸浓度作为无毒剂量的逆转浓度。
2.2.4无细胞毒浓度绿原酸逆转人肺腺癌耐药株PC9/ZD的作用
细胞培养以及实验方法同上。实验分组如下：PC9/ZD组、PC9/ZD+绿原酸8μg/ml组、PC9/ZD+绿原酸16μg/ml组，实验组分别加入吉非替尼使终浓度为0.1、0.2、0.5、1、5、10μmol/L，每个浓度3复孔，测定各孔的OD值，观察无细胞毒性绿原酸(5μg/ml、10μg/ml)联合吉非替尼作用于PC9/ZD后，吉非替尼对PC9/ZD细胞杀伤作用的变化，计算吉非替尼对人肺腺癌PC9/ZD的IC50。逆转倍数＝逆转前IC50值/逆转后IC50值。
2.3实验结果
2.3.1吉非替尼对PC9及PC9/ZD的IC50吉非替尼对人肺腺癌敏感细胞
PC9以及耐药细胞PC9/ZD的IC50分别为0.21μmol/L和5.45μmol/L。按 公式计算出耐药倍数为25.9。
表7 吉非替尼对人肺腺癌敏感细胞PC9以及耐药细胞PC9/ZD的IC50及耐药倍数
Figure PCTCN2018100603-appb-000008
2.3.2 MTT法测定无细胞毒的绿原酸浓度结果
绿原酸对人肺腺癌敏感细胞PC9以及耐药细胞PC9/ZD均有抑制增殖作用，IC50为190ug/ml。当绿原酸浓度低于16μg/ml时，对人肺腺癌PC9以及耐药细胞PC9/ZD的抑制率均＜10％，没有细胞毒性，结果如图1及图2所示。
2.3.3绿原酸对人肺腺癌耐药细胞PC9/ZD的逆转作用
绿原酸无细胞毒浓度(8μg/ml、16μg/ml)联合吉非替尼作用于人肺腺癌耐药细胞PC9/ZD之后，吉非替尼对PC9/ZD的IC50降低。8μg/ml绿原酸作为逆转剂时，吉非替尼对PC9/ZD的IC50为3.25，逆转倍数为1.67，以16μg/ml绿原酸作为逆转剂时，吉非替尼对PC9/ZD的IC50为2.03，逆转倍数为2.68。
表8 绿原酸对人肺腺癌耐药细胞PC9/ZD的逆转作用
Figure PCTCN2018100603-appb-000009
Figure PCTCN2018100603-appb-000010
实施例2表明，绿原酸联合吉非替尼应用于人肺腺癌耐药细胞PC9/ZD时，可以逆转吉非替尼的耐药性。
实施例3绿原酸与以EGFR靶点的药物联合体内抑瘤的增效减毒作用
3.1材料与仪器
受试药品：绿原酸；阿法替尼、厄洛替尼、帕尼单抗、尼妥珠单抗
受试细胞株：G422细胞、Lewis小鼠肺癌细胞株
受试动物：BABLc小鼠、C57BL/6小鼠
3.2实验方法
3.2.1实验动物肿瘤模型的建立
收集对数生长期细胞，1000rpm离心5min，细胞沉用PBS洗涤2次，计数后用无血清培养液调整细胞浓度为1x10 7/ml，无菌条件下送到动物室。在无菌实验条件下，小鼠右侧腋窝皮下注射1x10 7/ml的细胞，每只 0.1ml，注射局部出现明显皮丘，1周后裸鼠左侧腋窝处出现米粒大结节，说明移植模型成功建立，此时按照随机分组法分成6组。
BABLc小鼠G422脑胶质瘤移植瘤分组为：1、生理盐水组(NS组)；2、尼妥珠单抗组；3、绿原酸组；4、帕尼单抗组；5、绿原酸+尼妥珠单抗组；6、绿原酸+帕尼单抗组，每组各5只。
C57BL/6小鼠Lewis肺癌分组为：7、生理盐水组(NS组)；8、阿法替尼组；9、绿原酸组；10、厄洛替尼组；11、绿原酸+阿法替尼组；12、绿原酸+厄洛替尼组，每组5只
3.2.2给药方法
待肿瘤平均直径达100mm 3后开始治疗，每天腹腔注射一次，生理盐水对照组给0.2ml无菌生理盐水。
3.2.3抗肿瘤作用评价
观察给药后每天测动物体重；待阴性组瘤体积约为0.5cm 3时停止实验，眼球摘除取血测定WBC、HBC及HGB含量，脱颈椎处死小鼠并称重，剥取肿瘤并称重。
抑瘤率＝(对照组瘤重-实验组瘤重)/对照组瘤重x100％。
两药合并Q＝E(a+b)/(Ea+Eb-Ea×Eb)，其中E(a+b)为两药合用的抑制率，即实测合并效应，Ea和Eb为两药单用时的抑制率，分母(Ea+Eb-Ea×Eb)为期望合并效应，Q为两者比值。Q值在0.85～1.15时，两药合并效应为相加(+)，Q值在1.15～20时为协同(++)，Q值＞20为明显协同(+++)，Q值在0.05～0.85时为拮抗，Q值＜0.05为明显拮抗。
3.2.4统计学分析
实验数据用SPSS 17.0统计软件进行处理，所有数据均以均数士标准差(mean±SD)表示。组间比较采用单因素方差分析进行统计学处理，P＜0.05认为有差异，P＜0.01认为有显著性差异。
3.3实验结果
表9 绿原酸对G422脑胶质瘤BABLC小鼠移植瘤的抑瘤率
Figure PCTCN2018100603-appb-000011
注：**与绿原酸组比较P＜0.01，##与尼妥珠组比较P＜0.01，&&与帕尼组比较P＜0.01
如表9所示，绿原酸10mg/kg，尼妥珠单抗20mg/kg，以及帕尼单抗20mg/kg对G422脑胶质瘤移植瘤有较弱的抑制作用，与生理盐水组比较无显著性差异。但绿原酸与尼妥珠单抗，以及绿原酸与帕尼单抗联用有较好的抑瘤率，与单用相比有显著性差异。联合用药Q值计算结果表明，绿原酸与帕尼单抗以及尼妥珠单抗联用有协同效应。
表10 绿原酸对Lewis肺癌C57BL/6小鼠移植瘤的抑瘤率
Figure PCTCN2018100603-appb-000012
注：**与绿原酸组比较P＜0.01，##与阿法替尼组比较P＜0.01，&&与厄 洛替尼组比较P＜0.01
如表10所示，绿原酸与阿法替尼，以及绿原酸与厄洛替尼联用有较好的抑瘤率，与单用相比有显著性差异。联合用药Q值计算结果表明，绿原酸与阿法替尼以及厄洛替尼联用有协同效应。
3.4联合用药的对小鼠血常规的影响
表11 联合用药对G422脑胶质瘤BABLC小鼠血常规的影响
Figure PCTCN2018100603-appb-000013
Figure PCTCN2018100603-appb-000014
与阴性组比较*p＜0.05，**p＜0.01；与阳性组比较Δp＜0.05，ΔΔp＜0.01
表12 联合用药对Lewis肺癌C57BL/6小鼠血常规的影响
Figure PCTCN2018100603-appb-000015
Figure PCTCN2018100603-appb-000016
与阴性组比较*p＜0.05，**p＜0.01；与阳性组比较Δp＜0.05，ΔΔp＜0.01
结果显示：绿原酸(10mg/kg)，能够显著改善G422脑胶质瘤小鼠使用尼妥珠单抗、帕尼单抗后血常规中WBC、RBC、HGB的降低现象；此外，对Lewis肺癌小鼠使用阿法替尼、厄洛替尼后血常规中WBC、RBC、HGB降低的现象也得到明显改善，白细胞记数的增加，说明绿原酸能缓解VEGF通路抑制剂产生的骨髓抑制作用，血小板记数的增加可降低出 血性不良反应的发生，同时血红蛋白增加可预防贫血及贫血相关性不良反应的发生，表明绿原酸从诸多方面降低了以EGFR为靶点的药物的毒副作用。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (12)

  1. 一种抗肿瘤联合用药物，其特征在于，包括绿原酸、抗肿瘤药物和药学上可接受的辅剂。
  2. 根据权利要求1所述的抗肿瘤联合用药物，其特征在于，所述抗肿瘤药物为以EGFR为靶点的药物。
  3. 根据权利要求2所述的抗肿瘤联合用药物，其特征在于，所述抗肿瘤联合用药物包括：绿原酸和药学上可接受的辅剂形成的第一制剂，及以EGFR为靶点的药物和药学上可接受的辅剂形成的第二制剂。
  4. 根据权利要求3所述的抗肿瘤联合用药物，其特征在于，所述第一制剂10-40重量份，所述第二制剂2-40重量份。
  5. 根据权利要求3所述的抗肿瘤联合用药物，其特征在于，所述以EGFR为靶点的药物为EGFR酪氨酸激酶抑制剂和抗EGFR抗体中的一种或两种。
  6. 根据权利要求5所述的抗肿瘤联合用药物，其特征在于，所述EGFR酪氨酸激酶抑制剂为吉非替尼、厄洛替尼、埃克替尼、阿法替尼和拉帕替尼中的一种或几种。
  7. 根据权利要求5所述的抗肿瘤联合用药物，其特征在于，所述抗EGFR抗体为西妥昔单抗、曲妥珠单抗、尼妥珠单抗和帕尼单抗中的一种或几种。
  8. 根据权利要求3-7任意一项所述的抗肿瘤联合用药物，其特征在于，所述第一制剂的剂型为注射剂或口服制剂；
    所述第二制剂的剂型为注射剂或口服制剂。
  9. 一种权利要求1～7任意一项所述的抗肿瘤联合用药物在制备抗癌药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述癌症为非小细胞癌，乳腺癌，恶性神经胶质瘤或鼻咽癌。
  11. 一种绿原酸在制备抗癌药物毒副作用抑制剂中的用途。
  12. 根据权利要求11所述的用途，其特征在于，所述抗癌药物为以EGFR为靶点的药物。
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