Application of PQQ and quinone compound composition

Abstract

The invention discloses an application of a PQQ and quinone compound composition, and provides an application of pyrroloquinoline quinone or pharmaceutically acceptable salts and quinone compounds thereof in preparation of medicines for preventing and / or treating NQO1 high-expression malignant tumors. When the pyrroloquinoline quinone or the pharmaceutically acceptable salt thereof is combined with the quinone compound for use, the killing sensitivity of NQO1 high-expression malignant tumor cells to the quinone compound is obviously enhanced, explosive Coalism is initiated, a synergistic or synergistic anti-tumor effect is achieved, and the compound has a good application prospect. The killing effect of various quinone compounds on NQO1 high-expression tumor cells is improved in an outbreak mode, so that the application dosage of the NQO1 high-expression tumor cells is remarkably reduced to reduce side effects, and the effect of killing the NQO1 high-expression tumor cells with low dosage and high efficiency is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of a PQQ and quinone compound composition. BACKGROUND

[0002] NAD(P)H: quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) is an important cytosolic flavoenzyme. The enzyme was first isolated and identified from rat liver homogenate in the 1950s, and was named DT-diaphorase due to its unique two-electron reduction property. The human NQO1 gene is located on chromosome 16, with a full length of about 20 kb. The NQO1 protein encoded by the gene has a molecular weight of about 30 kDa and exists in a homodimeric form in the functional state, and specifically binds a flavin adenine dinucleotide (FAD) coenzyme near the catalytic site.

[0003] The core function of NQO1 is to catalyze a NAD(P)H-dependent two-electron reduction reaction, directly reducing a variety of quinones to the corresponding hydroquinones, which however have different effects depending on the stability of the hydrogenated quinones.

[0004] When stable hydroquinones are generated, the catalytic process of NQO1 is considered as an important detoxification pathway. It effectively avoids the generation of toxic semiquinone radicals and subsequent burst of Reactive Oxygen Species (ROS) through one-electron reduction pathway by two-electron reduction of quinones, thereby protecting cells from oxidative damage. On the contrary, when unstable hydroquinones are generated from the catalysis of certain specific quinones (especially some antitumor quinones such as β-lapachone), NQO1 plays the role of a bioactivating enzyme, converting low-toxic or non-toxic quinone prodrugs into compounds with strong cytotoxicity. The activated products not only can directly alkylate DNA, but also can continuously generate a large amount of ROS through redox cycling due to their own instability, causing serious DNA damage and damage to other cell structures, and finally selectively inducing tumor cell death.

[0005] A large number of studies have shown that the expression level of NQO1 in various human malignancies is significantly higher than that in the corresponding normal tissues, including non-small cell lung cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, and colorectal cancer. Clinical correlation analysis further shows that high expression of NQO1 in tumor tissues is often negatively correlated with poor patient prognosis. Notably, overexpression of NQO1 does not only occur in advanced tumors, but can also occur in the early stages of cancer progression or even in precancerous lesions.

[0006] Due to the specific high expression of NQO1 in various tumors and its key role in activating certain quinone prodrugs, it has become a highly potential tumor treatment target. Using NQO1-activated prodrugs such as beta-lapachone, BBI608, etc. can achieve selective killing of NQO1 high expression tumor cells. However, the current basic research and clinical experiments on NQO1 targeted compounds show that NQO1 activated prodrug treatment of tumors still has many application problems such as low drug efficacy, drug resistance, strong toxic and side effects, which restricts its clinical application potential and related drug development. SUMMARY

[0007] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide an application of a PQQ and quinone compound composition.

[0008] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is: In a first aspect of the present application, the application of pyrroloquinoline quinone or its pharmaceutically acceptable salt in the preparation of a sensitizing drug for preventing and / or treating NQO1 high expression malignant tumor is provided.

[0009] In a second aspect of the present application, the application of pyrroloquinoline quinone or its pharmaceutically acceptable salt and quinone compound in the preparation of a drug for preventing and / or treating NQO1 high expression malignant tumor is provided.

[0010] In a third aspect of the present application, the application of pyrroloquinoline quinone or its pharmaceutically acceptable salt and quinone compound in the preparation of at least one of the following drugs is provided: (1) a drug for activating NQO1 activity in NQO1 high expression malignant tumor cells; (2) a drug for increasing ROS production in NQO1 high expression malignant tumor cells; (3) a drug for reducing the activity of NQO1 high expression malignant tumor cells; (4) a drug for increasing the release of LDH in NQO1 high expression malignant tumor cells; (5) a drug for inducing cell membrane damage of NQO1 high expression malignant tumor cells; (6) a drug for inducing death of NQO1 high expression malignant tumor cells; (7) a drug for inhibiting the growth of NQO1 high expression malignant tumor cells.

[0011] In a fourth aspect of the present application, a pharmaceutical composition comprising pyrroloquinoline quinone or its pharmaceutically acceptable salt and quinone compound is provided.

[0012] The present application has the following beneficial effects: The present application can obviously enhance the killing sensitivity of NQO1 high expression malignant tumor cells to quinone compounds when pyrroloquinoline quinone or its pharmaceutically acceptable salt is used in combination with quinone compounds (for example, vitamin K3), trigger explosive Coalism, synergistic or synergistic anti-tumor effect, achieve explosive improvement of the killing effect of various quinone compounds on NQO1 high expression tumor cells, thereby significantly reducing the application dose of the latter to reduce side effects, and achieve the effect of killing NQO1 high expression tumor cells at low dose and high efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Toxicity of PQQ combined with different concentrations of VK3, VK4, MAM, KL1333, beta-LAP, Dunnione, DMNQ, TSA and TSB in NQO1 high expression pancreatic cancer MIA PaCa-2 cells; wherein A is PQQ combined with VK3, VK4, MAM; B is PQQ combined with KL1333, beta-LAP; C is PQQ combined with DMNQ, Dunnione; D is PQQ combined with TSA, TSB MTT result chart; *** P <0.001; Figure 2 The upper graph is the protein expression result of NQO1 in lung cancer cells A549 and NCI-H1975 cells; the lower graph is the MTT detection result of PQQ combined with VK3 in A549 (A) and NCI-H1975 (B) cells, respectively; *** P <0.001; Figure 3 The upper graph is the expression result of NQO1 in different pancreatic cancer cell lines; the lower graph is the MTT detection result of PQQ combined with VK3 in pancreatic cancer cells MIA PaCa-2 (A), AsPC-1 (B), SW1990 (C) and PANC-1 (D), respectively; *** P <0.001, ns P >0.05; Figure 4 The upper graph is the expression result of NQO1 in different breast cancer cells; the lower graph is the MTT detection result of PQQ combined with VK3 in breast cancer cells HCC1937 (A) and MDA-MB-231 (B), respectively; *** P <0.001, ns P >0.05; Figure 5The upper graph is the expression result of NQO1 in different glioma cell lines; the lower graph is the MTT detection result of PQQ and VK3 in combination in glioma cell U87 (A) and U251 (B); *** P <0.001; Figure 6 The upper graph is the expression result of NQO1 in different cancer cells; the lower graph is the MTT detection result of PQQ and VK3 in combination in hepatocellular carcinoma cell HepG2 (A), cervical cancer cell HeLa (B), colon cancer cell HT-29 (C) and prostate cancer cell PC-3 (D); *** P <0.001; Figure 7 The combination index of PQQ and VK3 on NQO1 high expression MIA PaCa-2 cells; A is the inhibition rate of PQQ and VK3 combination on MIA PaCa-2 cells, B is the calculation of combination index CI, and C is the HSA synergistic model score; Figure 8 A is the MTT detection result of PQQ and VK3 combination on the control group MIA PaCa-2 shNC (control interference RNA); B is the MTT detection result of PQQ and VK3 combination on MIA PaCa-2 shNQO1 cells stably knocked down NQO1; C is the effect of PQQ, VK3 alone and in combination on NQO1 enzyme activity; D is the effect of PQQ, VK3 alone, in combination, DIC and NAC on the generation of ROS in NQO1 high expression MIA PaCa-2 cells; *** P <0.001, ns P >0.05; Figure 9 The effects of PQQ, VK3 alone or in combination, and under the condition of adding NQO1 inhibitor dicoumarol (DIC) on the cytotoxicity (A), LDH release (B), PI penetration and morphological changes (C) of NQO1 high expression pancreatic cancer MIA PaCa-2 cells; Figure 10 7-AAD / Annexin V-PE double staining method for detecting the effects of PQQ, VK3, cisplatin (control anti-tumor drug) alone or in combination, and under the condition of adding DIC on cell death; Figure 11The study aimed to demonstrate the antitumor effects of PQQ and VK3 in a zebrafish tumor model xenografted with shNC-GFP Mia PaCa-2 cells. A represents the statistical results of fluorescent regions in the zebrafish experiment; B represents the morphological and fluorescence photographic results of zebrafish inoculated with shNC-GFP MIA PaCa-2 cells. *** P <0.001, ns P >0.05; Figure 12 The study investigated the antitumor effects of PQQ and VK3 in a NOD-SCID immunodeficient mouse tumor model in which Mia PaCa-2 cells were xenografted. Figures A, C, and D represent tumor size, morphology, and weight in tumor-bearing mice, respectively; and their effects on mouse body weight (D) and survival time (E). * P <0.05, ** P <0.01, *** P <0.001; Figure 13 The results of pathological staining of heart, liver, spleen, lung and kidney tissues of NOD-SCID tumor-bearing mice (A) and liver function test results are shown. Among them, B is the serum alanine aminotransferase (ALT) level of tumor-bearing mice and C is the serum aspartate aminotransferase (AST) level of tumor-bearing mice. * P <0.05, ** P <0.01, *** P <0.001. Detailed Implementation

[0014] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0015] The application of pyrroloquinoline quinone or its pharmaceutically acceptable salts and their combinations, as proposed in this invention, is described in detail below.

[0016] Some embodiments of the present invention provide the use of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof in the preparation of sensitizing drugs for the prevention and / or treatment of NQO1-overexpressing malignancies.

[0017] In some embodiments, the pyrroloquinoline quinone is PQQ (Pyrroloquinoline Quinone). The chemical structure of PQQ (CAS No.: 72909-34-3) is:

[0018] To solve the defects and deficiencies of the prior art in the development of quinone compounds for anti-tumor, the inventors, after a large amount of research and practice, finally provide that PQQ or a pharmaceutically acceptable salt thereof can be used as a chemosensitizing drug in combination with quinone compounds, especially naphthoquinone compounds. It is found through experiments that low-concentration (for example, 2.5 μM) PQQ and low-concentration quinones (such as VK3, β-lapachone, etc.) alone have no obvious killing effect on NQO1 high-expression malignant tumor cells. When PQQ is used in combination with quinone compounds, an explosive killing effect can be triggered in NQO1 high-expression malignant tumor cells, which can present: 1) Coalism effect: at a concentration at which neither PQQ nor quinone compounds alone has a killing effect, the combination of the two presents a significant killing effect; 2) Synergistic effect: at a concentration at which PQQ alone has no killing effect, the combination of PQQ with a quinone compound having a killing effect significantly increases the killing effect of the latter and significantly reduces the IC 50 value of the latter; 3) Synergistic effect: at a concentration at which both PQQ and quinone compounds alone have a killing effect, the combination of the two presents a significant synergistic killing effect. Therefore, the experimental results show that PQQ is an excellent chemosensitizing drug for NQO1 high-expression malignant tumors, which can greatly increase the sensitivity of NQO1 high-expression malignant tumor cells to quinone compounds. The use of PQQ in combination with quinone compounds for the preparation of NQO1 high-expression malignant tumor chemotherapeutic drugs can solve many clinical practical application problems such as low drug efficacy, drug insensitivity or resistance, and large toxic and side effects of quinone compounds. The application prospect of the combination of PQQ and quinone compounds is promising, and the potential commercial value is high.

[0019] It should be noted that the chemosensitizing drug is used to increase the sensitivity of NQO1 high-expression malignant tumor cells to anticancer drugs. PQQ can be used as a chemosensitizing drug in combination with anticancer drugs.

[0020] Some embodiments of the present application also provide the use of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds in the preparation of a drug for preventing and / or treating NQO1 high-expression malignant tumors.

[0021] Some embodiments of the present application also provide uses of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and a quinone compound in the preparation of at least one medicament for: (1) activating NQOl activity in NQOl high-expression malignant tumor cells; (2) increasing ROS production in NQOl high-expression malignant tumor cells; (3) reducing viability of NQOl high-expression malignant tumor cells; (4) increasing LDH release from NQOl high-expression malignant tumor cells; (5) inducing cell membrane damage of NQOl high-expression malignant tumor cells; (6) inducing death of NQOl high-expression malignant tumor cells; (7) inhibiting growth of NQOl high-expression malignant tumor cells.

[0022] Some embodiments of the present application also provide uses of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof in analytical reagents, biochemical reagents or detection reagents for sensitizing and / or killing NQOl high-expression malignant tumor cells.

[0023] In particular, the above-mentioned quinone compounds include benzoquinones, naphthoquinones and phenanthraquinones, preferably naphthoquinones.

[0024] In some embodiments, the quinone compound includes, but is not limited to, at least one of the following substances or their pharmaceutically acceptable salts or esters, derivatives thereof, pharmaceutically acceptable salts of their derivatives, or solvates thereof: vitamin K3 (VK3), vitamin K4 (VK4), KL1333, β-lapachone (β-LAP), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), 2-methoxy-1,4-naphthoquinone (MNQ), 2-methoxy-6-acetyl-7-methyljuglone (2-methoxystypandrone (MAM), shikonin, acetylshikonin, deoxyshikonin, isoshikonin, and tanshindiol A. A, TSA), Tanshindiol B (TSB), Tanshindiol C (TSC), Dunnione, NQ301 (CAS: 130089-98-4), NAT1-IN-1 (compound 350), YZ-35, LY5 (CAS: 1436382-03-4), QD394 (CAS: 2132411-21-1), NW16, STAT3-IN-4 (CAS: 825611-06-1), STAT3-IN-10 (CAS: 2499491-04-0), STAT3-IN-11 (CAS: 2503096-50-0), STAT3-IN-14 (CAS:123297-90-5), STAT3-IN-29 (CAS:2256751-16-1), STAT3-IN-35 (CAS:2849535-96-0), leucanthus quinone, isoleucanthus quinone, 6-hydroxyleucanthus quinone, 3-chloroleucanthus quinone, 3-bromoleucanthus quinone, leucanthus quinone, thymequinone, tocoquinone, sedge quinone, juglone, 7-methyljuglone, 2,3-dimethyljuglone, naphthoside (Nap hthazarin), Quinoclamine, Lapachol, Bupavaquinone, Aristolochic acid, 4-hydroxy- ...3-dimethyl-1,4-naphthoquinone, 2,7-dimethyl-1,4-naphthoquinone, 2,3-dicyano-1,4-naphthoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 2-amino-3-chloro-1,4-naphthoquinone, 2-benzoylamino-1,4-naphthoquinone, 2-hydroxy-3-methyl-1,4-naphthoquinone, 2-hydroxy-3-methoxy-1,4-naphthoquinone, 2-sulfonic acid sodium-1,4-naphthoquinone, 5-hydroxy-p-naphthoquinone, 6-methyl-1,4-naphthoquinone, 2,3,6-trimethyl-naphthoquinone, 7-methyl-5-hydroxy-naphthoquinone, β,β-dimethylacrylshikonin, 2-amino-3-carboxy-1,4-naphthoquinone, 2-carbamoyl-3-hydroxy-1,4-naphthoquinone, 2,7-dimethoxy-6-(1-acetoxyethyl)juglone, 6-ethyl-2,7-dimethoxyjuglone, 7-(hydroxymethyl)-2-methyl-1,4-naphthalenedione, 2-hydroxy-6-methoxy-1,4-dihydronaphthalene-1,4-dione), Alkannin, Aristolindiquinone, Aureoquinone, Deoxykalafungin, Droseron, Flaviolin, Herbarin, Isoplumbagin, Isobutylshikonin, Javanicin, KP372-1, Lavendamycin, Lawsone methyl ether, Majoranaquinone, Malvone A, Mitoquidone, Pronqodine, Ramentaceone, Salvisyrianone, SARS-CoV MPro-IN-2, Sarubicin B; preferably, the quinone compound comprises at least one of Vitamin K3, Vitamin K4, KL1333, β-LAP, MAM, DMNQ, MNQ, Dunnione, TSA, TSB, TSC, or a pharmaceutically acceptable salt or ester thereof, a derivative thereof, a pharmaceutically acceptable salt of the derivative thereof, a solvate thereof.

[0025] It should be noted that the quinone compound in the above embodiments can be replaced by a combination of one or more of its pharmaceutically acceptable salts or esters, derivatives thereof, pharmaceutically acceptable salts of the derivatives thereof, and nanoscale substances of the derivatives thereof, to achieve the same or similar therapeutic effect, which should all fall within the scope of protection of the present application.

[0026] Further, as stated above, the NQO1 high-expression malignant tumor includes at least one of non-small cell lung cancer, pancreatic cancer, breast cancer, brain glioma, liver cancer, colorectal cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, esophageal cancer, leukemia, malignant melanoma, renal cancer, bladder cancer, and head and neck squamous cell carcinoma.

[0027] In some embodiments, the NQO1 high-expression malignant tumor is further selected from at least one of non-small cell lung cancer, pancreatic cancer, breast cancer, brain glioma, liver cancer, colorectal cancer, prostate cancer, and cervical cancer. Further, some embodiments of the present application also provide a product for resisting NQO1 high-expression malignant tumors, which comprises pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and a quinone compound.

[0028] In particular, the product is a pharmaceutical composition, a kit, a health care drug or a health food, and the molar ratio of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds is 10:1-1:10, preferably 2.5:1-1:2.5.

[0029] In some embodiments, the product is a kit, and the use method of the kit is that the concentration of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof used is 0.25-50 μM. The killing effect of the combination of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds (such as VK3) on NQO1 high-expression malignant tumor cells can reach more than 80%. The use concentration of the above-mentioned combination of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds is far lower than the concentration of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds used alone in current experimental research.

[0030] In particular, some embodiments of the present application also provide a therapeutic drug for NQO1 high-expression malignant tumors, which comprises an effective amount of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and quinone compounds. The quinone compounds are vitamin K3, vitamin K4, KL1333, β-LAP, DMNQ, MNQ, MAM, TSA, TSB, TSC, a pharmaceutically acceptable salt or ester thereof, a derivative thereof, a pharmaceutically acceptable salt of the derivative thereof, or a combination of nanoscale substances of the derivative thereof.

[0031] The term "derivative" includes stereoisomers, isotopically labeled derivatives, deuterated compounds, prodrugs or solvates thereof. "Stereoisomers" include enantiomers and diastereomers, as well as cis-trans isomers and tautomers. The "deuterated compound" used in the present application refers to the form in which any hydrogen on the compound is replaced by deuterium. The term "pharmaceutically acceptable" means chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form, and physiologically compatible with the recipient.

[0032] The term "salt" refers to acid or base salts of the compound or its stereoisomers formed with inorganic acids, organic acids or bases, also includes zwitterionic salts (internal salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained by separation and purification during the preparation of the compound, or by mixing the above-mentioned compound or its stereoisomers with an appropriate amount (for example, equivalent amount) of acid or base. These salts can form a precipitate in solution and be collected by filtration, or be recovered after evaporation of the solvent, or be prepared by freeze-drying after reaction in an aqueous medium. The salt described in the present application can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt of the compound, etc.

[0033] The term "prodrug" as used herein denotes a compound which is rapidly transformed in vivo to afford the parent compound of the above formula, either directly by chemically or biologically reactivity or indirectly by solubilization and / or migration following administration to the site of action, for example by hydrolysis in blood.

[0034] The compounds of the present application can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present application.

[0035] In some embodiments, the above pharmaceutical is a pharmaceutical composition, further comprising a pharmaceutically acceptable excipient.

[0036] The pharmaceutical composition of the present application is suitable for various routes of administration and can be formulated into any dosage form which is pharmaceutically acceptable. For example, the above pharmaceutical composition can be administered orally, nasally, by inhalation, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially and intratumorally to a patient or subject in need of such treatment. When administered orally, the pharmaceutical composition can be formulated into oral preparations, for example, into conventional oral solid preparations such as tablets, capsules, pills, granules and the like; or into oral liquid preparations such as oral solutions, oral suspensions, syrups and the like. When formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants and the like can be added. When administered parenterally, the above pharmaceutical composition can also be formulated into injections, including injection solutions, sterile powders for injections and concentrated solutions for injections. When formulated into injections, they can be produced by using conventional methods in the field of pharmaceutical production, and when prepared, no additional agents can be added or suitable additional agents can be added depending on the nature of the pharmaceutical. When administered rectally, the pharmaceutical composition can be formulated into suppositories and the like. When administered via the lung, the pharmaceutical composition can be formulated into inhalation preparations, aerosol preparations, powder mist preparations or spray preparations and the like.

[0037] "Parenteral administration" includes administration by injection or infusion, and by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and sternal injection and infusion.

[0038] Pharmaceutically acceptable excipients are those that are nontoxic, biologically compatible, and otherwise biologically suitable for use in the body. The selection of a particular excipient will depend on the mode of administration or the type and state of the disease for which the particular patient is being treated. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersing agents, suspending agents, surface active agents, isotonic agents, thickening agents, emulsifying agents, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, coloring agents, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, among others, that are conventional in the art of pharmacy. If necessary, flavoring agents, preservatives, and sweetening agents, among others, can also be added to the pharmaceutical composition.

[0039] Oral administration can be the preferred mode of administration due to ease of administration, and tablets and capsules represent the most advantageous oral dosage unit forms in this regard as they obviously galenically employ solid pharmaceutical carriers. However, other dosage forms could be suitable depending on the clinical population (e.g., age and severity of the clinical condition), solubility of the particular disclosed compound being used, etc. Thus, the disclosed pharmaceutical compositions can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. In preparing the compositions in oral dosage forms, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease in administration, tablets and capsules are preferred oral dosage units in which solid pharmaceutical carriers are employed.

[0040] The pharmaceutical compositions of the disclosed oral dosage forms can include one or more pharmaceutical excipients and / or additives. Non-limiting examples of suitable excipients and additives include gelatin, natural sugars such as sucrose or lactose, lecithin, pectin, starches (e.g., corn starch or amylopectin), dextran, polyvinylpyrrolidone, polyvinyl acetate, gum arabic, alginic acid, talc, aspidium, silica gel (e.g., colloidal), cellulose, cellulose derivatives (e.g., cellulose ethers in which the hydroxyl groups of cellulose are partially etherified with lower saturated aliphatic alcohols and / or lower saturated aliphatic hydroxyl alcohols, such as methyl oxpropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids and magnesium, calcium or aluminum salts of fatty acids having 12 to 22 carbon atoms, especially saturated salts (e.g., stearates), emulsifiers, fats, especially vegetable (e.g., peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower seed oil, fish liver oil, in each case optionally also hydrated); saturated fatty acids C 12 H 24O2 to C 18 H 36 O2 glycerol esters and polyglycerol esters and mixtures thereof, the glycerol hydroxyl groups possibly being wholly or also only partially esterified (e.g. monoglycerides, diglycerides and triglycerides); pharmaceutically acceptable monovalent or polyvalent alcohols and polyglycols such as polyethylene glycol and derivatives thereof, esters of aliphatic saturated or unsaturated fatty acids (2 to 22 carbon atoms, especially 10 to 18 carbon atoms) with monovalent fatty alcohols (1 to 20 carbon atoms) or polyvalent alcohols (such as ethylene glycol, glycerol, diethylene glycol, pentaerythritol, sorbitol, mannitol and the like, which can also optionally be etherified), esters of citric acid with primary alcohols, acetic acid, urea, benzyl benzoate, dioxolane, glycerol formal, tetrahydrofurfuryl alcohol, C1-C4-alkyl esters of C1-C4-alkyl glycolate, dimethylacetamide, lactamide, lactate esters, ethyl carbonate, silicones (especially medium viscosity polydimethylsiloxanes), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like. 12 Polyethylene glycol ethers of alcohols, dimethylacetamide, lactamide, lactate esters, ethyl carbonate, silicones (especially medium viscosity polydimethylsiloxanes), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like.

[0041] Other auxiliary substances which can be used for the preparation of oral dosage forms are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, sodium carboxymethylcellulose or microcrystalline cellulose. Conventional coating substances can also be used for the preparation of oral dosage forms. Coating substances which can be considered, for example, are: polymerization products and copolymerization products of acrylic and / or methacrylic acid and / or esters thereof; copolymerization products of acrylic and methacrylic acid esters with a low content of ammonium groups (e.g. Eudragit R RS), copolymerization products of acrylic and methacrylic acid esters and trimethylammonium methacrylate (e.g. Eudragit R RL); polyvinyl acetate; fats, oils, waxes, fatty alcohols; hydroxypropyl methylcellulose phthalate or acetate succinate; cellulose acetate phthalate, starch acetate phthalate and polyvinyl acetate phthalate, carboxymethyl cellulose; methylcellulose phthalate, methylcellulose succinate, methylcellulose phthalate succinate and methylcellulose phthalate hemiester; zein; ethylcellulose and ethylcellulose succinate; shellac, gluten; ethylcarboxyethylcellulose; ethyl acrylate-maleic anhydride copolymer; maleic anhydride-vinyl methyl ether copolymer; styrene-maleic acid copolymer; 2-ethylhexyl acrylate maleic anhydride; crotonic acid-vinyl acetate copolymer; glutamic acid / glutamate copolymer; carboxymethyl ethylcellulose glycerol monocaprylate; cellulose acetate succinate; polyarginine and the like.

[0042] In some embodiments of the present application, plasticizers that can be considered as coating materials in oral dosage forms are: citric and tartaric acid esters (acetyl triethyl citrate, acetyl tributyl citrate, tributyl citrate, triethyl citrate); glycerol and glycerol esters (glycerol diacetate, glycerol triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl phthalate, dipentyl phthalate, diethyl phthalate, dimethyl phthalate, dipropyl phthalate), di(2-methoxy- or 2-ethoxyethyl) phthalate, ethyl phthalyl ethyl glycolate, butyl ethyl phthalyl glycolate and butyl glycolate; alcohols (propylene glycol, polyethylene glycols of various chain lengths), adipic acid esters (diethyl adipate, di-(2-methoxy- or 2-ethoxyethyl) adipate); benzophenone; diethyl sebacate and dibutyl sebacate, dibutyl succinate, dibutyl tartrate; diethylene glycol dipropionate; ethylene glycol diacetate, dibutyrate, dipropionate; tributyl phosphate, glyceryl tributyrate; polyethylene glycol sorbitan monooleate (polysorbate, such as polysorbate 50); sorbitan monooleate; and the like.

[0043] In some embodiments of the present application, suitable binders, lubricants, disintegrants, colorants, flavoring agents, flow-inducing agents, and melting agents can be included as carriers. The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include, but are not limited to, lactose, terra alba, sucrose, dextrose, methyl cellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0044] In some embodiments of the present application, binders can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. In another aspect, disintegrants can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0045] In some embodiments of the present application, oral dosage forms, such as solid dosage forms, can contain the disclosed compounds linked to polymers as targetable drug carriers or as prodrugs. Suitable biodegradable polymers for achieving controlled release of drugs include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactone, polyhydroxybutyric acid, polyortho esters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogels, preferably covalently crosslinked hydrogels.

[0046] In some embodiments of the application, tablets can include the active ingredient mixed with non-toxic and pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin, or acacia; and lubricating agents such as magnesium stearate, stearic acid, or talc. Tablets can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over an extended period of time. Examples of coating agents include, but are not limited to, sugar, shellac, or other enteric coating agents.

[0047] In some embodiments of the application, tablets comprising the disclosed compounds can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0048] In some embodiments of the application, solid oral dosage forms such as tablets can be coated with an enteric coating to prevent easy disintegration in the stomach. In various aspects, enteric coating agents include, but are not limited to, hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, polyvinyl acetate-phthalate, and cellulose acetate phthalate. In some embodiments of the application, the enteric coating can comprise hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, polyvinyl acetate-phthalate, and cellulose acetate phthalate.

[0049] In some embodiments of the application, oral dosage forms can be solid dispersions with water-soluble or water-insoluble carriers. Examples of water-soluble or water-insoluble carriers include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl methylcellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropyl methylcellulose phthalate, carboxymethyl ethylcellulose or hydroxypropyl methylcellulose, ethyl cellulose or stearic acid.

[0050] In some embodiments of the application, oral dosage forms can be liquid dosage forms, including those that are ingested or administered in the form of a mouthwash or gargle. For example, liquid dosage forms can include aqueous suspensions containing the active material suspended in a mixture with excipients suitable for preparing an aqueous suspension. Additionally, oily suspensions can be formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or mineral oil (such as liquid paraffin). Oily suspensions can also contain various excipients. The pharmaceutical compositions of the present disclosure can also be in the form of oil-in-water emulsions, which can also contain excipients such as sweetening and flavoring agents.

[0051] In some embodiments of the application, for the preparation of solutions or suspensions, for example, water, especially sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1,2-propanediol, polyethylene glycols and derivatives thereof, fatty alcohols, partial esters of glycerol), oils (for example, peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soybean oil, castor oil, neatsfoot oil), paraffins, dimethyl sulfoxide, triglycerides, etc. can be used.

[0052] In some embodiments of the application, in the case of liquid dosage forms such as drinkable solutions, the following substances can be used as stabilizers or solubilizers: lower aliphatic monovalent and polyvalent alcohols having 2-4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols having a molecular weight of between 200-600 (for example, 1-40% aqueous solution), diethylene glycol monoethyl ether, 1,2-propanediol, organic amides, for example amides of aliphatic C1-C6-carboxylic acids with ammonia or primary, secondary or tertiary C1-C4- amines or C1-C4-hydroxylamines, such as urea, urethane, acetamide, N-methylacetamide, N,N-diethylacetamide, N,N-dimethylacetamide, lower aliphatic amines and diamines having 2-6 carbon atoms, such as ethylenediamine, hydroxyethyl theophylline, tromethamine (for example, 0.1% to 20% aqueous solution), aliphatic amino acids.

[0053] In some embodiments of the application, in the preparation of the disclosed liquid dosage forms, solubilizers and emulsifiers can be included, for example, the following non-limiting examples can be used: polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phospholipids such as lecithin, gum arabic, gum tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated glyceryl trioleate, linoleylated glyceryl trioleate, polyethylene oxide condensates of fatty alcohols, alkylphenols or fatty acids or 1-methyl-3-(2-hydroxyethyl)imidazolidinone-(2). In this context, polyoxyethylation means that the substance in question contains a polyoxyethylene chain, the degree of polymerization of which is usually between 2 and 40, in particular between 10 and 20. Polyoxyethylated substances of this type can be obtained, for example, by reacting a hydroxyl-containing compound (for example, a monoglyceride or diglyceride or an unsaturated compound, such as those containing an oleic acid group) with ethylene oxide (for example, 40 moles of ethylene oxide per 1 mole of glyceride). Examples of glyceryl trioleate are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil.

[0054] In some embodiments of the present application, the liquid dosage form can also contain preservatives, stabilizing agents, buffering agents, flavoring agents, sweetening agents, coloring agents, antioxidants and complexing agents, etc. Complexing agents that can be considered, for example, are chelate-forming agents such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid and salts thereof.

[0055] In some embodiments of the present application, it can be necessary to stabilize the liquid dosage form at a pH range of about 6 to 9 with physiologically acceptable bases or buffers. It is preferable to have a pH value as neutral or weakly basic as possible (up to pH 8).

[0056] In some embodiments of the present application, to enhance the solubility and / or stability of the disclosed compounds of the disclosed liquid dosage form, parenteral injection dosage form or intravenous injectable dosage form, it can be advantageous to use a-, b- or g-cyclodextrin or derivatives thereof, in particular hydroxyalkyl substituted cyclodextrins, such as 2-hydroxypropyl- b-cyclodextrin or sulfobutyl-b-cyclodextrin. Co-solvents such as alcohols can also improve the solubility and / or stability of the compounds according to the present disclosure in pharmaceutical compositions.

[0057] In some embodiments of the present application, the disclosed liquid dosage form, parenteral injection dosage form or intravenous injectable dosage form can also contain liposomal delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine.

[0058] In some embodiments of the present application, the pharmaceutical composition is suitable for injection, e.g. parenteral administration, e.g. intravenous, intramuscular or subcutaneous administration. The pharmaceutical composition for injection can be prepared as a solution or suspension of the active compound in water. Suitable surfactants such as hydroxypropylcellulose can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Moreover, preservatives can be included to prevent the harmful growth of microorganisms.

[0059] In some embodiments of the present application, the pharmaceutical composition suitable for parenteral administration can include sterile aqueous or nonaqueous solutions, suspensions, or emulsions. Moreover, the composition can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable dosage form is sterile and must be fluid when used in a syringe; the pharmaceutical composition should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0060] For example, injectable solutions can be prepared in which the carrier comprises saline, dextrose, or a mixture of saline and dextrose. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents, and the like can be employed. In some aspects, the disclosed parenteral formulations can comprise about 0.01 M to 0.1 M, such as about 0.05 M, of a phosphate buffer. In another aspect, the disclosed parenteral formulations can comprise about 0.9% saline.

[0061] In some embodiments of the present application, the disclosed parenteral pharmaceutical compositions can comprise a pharmaceutically acceptable carrier, such as aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, but are not limited to, water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as antimicrobials, antioxidants, agents for the adjustment of tonicity, inert gases, and the like can also be present. In another aspect, the disclosed parenteral pharmaceutical compositions can contain a small amount of additives such as substances that enhance isotonicity and chemical stability, for example, buffers and preservatives. For injectable pharmaceutical compositions, formulations in solid form that will be converted to liquid form just prior to use are also contemplated. In addition, other adjuvants can be included to make the formulation isotonic with the blood of the subject or patient.

[0062] It should be noted that although the therapeutic effect of PQQ combined with VK3 is only listed in the examples of the present application, other quinone compounds combined with PQQ for anti-NQO1 high expression malignant tumors are also within the protection scope of the examples of the present application.

[0063] Experimental materials: 1. Experimental cell strains: A549 human non-small cell lung cancer cell line, HepG2 human hepatoma cell line and HeLa human cervical cancer cell line were purchased from ATCC cell bank in the United States. HT-29 human colon cancer cell line, MDA-MB-231 human breast cancer cell line, U87 human brain astrocytoma cell line, U251 human brain astrocytoma cell line and SW1990 human pancreatic cancer cell line were purchased from Shanghai Cell Bank of Chinese Academy of Sciences. NCI-H1975 human non-small cell lung adenocarcinoma cells were donated by Professor Lujinjian of Chinese Medicine Research Institute of University of Macau. MIA PaCa-2 human pancreatic ductal carcinoma cell line, AsPC-1 human metastatic pancreatic adenocarcinoma cells and PANC-1 human pancreatic cancer cell line were donated by Professor Chen Qiang of Health Science College of University of Macau. HCC1937 human breast cancer cell line was donated by Professor Deng Chixia of Health Science College of University of Macau. PC-3 human prostate cancer cell line was donated by Professor Wang Ying of Chinese Medicine Research Institute of University of Macau.

[0064] 2. Cell culture and NQO1 protein expression: A549, NCI-H1975, HCC1937 and HeLa cells were routinely cultured with complete medium (RPMI-1640 medium containing 10% FBS and 1% double-antibiotic). AsPC-1, SW1990, MDA-MB-231, PANC-1, HepG2, U251 and U87 cells were routinely cultured with complete medium (DMEM medium containing 10% FBS and 1% double-antibiotic). MIA PaCa-2 cells were routinely cultured with complete medium (DMEM medium containing 10% FBS, 2.5% horse serum and 1% double-antibiotic). PC-3 cells were routinely cultured with complete medium (F-12K medium containing 10% FBS and 1% double-antibiotic). Cell culture was carried out at 37℃, 5% CO2. The expression level of NQO1 in the above tumor cell lines was detected by Western Blotting method.

[0065] 3. Preparation of drug mother liquor: PQQ (purchased from Shanghai Microlin Biochemical Technology Co., Ltd., purity >98%), vitamin K3 and vitamin K4 (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity >98%), β-LAP, DMNQ (purchased from Selleck Chemicals Co., Ltd., purity >98%), KL1333 and Dunnione (purchased from Shanghai Pharmaron New Drug Development Co., Ltd., purity >98%), MAM (purchased from Pharmalink (Wuhan) Chemical Technology Co., Ltd., purity >97%), TSA and TSB (purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., purity >98%) powders were weighed and dissolved in DMSO solution to a final concentration of 10 mmol.

[0066] Example 1 This example tests the killing effect of PQQ combined with quinone compounds on NQOl high expression cell line MIA PaCa-2. The specific process is: using MTT detection method to detect the effect of PQQ combined with different concentrations of VK3, VK4, MAM, KL1333, β-LAP, DMNQ, Dunnione, TSA and TSB on the cell viability of NQOl high expression pancreatic cancer cells MIA PaCa-2.

[0067] MTT test steps: take the logarithmic growth period cells according to the density of 9x10 3 Cells / well in 96-well plates, 5% CO2, 37℃ incubate overnight. When drug treatment, MIA PaCa-2 cells use DMEM medium containing 1% FBS. 2.5 μM PQQ combined with different concentrations of VK3 (0.625 μM, 1.25 μM, 2.5 μM), VK4 (1.25 μM, 2.5 μM, 5 μM), MAM (0.625 μM, 1.25 μM, 2.5 μM), KL1333 (0.625 μM, 1.25 μM, 2.5 μM), β-LAP (0.625 μM, 1.25 μM, 2.5 μM), DMNQ (0.625 μM, 1.25 μM, 2.5 μM), Dunnione (0.25 μM, 0.5 μM, 1 μM), TSA (1.25 μM, 2.5 μM, 5 μM), TSB (1.25 μM, 2.5 μM, 5 μM), after 24 h treatment, aspirate the cell culture solution, add 20 μL MTT solution (5 mg / mL) per well, continue to incubate in 37℃ incubator for 4 h, aspirate the MTT solution, add 100 μL DMSO solution per well to dissolve the formazan crystals, incubate at room temperature for 10 min, use the enzyme label instrument to detect the absorbance value at 570 nm, calculate the cell viability. The detection results are shown in Figure 1 . Among them, Figure 1 MTT results of A, B, C and D in which A is PQQ combined with VK3, VK4 and MAM; B is PQQ combined with KL1333 and β-LAP; C is PQQ combined with DMNQ and Dunnione; D is PQQ combined with TSA and TSB.

[0068] According to Figure 1It can be seen that PQQ, VK3, VK4, MAM, DMNQ, TSA, TSB alone have no significant toxicity to MIA PaCa-2, but after combination, they show significant killing effect on MIA PaCa-2. KL1333, β-LAP and Dunnione show certain cytotoxicity when used alone, and PQQ combined with them significantly enhances their killing effect on MIA PaCa-2. This shows that in NQO1 high expression pancreatic cancer cell MIA PaCa-2, PQQ combined with Figure 1 The combination of traditional Chinese medicines shows Coalism, synergism or synergistic effect.

[0069] Example 2 This example studies the toxicity of PQQ combined with VK3 on NQO1 high expression lung cancer cells. The specific process is: the effect of PQQ combined with VK3 on the cell viability of lung cancer cell lines (A549 and NCI-H1975 cells) is detected by MTT detection method.

[0070] MTT test steps: take the logarithmic growth period cells, inoculate in 96-well plates at a density of 8×10 3 Cells / well, 5% CO2, 37℃ incubate overnight. In 1% FBS RPMI-1640 medium, PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) is combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM), and after 24 h treatment, the cell culture solution is aspirated, 20 μL MTT solution (5 mg / mL) is added to each well, and incubation is continued in a 37℃ incubator for 4 h. The MTT solution is aspirated, 100 μL DMSO solution is added to each well to dissolve the formazan crystals, and incubation is carried out at room temperature for 10 min. The absorbance value at 570 nm is detected by an enzyme-labeled instrument, and the cell viability is calculated. The detection results are shown in Figure 2 . The upper graph shows the protein expression results of NQO1 in A549 and NCI-H1975 cells; the lower graph shows the MTT detection results of PQQ combined with VK3 on A549 (A) and NCI-H1975 (B) cells.

[0071] According to Figure 2 It can be seen that PQQ or VK3 alone has no significant toxic effect on the cell viability of A549 and NCI-H1975 cells. After PQQ is combined with VK3, the toxicity to A549 and NCI-H1975 cells is significantly enhanced. This shows that PQQ combined with VK3 on NQO1 expressing lung cancer cells shows Coalism effect.

[0072] Example 3 This example investigates the toxicity of PQQ combined with VK3 on pancreatic cancer cells. The procedure is as follows: the effect of PQQ combined with VK3 on the viability of pancreatic cancer cell lines (MIA PaCa-2, AsPC-1, SW1990 and PANC-1 cells) is detected by MTT assay.

[0073] The MTT assay is basically the same as the operation provided in Experimental Example 2, except that: logarithmic growth phase cells are inoculated at a density of 9 x 104 3 Cells are inoculated in 96-well plates and cultured overnight at 37°C in 5% CO2. When drug treatment is performed, MIA PaCa-2, AsPC-1, SW1990 and PANC-1 cells are treated with PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM) in DMEM medium containing 1% FBS. The detection results are shown in Figure 3 . The upper panel shows the expression of NQO1 in different pancreatic cancer cell lines, and the lower panel shows the MTT detection results of PQQ combined with VK3 in MIA PaCa-2 (A), AsPC-1 (B), SW1990 (C) and PANC-1 (D) cells.

[0074] As can be seen from Figure 3 , in MIA PaCa-2, AsPC-1 and SW1990 cells with high expression of NQO1, PQQ or VK3 alone has no significant effect on cell viability, but PQQ combined with VK3 produces significant cytotoxicity. In PANC-1 cells with no significant expression of NQO1, PQQ or VK3 alone, as well as PQQ combined with VK3, has no significant effect on cell viability. This indicates that PQQ combined with VK3 in NQO1 high expression pancreatic cancer cells presents Coalism effect, while has no effect on NQO1 non-expression pancreatic cancer cells.

[0075] Example 4 This example investigates the toxicity of PQQ combined with VK3 on breast cancer cells. The procedure is as follows: The MTT assay is basically the same as the operation provided in Experimental Example 2, except that: when drug treatment is performed, HCC1937 cells are treated with PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM) in RPMI-1640 medium containing 1% FBS, and MDA-MB-231 cells are treated with PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM) in DMEM medium containing 1% FBS. The detection results are shown in Figure 4. Wherein, the upper graph is the expression result of NQO1 in different breast cancer cells; the lower graph is the MTT detection result of PQQ and VK3 combined in HCC1937 (A) and MDA-MB-231 (B) cells.

[0076] According to Figure 4 It can be seen that in the HCC1937 cells with high expression of NQO1, PQQ or VK3 alone has no significant effect on cell viability, and the combination of PQQ and VK3 produces significant cytotoxicity. In MDA-MB-231 cells with no significant expression of NQO1, PQQ or VK3 alone, and the combination of PQQ and VK3, have no significant effect on cell viability. This shows that PQQ and VK3 in NQO1 high expression breast cancer cells present Coalism effect, and have no effect on NQO1 non-expression breast cancer.

[0077] Example 5 This example studies the toxicity of PQQ and VK3 on NQO1 high expression glioma cells. The specific process is: using MTT detection method to detect the effect of PQQ and VK3 combined on the cell viability of NQO1 high expression glioma cell lines (U87 and U251 cells).

[0078] The MTT detection method is basically the same as the operation provided in Example 2, the difference is that U87 and U251 cells use DMEM medium containing 1% FBS, and PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) is combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM). The detection result is shown in Figure 5 . Wherein, the upper graph is the expression result of NQO1 in different brain glioma cell lines; the lower graph is the MTT detection result of PQQ and VK3 combined in U87 (A) and U251 (B) cells.

[0079] According to Figure 5 It can be seen that in the U87 and U251 cells with high expression of NQO1, PQQ or VK3 alone has no significant effect on cell viability, and the combination of PQQ and VK3 produces significant cytotoxicity. This shows that PQQ and VK3 in NQO1 high expression glioma cells present Coalism effect.

[0080] Example 6 This example studies the toxicity of PQQ and VK3 on NQO1 high expression hepatocellular carcinoma cells, cervical cancer cells, colorectal cancer cells and prostate cancer cells. The specific process is: The MTT detection method is basically the same as the operation provided in Example 2, except that when the drugs are treated, HepG2 and HeLa cells use DMEM medium containing 1% FBS, HT-29 cells use 5A medium containing 1% FBS, and PC-3 cells use F-12K medium containing 1% FBS. PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) is combined with different concentrations of VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM). The detection results are shown in Figure 6 , wherein the upper graph is the expression of NQO1 in different cancer cells; the lower graph is the MTT detection results of PQQ and VK3 combined in HepG2 (A), HeLa (B), HT-29 (C) and PC-3 (D) cells.

[0081] According to Figure 6 , it can be seen that PQQ or VK3 alone has no significant effect on cell viability, and the combination of PQQ and VK3 produces significant cytotoxicity. This indicates that PQQ and VK3 exhibit Coalism effect in liver cancer cells, cervical cancer cells, colorectal cancer cells and prostate cancer cells with high expression of NQO1.

[0082] Example 7 This example calculates the combination index of PQQ and VK3 on NQO1 high expression MIA PaCa-2 cells, and the specific process is as follows: The effect of PQQ and VK3 combined on the cell viability of NQO1 high expression MIA PaCa-2 cells in Example 3 is converted into inhibition rate for calculation, and the combination effect of PQQ and VK3 is evaluated based on the inhibition rate of combination, respectively using combination index CI (Combination Index) value and HSA (Highest Single Agent) synergy score. Among them, the combination index CI is calculated by CompuSyn 1.0 software, and the HSA synergy score is calculated by Synergy Finder 3.0 tool. The calculation results are shown in Figure 7 , wherein A is the inhibition rate of PQQ and VK3 combined on MIA PaCa-2 cells, B is the calculation of combination index CI, and C is the HSA synergy model score.

[0083] According to Figure 7It can be seen that the combination index of PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and VK3 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM) at different concentrations is generally less than 0.5, and is concentrated near 0.1 ~ 0.3. The lower the CI value, the stronger the synergistic effect (CI value less than 1 is synergistic effect, wherein CI value less than 0.3 is strong synergy; CI value equal to 1 is additive effect; CI value greater than 1 is antagonistic effect). The results show that PQQ and VK3 combined on MIA PaCa-2 cells show very strong multi-synergistic effect.

[0084] In addition, the overall effect of PQQ and VK3 combination was evaluated by using HSA synergy model. The results show that the overall HSA synergy score of PQQ and VK3 combination is as high as 55.526 points in this concentration range, indicating that PQQ and VK3 combined on MIA PaCa-2 cells show strong synergistic effect (HSA score greater than 10 is synergistic effect, HSA score between -10 and 10 is additive effect, and HSA score less than -10 is antagonistic effect).

[0085] Example 8 This example studies the dependence of PQQ and VK3 combination effect on NQO1, the influence on NQO1 enzyme activity and the influence on ROS generation in NQO1 high expression cells. The specific process is: the influence of PQQ and VK3 combination on the viability of pancreatic cancer cell line MIA PaCa-2 shNC (control group) and MIA PaCa-2 shNQO1 cells stably knocked down NQO1 was detected by MTT detection method.

[0086] The MTT detection method is basically the same as the operation provided in Example 2, the difference is that during drug treatment, PQQ (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) is combined with different concentrations of VK3 (0 μM, 0.25 μM, 0.5 μM, 1 μM).

[0087] NQO1 activity detection step: dissolve the drug in 100 μL ddH2O according to the experimental concentration, then prepare 100 μL reaction solution, which includes human NQO1 recombinant protein (50 ng), FAD (5 μM), NADH (200 μM), dissolved in Tris-HCl buffer. Mix the drug solution and the reaction solution into a 200 μL reaction system, detect the maximum absorption value of NADH at 340 nm, and monitor the absorption value change within 20 min to reflect the change of NQO1 catalytic activity. Among them, DMSO, PQQ (5 μM), VK3 (5 μM), Comb. (5 μM PQQ + 5 μM VK3), Comb. + DIC (5 μM PQQ + 5 μM VK3 + 5 μM DIC), β-LAP (5 μM) are grouped.

[0088] ROS generation detection step: take the logarithmic growth phase cells according to the density of 15×10 4 Cells were seeded in 12-well plates at 5% CO2, 37°C overnight. When the drug was treated, MIA PaCa-2 cells used DMEM medium containing 1% FBS. Before drug treatment, load ROS fluorescent probe DCFH-DA and incubate for 30 min. When the drug was treated, PQQ (2.5 μM) and VK3 (2.5 μM) were treated for 4 h respectively, Comb. group (2.5 μM PQQ + 2.5 μM VK3) was treated for 1 h, 2 h and 4 h respectively, Comb. + DIC (5 μM) was treated for 4 h, Comb. + NAC (1 mM) was treated for 4 h, and tert-butyl hydrogen peroxide (TBHP) was treated for 30 min. After incubation, wash with PBS for 3 times, trypsinize, terminate digestion with complete medium, and detect with flow cytometry FITC-A channel. NAC is N-acetylcysteine, a commonly used ROS scavenger.

[0089] The detection results are shown in Table 1 Figure 8 , wherein A is the combined MTT detection result of PQQ and VK3 combination on the control group MIA PaCa-2 shNC; B is the combined MTT detection result of PQQ and VK3 combination on the MIA PaCa-2 shNQO1 cells stably knocked down NQO1; C is the effect of PQQ, VK3 alone and combination on NQO1 enzyme activity; D is the effect of PQQ, VK3 alone, combination and DIC, NAC on ROS generation in NQO1 high expression MIA PaCa-2 cells.

[0090] According to the above results, it can be seen that PQQ and VK3 can effectively inhibit the growth of MIA PaCa-2 cells, and the combination of PQQ and VK3 has a better effect than PQQ and VK3 alone. The combination of PQQ and VK3 can effectively inhibit the growth of MIA PaCa-2 cells, and the combination of PQQ and VK3 has a better effect than PQQ and VK3 alone. The combination of PQQ and VK3 can effectively inhibit the growth of MIA PaCa-2 cells, and the combination of PQQ and VK3 has a better effect than PQQ and VK3 alone. Figure 8It can be seen that the combination of the two shows a significant killing effect on MIA PaCa-2 shNC cells in the control group (high expression of NQO1); while in the MIA PaCa-2 shNQO1 cells stably knocked down NQO1, the effect of the combination of PQQ and VK3 completely disappears, which indicates that the killing effect of the combination of PQQ and VK3 on cells is highly dependent on NQO1. In the NQO1 enzyme activity experiment results, PQQ or VK3 alone has no significant effect on NQO1 enzyme activity, while after being combined, NQO1 is significantly activated, and this effect can be inhibited by the specific inhibitor DIC of NQO1, suggesting that the combination of the two can play a role by activating NQO1 enzyme activity. In MIA PaCa-2 cells, the combination of PQQ and VK3 promotes the generation and accumulation of ROS in a time-dependent manner, while the antioxidants NAC and NQO1 inhibitor DIC can significantly reduce the generation of ROS induced by the combination of the two, which indicates that the ROS generated by the combination of PQQ and VK3 is dependent on NQO1.

[0091] In summary, the combined effect of PQQ and VK3 depends on NQO1, which is manifested as significantly activating NQO1 enzyme activity and inducing a large amount of ROS generation in NQO1 high expression cells.

[0092] Example 9 This example studies the killing effect of the combination of PQQ and VK3 on NQO1 high expression MIA PaCa-2 cells, and the specific process is: using MTT, LDH release and PI staining detection methods to detect the effects of PQQ (2.5 μM), VK3 (2.5 μM), the combination group Comb. (2.5 μM PQQ + 2.5 μM VK3), the combination + NQO1 inhibitor group (2.5 μM PQQ + 2.5 μM VK3 + 5 μM DIC) on MIA PaCa-2 cell viability and morphology, and the NQO1 inhibitor is dicoumarol (DIC).

[0093] The MTT detection method is basically the same as the operation provided in Example 2, the difference is that: when the drug is treated, MIA PaCa-2 cells use DMEM medium containing 1% FBS. PQQ (2.5 μM), VK3 (2.5 μM), the combination group Comb. (2.5 μPQQ + 2.5 μM VK3), the combination + NQO1 inhibitor group (2.5 μM PQQ + 2.5 μM VK3 + 5 μM DIC) are used to treat cells for 24 h.

[0094] LDH release detection method steps: take the logarithmic growth period cells according to the density of 9×10 3Cells were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2. Cells were treated for 12 h in DMEM medium containing 1% FBS with PQQ (2.5 μM), VK3 (2.5 μM), a combination group (2.5 μM PQQ + 2.5 μM VK3), and a combination group with NQO1 inhibitor (2.5 μM PQQ + 2.5 μM VK3 + 5 μM DIC). One h before the scheduled detection time, 10% of the original culture medium volume of LDH release reagent was added to the control wells for maximum enzyme activity, mixed thoroughly, and incubated for another h. The culture plates were then plated at 400°C. g Centrifuge for 5 min, aspirate 40 μL of supernatant into a new 96-well plate, add 40 μL of LDH detection working solution to each well, mix well, incubate at room temperature in the dark for 30 min, measure the absorbance at 490 nm using a microplate reader, and calculate the LDH release ratio.

[0095] PI staining procedure: Take cells in logarithmic growth phase and stain them at a density of 9 × 10⁻⁶. 3 Cells were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2. Cells were then treated for 12 h in DMEM medium containing 1% FBS with PQQ (2.5 μM), VK3 (2.5 μM), a combination group (2.5 μM PQQ + 2.5 μM VK3), or a combination group with NQO1 inhibitor (2.5 μM PQQ + 2.5 μM VK3 + 5 μM DIC). The original medium was discarded, and 50 μL of PI staining working solution (50 μg / mL) was added to each well. Cells were photographed in the dark after 10 min.

[0096] See test results Figure 9 Among them, A represents the results of cell viability detection by the MTT assay; B represents the results of LDH release detection; and C represents the results of PI staining and morphological photography.

[0097] according to Figure 9 It was found that the use of 2.5 μM PQQ or 2.5 μM VK3 alone had no significant effect on cell viability, LDH release, and the number of PI-positive cells in MIA PaCa-2 cells. However, the combined use of PQQ and VK3 produced significant cytotoxicity to MIA PaCa-2 cells, significantly increasing LDH release and the number of PI-positive cells, and causing significant changes in cell morphology. Co-treatment with the NQO1 inhibitor dicumarol significantly reversed the combined effect of PQQ and VK3.

[0098] Example 10 This example investigates the PQQ and VK3 combined induction of NQO1 high expression tumor cells to undergo NQO1 dependent necrosis and apoptosis. The specific process is as follows: In step 10 of the experiment, according to the method in the 7-AAD / AnnexinV-PE double staining cell apoptosis and necrosis detection kit (purchased from BD Biosciences company), the pancreatic cancer cell MIA PaCa-2 was detected.

[0099] The cell death of 2.5 μM PQQ, 2.5 μM VK3, combined group Comb. (2.5 μM PQQ + 2.5 μM VK3), combined + NQO1 inhibitor group (2.5 μM PQQ + 2.5 μM VK3 + 5 μM DIC), and positive control cisplatin group (100 μM Cisplatin) was detected, respectively. Among them, the PQQ group, the VK3 group, the combined group Comb. and the combined + NQO1 inhibitor group were treated for 12 h, and the positive control cisplatin group was treated for 24 h. Staining was performed according to the instructions in the kit, and then the flow cytometer (BD LSR Fortessa) was used for detection.

[0100] The detection results are shown in Figure 10 . When PQQ or VK3 was used alone, there was no obvious apoptotic cell and necrotic cell population (Q1, Q2 and Q3 area), and a large number of apoptotic cell population (Q2 and Q3 area) was observed in the positive control cisplatin group. In the combined treatment group, a large number of necrotic and apoptotic cell populations (Q1, Q2 and Q3 area) were sorted out, indicating that the combined group induced a large number of necrosis and apoptosis of MIA PaCa-2 cells, and the effect was reversed by NQO1 inhibition dicumarol (Comb. + DIC group), indicating that the effect is highly dependent on NQO1.

[0101] Example 11 This example investigates the anti-tumor effect of PQQ and VK3 in the zebrafish tumor model of xenotransplantation shNC-GFP Mia PaCa-2 cells. The specific process is as follows: Zebrafish animal experiment steps: The shNC-GFP MIA PaCa-2 cells stably expressing green fluorescent protein (GFP) were inoculated into the yolk sac of zebrafish larvae, and 5 μM PQQ, 1 μM VK3, Comb. (5 μM PQQ + 1 μM VK3) combined treatment and 1 μM Gemcitabine (positive drug) were given after 12 h; observe the size of tumor cell mass, take photos with inverted fluorescence microscope after drug treatment for 1 day and calculate the fluorescence area.

[0102] The detection results are shown in Figure 11A is the statistical result of the fluorescence region in the zebrafish experiment; B is the morphological and fluorescence photographing result of the zebrafish experiment of inoculating shNC-GFP MIA PaCa-2 cells.

[0103] According to Figure 11 As shown in A and B, 5 μM PQQ has no significant effect on the growth of shNC-GFP MIA PaCa-2 transplants, while the 1 μM VK3 group partially inhibits the size of the transplanted tumors; the combination group (5 μM PQQ + 1 μM VK3) and the positive drug Gemcitabine group can significantly inhibit the tumor size. This shows that PQQ combined with VK3 shows good synergistic anti-tumor effect in the zebrafish animal experiment in vivo.

[0104] Example 12 This example studies the anti-tumor effect of PQQ combined with VK3 in the NOD-SCID immunodeficient mouse tumor model of xenotransplanted Mia PaCa-2 cells, and the specific process is as follows: 1. Establishing a MIA PaCa-2 cell NOD-SCID mouse subcutaneous pancreatic cancer tumor model SPF level NOD-SCID immunodeficient mice were purchased from the Experimental Animal Center of the University of Macau, 8-12 weeks old, weighing 20-22 g, female mice. Each mouse was subcutaneously injected with 4×10 6 MIA PaCa-2 cells in the PBS suspension and basement glue 1:1 mixed solution. The injection site was observed every 3 days. After two weeks, the number of tumor-bearing mice was counted and caged for subsequent experiments.

[0105] 2. Experimental grouping and drug administration The successfully inoculated mice were randomly divided into Vehicle group (vehicle PBS), PQQ group (10 mg / kg), VK3 group (5 mg / kg), combined low-dose group (Comb. L, 2.5 mg / kg PQQ + 1.25 mg / kg VK3), combined high-dose group (Comb. H, 10 mg / kg PQQ + 5 mg / kg VK3), and positive drug Gemcitabine group (15 mg / kg), 6 mice / group. The first day of drug administration after successful modeling was recorded as D1, and so on. Among them, the Vehicle group, PQQ group, VK3 group, Comb. L group and Comb. H group were administered intraperitoneally once a day, and the Gemcitabine group was administered intraperitoneally once every three days. The specific dose of drug administration was 0.2 mL per mouse for a 20 g mouse, adjusted according to the weight of the mouse, a total of 21 days. The body weight and tumor size of the mice were recorded every two days during the drug administration period. 3. Animal sacrifice, sampling and sample processing After the end of the experiment, the mice were euthanized by carbon dioxide, and the heart, liver, spleen, lung and kidney tissues were taken after dissection and fixed in 4% paraformaldehyde (4% PFA) fixing solution. The subcutaneous tumor tissue of the mice was weighed, photographed and then fixed in 4% PFA fixing solution. The mouse eyeball was taken for blood, and the supernatant was obtained after centrifugation, which was the mouse serum.

[0106] 4. Histopathological detection After the heart, liver, spleen, lung and kidney tissues of the mice were fixed in 4% paraformaldehyde, they were routinely paraffin-embedded, cut into 6μm thick sections, and routinely H&E stained. They were observed, photographed and analyzed under a light microscope.

[0107] 5. Liver function detection The collected mouse serum was detected for ALT and AST levels in the serum according to the methods in the alanine aminotransferase (glutamic-pyruvic transaminase / ALT / GPT) test kit and aspartate aminotransferase (glutamic-oxaloacetic transaminase / AST / GOT) test kit purchased from Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd.

[0108] The detection results are shown in Figure 12 , wherein A-C are tumor size statistics, morphological photographs and tumor weight of tumor-bearing mice, D is the body weight of the mice, and E is the survival curve of the mice.

[0109] The detection results are shown in Figure 13 , A is the H&E staining of the heart, liver, spleen, lung and kidney tissues of tumor-bearing mice, B is the ALT level in the serum of tumor-bearing mice, and C is the AST level in the serum of tumor-bearing mice.

[0110] The results show that the PQQ group and the VK3 group have no significant effect on the tumor growth of the NOD-SCID tumor-bearing mice, while the high-dose and low-dose groups of PQQ combined with VK3, and the positive drug gemcitabine group all show significant anti-tumor effects. During the experiment, gemcitabine caused a significant decrease in the body weight of the tumor-bearing mice, and the general state of the mice was observed to be poor, and the activity of the mice was significantly weakened, and finally the survival rate of the group was only 83%; while the tumor-bearing mice in the combined group had no significant change in body weight, the general state of the mice was good, the activity of the mice was strong and 100% survival, which indicates that compared with the gemcitabine group, the combined group shows higher safety and fewer toxic side effects. In addition, H&E staining observed that the liver tissue of the mice in the gemcitabine group had inflammatory infiltration around the blood vessels, the alveolar structure in the lung tissue was obviously vacuolated, and the levels of liver function indicators ALT and AST were significantly increased, which indicates that gemcitabine has certain toxic side effects on the liver and lungs. The H&E staining of the heart, liver, spleen, lung and kidney tissue sections of the PQQ combined with VK3 group has no obvious lesions, and compared with the blank control group, the levels of ALT and AST also have no obvious increase, which indicates that the combined group has no obvious toxicity to the main organs under the condition of significantly inhibiting tumor growth, which indicates that the safety of PQQ combined with VK3 is good.

[0111] In summary, the embodiments of the present application provide the application of PQQ in the preparation of NQO1 high-expression malignant tumor chemotherapy sensitization drugs, and the compatibility of PQQ and quinones can explosively improve the killing effect of the latter on NQO1 high-expression malignant tumors, and realize the high-efficiency and low-toxicity treatment of NQO1 high-expression tumors. PQQ is a new food raw material, which has been used in the field of food additives and nutritional health products, and has good safety. The high expression of NQO1 in malignant tumors is very common, and there is significant high expression in lung cancer, pancreatic cancer, brain glioma, liver cancer and colorectal cancer. Vitamin K3 is a drug that has been clinically used for many years, and also has certain anti-tumor effect. The anti-tumor research of quinones such as β-LAP has also entered the clinical trial stage. Quinones such as β-LAP have selective killing effect on NQO1 high-expression malignant tumors, but have great toxic side effects, which is the key to limit the dose and drug effect. The chemotherapy sensitization drug prepared by the new food raw material PQQ in the embodiments of the present application is high-efficiency and low-toxicity, and has important significance and wide application prospect in the preparation of NQO1 high-expression malignant tumor treatment drugs or drugs for preventing recurrence. The present application provides a new idea for targeted treatment of NQO1 high-expression malignant tumors, and may provide new technology, new method and new means for clinically efficient treatment of NQO1 high-expression malignant tumors.

[0112] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. Use of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating NQOl high expression malignant tumor sensitization.

2. Use of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and a quinone compound in the preparation of a drug for preventing and / or treating NQOl high expression malignant tumor.

3. Use of pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and a quinone compound in the preparation of at least one drug selected from the group consisting of: (1) a drug for activating NQOl activity in NQOl high expression malignant tumor cells; (2) a drug for increasing ROS production in NQOl high expression malignant tumor cells; (3) a drug for reducing the viability of NQOl high expression malignant tumor cells; (4) a drug for increasing the release of LDH from NQOl high expression malignant tumor cells; (5) a drug for inducing cell membrane damage in NQOl high expression malignant tumor cells; (6) a drug for inducing cell death in NQOl high expression malignant tumor cells; (7) a drug for inhibiting the growth of NQOl high expression malignant tumor cells.

4. A product for use against a malignant tumor with high expression of NQOl, characterized in that: The drug comprises pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and a quinone compound.

5. The use according to claim 2 or 3 or the product against malignant tumors with high NQOl expression according to claim 4, characterized in that: The quinone compound includes at least one of the following substances or pharmaceutically acceptable salts thereof: Vitamin K3, Vitamin K4, KL1333, beta-lapachone, 2,3-dimethoxy-1,4-naphthoquinone, 2-methoxy-1,4-naphthoquinone, 2-methoxy-6-acetyl-7-methyljuglone, shikonin, acetylshikonin, deoxyshikonin, isoshikonin, danshenspirodiol A, danshenspirodiol B, danshenspirodiol C, dunnione, NQ301, NAT1-IN-1, YZ-35, LY5, QD394, NW16, STAT3-IN-4, STAT3-IN-10, STAT3-IN-11, STAT3-IN-14, STAT3-IN-29, STAT3-IN-35, plumbagin, isolplumbagin, 6-hydroxyplumbagin, 3-chloro plumbagin, 3-bromo plumbagin, leucoplumbagin, thymoquinone, tocoquinone, scirpusin, juglone, 7-methyljuglone, 2,3-dimethyljuglone, naphthazarin, fischerin, lapachol, buparvaquone, aristolactam, 4-hydroxyerysodine, deoxylapachone, meissenin, 3-hydroxymeissenin, rhizoxin, isorhizoxin, Lawsone, 1,4-naphthoquinone-2-sulfonic acid potassium, 2-amino-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 2-methoxy-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone, 2,3-dimethyl-1,4-naphthoquinone, 2,7-dimethyl-1,4-naphthoquinone, 2,3-dicyano-1,4-naphthoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 2-amino-3-chloro-1,4-naphthoquinone, 2-benzoylamino-1,4-naphthoquinone, 2-hydroxy-3-methyl-1,4-naphthoquinone, 2-hydroxy-3-methoxy-1,4-naphthoquinone, 2-sodium sulfonate-1,4-naphthoquinone, 5-hydroxy p-naphthoquinone, 6-methyl-1,4-naphthoquinone, 2,3,6-trimethyl naphthoquinone, 7-methyl-5-hydroxy naphthoquinone, beta,beta-dimethylacrylshikonin, 2-amino-3-carboxy-1,4-naphthoquinone, 2-carbamoyl-3-hydroxy-1,4-naphthoquinone, 2,7-dimethoxy-6-(1-acetyloxyethyl)juglone, 6-ethyl-2,7-dimethoxyjuglone, 7-(hydroxymethyl)-2-methyl-1,4-naphthalenedione, 2-hydroxy-6-methoxy-1,4-dihydronaphthalene-1,4-dione, shikonin, aristolochic acid, menaquinone, deoxykaraniphenin, lychnin, xanthotoxol, isatidin, isobutylshikonin, javanicin, KP372-1, lavandustin A, hancoside A, malpuroquinone, mitoquinolone, Pronqodine, Ramentaceone, syriacitrin, SARS-CoV MPro-IN-2, Sarubicin B.

6. The use according to any one of claims 1 to 3 or the product against malignant tumors with high NQOl expression according to claim 4, characterized in that: The NQO1 high-expression malignant tumor includes at least one of non-small cell lung cancer, pancreatic cancer, breast cancer, brain glioma, liver cancer, colorectal cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, esophageal cancer, leukemia, malignant melanoma, kidney cancer, bladder cancer, and head and neck squamous cell carcinoma.

7. The product for use against NQOl-high malignant tumors according to claim 4, characterized in that: The product is a pharmaceutical composition, and further includes a pharmaceutically acceptable excipient.

8. The product for the treatment of malignant tumor with high expression of NQOl according to claim 4, characterized by: The molar ratio of the pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof and the quinone compound is 10:1 to 1:

10.

9. The product for use against NQOl-high malignant tumors according to claim 7, characterized in that: The dosage form of the pharmaceutical composition includes any one of a tablet, a capsule, a pill, a granule, an oral solution, an oral suspension, a syrup, an injection solution, a sterile powder for injection, a concentrated solution for injection, a suppository, an inhalation preparation, an aerosol, a powder mist, or a spray.

10. The product for use against NQOl-high malignant tumors according to claim 4, characterized in that: The product is a kit, and the use concentration of the pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof in the kit is 0.25 to 50 μM.

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