WO2022237293A1 - 烟酰胺单核苷酸及其在抗肿瘤药物心肌损伤中的保护应用 - Google Patents

烟酰胺单核苷酸及其在抗肿瘤药物心肌损伤中的保护应用 Download PDF

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WO2022237293A1
WO2022237293A1 PCT/CN2022/080080 CN2022080080W WO2022237293A1 WO 2022237293 A1 WO2022237293 A1 WO 2022237293A1 CN 2022080080 W CN2022080080 W CN 2022080080W WO 2022237293 A1 WO2022237293 A1 WO 2022237293A1
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drug
nmn
nicotinamide mononucleotide
anthracycline
caused
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PCT/CN2022/080080
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French (fr)
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邢东明
原阳
邹林峰
高远真
李梦娇
卢琦
叶婷
张钰坤
柳欣林
张仁帅
王超
钟英杰
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青岛大学附属医院
青岛肿瘤研究院
清华大学
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

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  • the invention belongs to the technical field of drugs for preventing and treating cardiotoxicity of chemotherapeutic drugs, and specifically relates to nicotinamide mononucleotide and its protective application in myocardial injury caused by anti-tumor drugs.
  • anthracycline chemotherapy drugs still have cornerstone value in the chemotherapy of breast cancer, lymphoma, head and neck cancer, multiple myeloma and other tumors.
  • Clinically commonly used anthracyclines such as doxorubicin (doxorubicin), daunorubicin, aclarubicin, idarubicin, epirubicin, etc., all contain a tetracyclic ring as follows Chromophores that differ only in the substituents on the tetracyclic chromophores. Among them, the most classic and representative drug is doxorubicin (doxorubicin).
  • anthracycline chemotherapy drugs are constantly being introduced, such as epirubicin, pirarubicin, daunorubicin, aclarubicin, idarubicin, etc.
  • cardiotoxicity is still a common adverse reaction of anthracyclines
  • the minimum cumulative doses of doxorubicin and epirubicin causing chemotherapy-induced heart failure are: 500mg/m 2 and 1000mg/m 2 (Epirubicin versus doxorubicin: which is the anthracycline of choice for the treatment of breast cancer? [J]. Clin Breast Cancer, 2003, 4 Suppl 1 (S26-33.).
  • anthracycline chemotherapy drugs is consistent and the active groups are close (Theoretical ground for adsorptive therapy of anthracyclines cardiotoxicity[J] .Exp Oncol,2012,34(4):314-322.).Meanwhile, the common mechanism of anthracycline cardiotoxicity is equal to oxygen free radical generation, apoptosis etc. (Irbesartan suppresses cardiac toxicity induced by doxorubicin via regulating the p38-MAPK/NF-kappaB and TGF-beta1 pathways[J].
  • Dexrazoxane is currently the only drug included in clinical guidelines for the treatment of myocardial injury caused by anthracyclines. It is expensive, has a large dosage (the recommended dosage is 5-10 times that of doxorubicin), and tends to aggravate myelosuppression. More stringent control over medication. Therefore, it is very important to develop a new generation of cardioprotective drugs.
  • Nicotinamide mononucleotide is an exogenous supplementary form of cellular oxidized nicotinamide adenine dinucleotide (NAD + ), which essentially consists of the nitrogenous base nicotinamide and the C-1 position of D-ribose Nucleotides with ⁇ -N-glycosidic linkages.
  • NAD + nicotinamide adenine dinucleotide
  • Oral administration is a commonly used safe method at present. Long-term intake of NMN for 60kg adults is safe at 200-300mg/day. The daily upper limit recommended by WHO is 900mg.
  • the invention provides the application of nicotinamide mononucleotide as an anthracycline antitumor drug for protecting myocardial injury.
  • Nicotinamide mononucleotide exerts its physiological functions by converting into NAD + in the human body, such as activating NAD + substrate-dependent enzyme Sirtuins (histone deacetylase, also known as sirtuin), regulating cell survival and death, maintain the redox state.
  • Sirtuins histone deacetylase, also known as sirtuin
  • NMN has a protective effect on myocardial injury caused by anthracycline chemotherapy drugs such as doxorubicin (Dox), and reduces the side effects of Dox in treating tumors.
  • Dox doxorubicin
  • the invention provides the protective application of nicotinamide mononucleotide in myocardial injury caused by anti-tumor drugs.
  • the present invention also provides the application of nicotinamide mononucleotide in the prevention and/or treatment of cardiotoxicity of anthracycline antineoplastic drugs.
  • the antineoplastic drug includes at least one of doxorubicin, daunorubicin, aclarubicin, idarubicin, epirubicin or mitoxantrone.
  • the present invention is used for preparing medicines, medicine mixtures and medicine compositions for inhibiting myocardial injury caused by antineoplastic drugs.
  • the drug, drug mixture or drug composition includes nicotinamide mononucleotide.
  • the present invention also provides a medicine, a medicine mixture or a medicine composition, the active ingredient of which comprises nicotinamide mononucleotide,
  • the drug, drug mixture or pharmaceutical composition has at least one function in the following 1)-5):
  • the drug, drug mixture or pharmaceutical composition is in any pharmaceutically acceptable dosage form, including at least one of tablet, capsule, injection, granule, suspension and solution.
  • nicotinamide mononucleotide (NMN, ⁇ -Nicotinamide Mononucleotide) is the product of nicotinamide phosphoribosyltransferase reaction and one of the key precursors of NAD + .
  • NMN is produced by nicotinamide (Nicotinamide, Nam) under the catalysis of Nampt, and then NMN is catalyzed by nicotinamide mononucleotide adenylyltransferase to generate NAD + .
  • Extracellular NMN needs to be dephosphorylated and transformed into nicotinamide riboside (NR) to enter the interior of liver cells.
  • NR nicotinamide riboside
  • NR nicotinamide riboside kinase 1
  • NMN NMN
  • ATP nicotinamide riboside kinase 1
  • NMN exerts its physiological functions by converting into NAD + in the human body, such as activating NAD + substrate-dependent enzyme Sirt1, regulating cell survival and death, maintaining redox state, etc.
  • NMN In the experiment of NMN interfering with doxorubicin-induced myocardial injury in H9c2 cardiomyocytes, it was found that NMN increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH) better than dexrazoxane; Hydrogenase (LDH) production and malondialdehyde (MDA) levels were also superior to dexrazoxane; inhibition of reactive oxygen species (ROS) production was comparable to dexrazoxane.
  • SOD superoxide dismutase
  • GSH glutathione peroxidase
  • MDA malondialdehyde
  • ROS reactive oxygen species
  • NMN is superior to dexrazoxane in improving myocardial injury-related characteristics, the cost of raw materials is low, and there are already marketed oral products. Therefore, it is a potential candidate drug for improving myocardial injury caused by anthracyclines, and the new value of NMN has also been discovered application.
  • Fig. 1 is the comparison chart of the influence of NMN on the survival rate of doxorubicin and its analogue epirubicin (EPI) injury cardiomyocyte in embodiment 1, wherein, C: blank control group; DO: model group 1; EP: Model group 2; DO+DE: Positive control group; &: Compared with the control group, there is a statistical difference between the model group, P ⁇ 0.05; *: Compared with the model group 1 (DOX), there is a statistical difference, P ⁇ 0.05; #: Compared with model group 2 (EPI), P ⁇ 0.05;
  • Figure 3 is a comparison chart of the effect of NMN on the LDH leakage rate of adriamycin-injured cardiomyocytes in Example 2, wherein, C: blank control group; **: compared with the model group, P ⁇ 0.05;
  • Figure 4 is a comparison chart of the effect of NMN on MDA of cardiomyocytes injured by doxorubicin in Example 3, wherein, C: blank control group; **: compared with the model group, P ⁇ 0.05;
  • Figure 5 is a comparison chart of the effect of NMN on the ROS of adriamycin-injured cardiomyocytes in Example 4, wherein, Control: blank control group; **: compared with the model group, P ⁇ 0.05;
  • Figure 6 is a comparison chart of the effect of NMN on the ATP level of adriamycin H9c2 cardiomyocytes in Example 5; wherein, C: blank control group; **: compared with the model group, P ⁇ 0.05;
  • Figure 8 is a comparison chart of the influence of NMN on the mitochondrial membrane potential of adriamycin H9c2 cardiomyocytes in Example 6; wherein, C: blank control group; **: compared with the model group, P ⁇ 0.05;
  • Figure 10 is a comparison chart of the effect of NMN on the apoptosis of adriamycin H9c2 cardiomyocytes in Example 7; wherein, C: blank control group; **: compared with the model group, P ⁇ 0.05.
  • the anthracycline antibiotics described in the present invention preferably include one of doxorubicin, daunorubicin, aclarubicin, idarubicin, epirubicin or mitoxantrone, in the embodiments of the present invention as Doxorubicin and epirubicin were used as examples to carry out corresponding experiments.
  • the H9c2 cell line is a rat cardiomyocyte, which was purchased from the ATCC cell bank in the United States. H9c2 cells were cultured in complete cardiomyocyte medium solution at a temperature of 37° C. in an incubator containing 95% air and 5% carbon dioxide (CO 2 ).
  • Cardiomyocyte complete culture medium solution the volume ratio is 89% DMEM+10% FBS (FBS: fetal bovine serum, purchased from the company Dalian Meilun Biotechnology Co., Ltd.; item number PWL001)+1% double antibody (double antibody: penicillin/chain Mycin solution was purchased from the company Dalian Meilun Biotechnology Co., Ltd.; product number MA0110).
  • FBS fetal bovine serum, purchased from the company Dalian Meilun Biotechnology Co., Ltd.; item number PWL001
  • double antibody double antibody: penicillin/chain Mycin solution was purchased from the company Dalian Meilun Biotechnology Co., Ltd.; product number MA0110
  • Dexrazoxane purchased from the company Hubei Weideli Chemical Technology Co., Ltd.; product number Y694.
  • Nicotinamide mononucleotide chemical formula C 11 H 15 N 2 O 8 P, CAS number 1094-61-7.
  • DCFH-DA active oxygen ROS fluorescent probe purchased from the company Dalian Meilun Biotechnology Co., Ltd.; product number MB4682; dosage 1ml; incubation time 15-60min; fluorescence band: excitation wavelength 504nm, emission wavelength 529nm.
  • CCK-8 kit was purchased from the company Dalian Meilun Biotechnology Co., Ltd.; product number MA0218-L-10000T.
  • the LDH detection kit was purchased from the company Beijing Suolaibao Technology Co., Ltd.; item number BC0685.
  • TUNEL Cell Apoptosis Detection Kit was purchased from the company Shanghai Biyuntian Biotechnology Co., Ltd.; product number C1088.
  • ATP detection kit was purchased from the company Shanghai Biyuntian Biotechnology Co., Ltd.; item number, S0026B.
  • Lipid oxidation (MDA) detection kit was purchased from the company Shanghai Biyuntian Biotechnology Co., Ltd.; item number S0131S.
  • Mitochondrial Membrane Potential Detection Kit (JC-1) was purchased from the company Beijing Suo Laibao Technology Co., Ltd.; product number M8650-100T.
  • H9c2 cells were exposed to doxorubicin (DOX) concentrations of 0.5/1/2/10/20 ⁇ M, and the optimal concentration of doxorubicin was selected as 5 ⁇ M for subsequent experiments.
  • H9c2 cells were exposed to 5 ⁇ M Dox+20 ⁇ M Dextra and 5 ⁇ M Dox+20 ⁇ M NMN for 1 day. Cells treated with DMEM were used as blank control, and cells treated with dexrazoxane were used as positive control.
  • DOX doxorubicin
  • the myocardial injury model group was given adriamycin 5 ⁇ M
  • the positive control group was given dexrazoxane 20 ⁇ M
  • the NMN treatment group was given NMN 20 ⁇ M
  • the other two groups were given 5 ⁇ M DOX+20 ⁇ M Dexra and 5 ⁇ M DOX+20 ⁇ M NMN, respectively.
  • Example 1 the protective effect of NMN on H9c2 cardiomyocyte injury caused by doxorubicin and its analogs
  • H9c2 cardiomyocytes were diluted with medium to 10 6 /ml cell suspension, and inoculated in 96-well plate, 100 ⁇ L of cell suspension was added to each well, cultured for 24 hours, and then treated with drugs.
  • CCK-8 cell viability was used to measure the absorbance at 450 nm using a microplate reader to calculate the cardiomyocyte activity. Cell status was observed by an inverted light microscope.
  • NMN has a protective effect against two typical anthracyclines, and has a better protective effect against epirubicin, which is less cardiotoxic, and significantly improves cell survival.
  • NMN reduces the impact of doxorubicin H9c2 cardiomyocyte lactate dehydrogenase (LDH) leakage
  • the LDH in the cells of the control group was 100%.
  • the difference between the normal control group is the LDH release rate, and 1 ⁇ mol of pyruvate produced in the reaction system is regarded as 1 unit, calculated per unit/mg protein.
  • the LDH detection kit was used for determination. The results showed ( Figure 3) that the leakage rate of LDH in the different medication groups was significantly lower than that in the model group.
  • Example 3 NMN inhibits the influence of doxorubicin H9c2 cardiomyocyte lipid peroxidation level
  • MDA lipid peroxide malondialdehyde
  • TSA thiobarbituric acid
  • Embodiment 4 the influence of NMN on the reactive oxygen species (ROS) level of doxorubicin H9c2 cardiomyocytes
  • the fluorescent probe DCFH-DA freely passes through the cell membrane, and after entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH. However, DCFH cannot permeate the cell membrane, so the probe can be easily loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. The level of reactive oxygen species in cells can be known by detecting the fluorescence of DCF. Using the same treatment method as in Example 2, after culturing for 24 hours, each group was loaded with DCFH-DA probes, photographed with a laser confocal microscope, and the image was processed with ImageJ image processing software ( FIG. 5 ).
  • Embodiment 5 the influence of NMN on the ATP level of adriamycin H9c2 cardiomyocytes
  • ATP plays an important role in various physiological and pathological processes of cells. Changes in ATP levels can affect cell function. Usually, the ATP level will decrease when the cells are in apoptosis, necrosis or in some toxic state. The same treatment method was adopted as in Example 2. After culturing for 24 hours, the operation was performed according to the instructions of the ATP detection kit.
  • the ATP content of the myocardial injury model group was significantly lower than that of the control group.
  • the DOX+NMN group was significantly higher than the model group, confirming that NMN can increase ATP production and maintain mitochondrial function.
  • Example 2 The same treatment method was adopted as in Example 2. After culturing for 24 hours, the operation was performed according to the instructions of the mitochondrial membrane potential detection kit (JC-1). After the cells were resuspended in JC-1 staining buffer (1 ⁇ ), the images were photographed with a laser confocal microscope ( Figure 7), and the images were processed with ImageJ image processing software ( Figure 8). The red fluorescence is the aggregated JC-1 probe, indicating that the mitochondrial potential is normal. Green is the monomeric JC-1 probe indicating abnormal mitochondrial membrane potential, suggesting potential mitochondrial damage. The green fluorescence intensity of the model group was significantly higher than that of the control group, P ⁇ 0.05. The DOX+NMN group was significantly lower than the model group, confirming that NMN can improve mitochondrial membrane depolarization and improve mitochondrial function.
  • JC-1 mitochondrial membrane potential detection kit
  • TUNEL staining was performed, and the damaged DNA of the cells was mainly displayed as green fluorescence, and the blue fluorescence was Dapi-labeled nuclei ( FIG. 9 ). After staining, it was processed and photographed with a laser confocal microscope. The number of TUNEL-positive cells was counted using the image analysis software Image-Pro plus 5.0 (Media Cybemetrics Inc, USA), and the number of TUNEL-positive cells/field of view was expressed ( Figure 10).
  • the fluorescence intensity of the model group was significantly enhanced compared with the control group, and the fluorescence intensity of the DOX+NMN group was significantly lower than that of the model group, confirming that NMN can improve the apoptosis induced by doxorubicin.

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Abstract

烟酰胺单核苷酸在蒽环类抗肿瘤药心肌毒性预防和/或治疗中的应用，其中烟酰胺单核苷酸在体内通过转化为NAD+来发挥其生理功能，如激活NAD+底物依赖性酶Sirtuins(组蛋白脱乙酰酶，又称沉默调节蛋白)、调节细胞存活和死亡、维持氧化还原状态，其对阿霉素等蒽环类化疗药引发的心肌损伤产生保护作用，减轻阿霉素治疗肿瘤的副作用。

Description

烟酰胺单核苷酸及其在抗肿瘤药物心肌损伤中的保护应用 技术领域
本发明属于防治化疗药心脏毒性的药物技术领域，具体涉及烟酰胺单核苷酸及其在抗肿瘤药物心肌损伤中的保护应用。
背景技术
随着癌症发病率日趋上升，包括蒽环类化疗药在内的抗肿瘤药物用药周期以及累积用量不断增加，化疗药物的不良反应越加凸显，其中以心肌损伤带来的危害最受关注，因而就化疗药物的心肌损伤新临床需求以及学科趋势，近年来全球肿瘤心脏病这一新学科得以迅速发展。其宗旨是在确保化疗药物抗肿瘤作用的同时，如何评估、预防、治疗化疗药带来的心血管不良反应。
目前，蒽环类化疗药物在乳腺癌、淋巴瘤、头颈部肿瘤、多发性骨髓瘤等肿瘤的化疗中依然具有基石性价值。临床常用的蒽环类药物如多柔比星(阿霉素)、柔红霉素、阿克拉霉素、伊达比星、表柔比星等，其化学结构均含有如下的1个四环发色团，不同之处只是四环发色团上的取代基变化而已。其中，最经典且具代表性的药物是多柔比星(阿霉素)。
Figure PCTCN2022080080-appb-000001
尽管蒽环类化疗药物不断推陈出新，如表柔比星、吡柔比星、柔红霉素、阿克拉霉素、伊达比星等，但心脏毒性仍然是蒽环类药物具有共性的不良反应，如多柔比星、表柔比星引起化疗性心衰的最低累积剂量分别为：500mg/m 2，1000mg/m 2(Epirubicin versus doxorubicin:which is the anthracycline of choice for the treatment of breast cancer？[J].Clin Breast Cancer,2003,4 Suppl 1(S26-33.)。主要原因在于蒽环类化疗药物的母核结构一致，活性基团接近(Theoretical ground for adsorptive therapy of anthracyclines cardiotoxicity[J].Exp Oncol,2012,34(4):314-322.)。同时，蒽环类药物心脏毒性的共性机制多与氧自由基生成、细胞凋亡等相等(Irbesartan suppresses cardiac toxicity induced by doxorubicin via regulating the p38-MAPK/NF-kappaB and TGF-beta1 pathways[J].Naunyn Schmiedebergs Arch Pharmacol,2019；PTEN enhances nasal epithelial cell resistance to TNFalphainduced inflammatory injury by limiting mitophagy via repression of the TLR4JNKBnip3 pathway[J].Mol Med Rep,2018,18(3):2973-2986)。
右雷佐生是目前被纳入临床指南用于治疗蒽环类药物心肌损伤的唯一药物，价格昂贵、给药剂量大(建议剂量为阿霉素的5～10倍)，且易加重骨髓抑制，临床对于用药控制较为严格。因此，研发新一代心肌损伤保护性药物非常重要。
烟酰胺单核苷酸(NMN)是细胞氧化型烟酰胺腺嘌呤二核苷酸(NAD +)的可外源补充形式，本质是含氮碱基烟酰胺与D-核糖的C-1位处于β-N-糖苷键的核苷酸。尽管NMN已成为目前抗衰老领域的热点物质，但其确切功效以及在组织器官层面的抗衰老价值并未得到令人信服的深入阐述。口服是目前常用的安全方式，60kg成人长期摄入NMN在200～300mg/日是安全的，WHO建议的每日上限量是900mg，进一步大鼠动物实验认为该上限可提高到1500mg/kg/day的用量，表明其具有稳定的安全性(CROS C,CANNELLE H,LAGANIER L,et al.Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide
Figure PCTCN2022080080-appb-000002
in Sprague-Dawley rats[J].Food Chem Toxicol,2021,150(112060.)。迄今，尚无NMN对于其在化疗药物心肌损伤治疗价值的研发报道。NMN的化学结构式如下：
Figure PCTCN2022080080-appb-000003
发明内容
本发明提供了烟酰胺单核苷酸作为蒽环类抗肿瘤药物心肌损伤的保护性药物的应用。烟酰胺单核苷酸在人体内通过转化为NAD +来发挥其生理功能,如激活NAD +底物依赖性酶Sirtuins(组蛋白脱乙酰酶,又称沉默调节蛋白)、调节细胞存活和死亡、维持氧化还原状态。NMN作为右雷佐生(Dex)的替代品，对阿霉素(Dox)等蒽环类化疗药引发的心肌损伤产生保护作用，减轻Dox治疗肿瘤的副作用。
本发明是采用以下的技术方案实现的：
本发明提供了烟酰胺单核苷酸在抗肿瘤药物心肌损伤中的保护应用。
本发明还提供了烟酰胺单核苷酸在蒽环类抗肿瘤药心肌毒性预防和/或治疗中的应用。
具体的，所述抗肿瘤药包括阿霉素、柔红霉素、阿克拉霉素、伊达比星、表柔比星或米托蒽醌中的至少一种。
进一步地，作为本发明的一个优选方案，用于制备抑制抗肿瘤药所致的心肌损伤的药物、药物混合物、药物组合物。
进一步地，作为本发明的一个优选方案，所述药物、药物混合物或药物组合物中，包括烟酰胺单核苷酸。
本发明还提供一种药物、药物混合物或药物组合物，其活性成分包括烟酰胺单核苷酸，
所述药物、药物混合物或药物组合物具有下述1)-5)中至少一种功能：
1)预防和/或治疗蒽环类肿瘤化疗药的心肌毒性；
2)缓解蒽环类化疗药引起的心肌细胞乳酸脱氢酶异常；
3)降低蒽环类化疗药引起的活性氧自由基升高；
4)缓解蒽环类化疗药引起的心肌细胞脂质过氧化；
5)缓解蒽环类化疗药引起的线粒体膜去极化及细胞凋亡。
具体的，所述药物、药物混合物或药物组合物为药学上可接受的任意剂型，包括片剂、胶囊剂、注射剂、颗粒剂、混悬剂和溶液剂中的至少一种。
本技术的核心在于，烟酰胺单核苷酸(NMN,β-Nicotinamide Mononucleotide)是烟酰胺磷酸核糖转移酶反应的产物,是NAD +的关键前体之一。在哺乳动物体内,NMN由烟酰胺(Nicotinamide,Nam)在Nampt的催化下生成,随后NMN在烟酰胺单核苷酸腺苷转移酶的催化下生成NAD +。细胞外NMN需要去磷酸转化为烟酰胺核苷(Nicotinamide riboside,NR)才能进入肝细胞内部,进入胞内后,NR在烟酰胺核苷激酶1(Nicotinamide riboside kinase,NRK1)的作用下磷酸化生成NMN,随后NMN和ATP结合生成NAD +。NMN在人体内通过转化为NAD +来发挥其生理功能,如激活NAD +底物依赖性酶Sirt1、调节细胞存活和死亡、维持氧化还原状态等。
通过NMN干扰阿霉素诱导的H9c2心肌细胞的心肌损伤实验中发现，NMN提高超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH)活性优于右雷佐生；降低乳酸脱氢酶(LDH)产生和丙二醛(MDA)水平也优于右雷佐生；抑制活性氧自由基(ROS)产生与右雷佐生相媲美。NMN可有效缓解阿霉素引起的线粒体膜去极化及细胞凋亡。因此可证明NMN通过抗氧化及减轻氧化应激和细胞凋亡达到抗阿霉素心肌损伤的作用。
与现有技术相比，本发明的有益效果：
NMN在改善心肌损伤相关特征上优于右雷佐生，原料成本低廉，且已有上市口服产品，因此是作为改善蒽环类药物心肌损伤具有潜力的备选药物，同时也发现了NMN的新价值应用。
附图说明
图1为实施例1中NMN对阿霉素及其类似物表柔比星(EPI)损伤心肌细胞的存活率的 影响对比图，其中，C：空白对照组；DO：模型组1；EP：模型组2；DO+DE：阳性对照组；；&：模型组与对照组相比，存在统计学差异，P<0.05；*：表示与模型组1(DOX)相比，存在统计学差异，P<0.05；#：与模型组2(EPI)相比，P<0.05；
图2为实施例1中NMN对阿霉素损伤心肌细胞影响的光学显微镜图像示例，原始放大倍数＝400×，标尺＝100μm；其中，C：空白对照组；DO：模型组1；DO+DE：阳性对照组；DO+NMN组对比模型组，细胞数量多，形态正常；
图3为实施例2中NMN对阿霉素损伤心肌细胞LDH漏出率的影响对比图，其中，C：空白对照组；**：与模型组相比，P<0.05；
图4为实施例3中NMN对阿霉素损伤心肌细胞MDA的影响对比图，其中，C：空白对照组；**：与模型组相比，P<0.05；
图5为实施例4中NMN对阿霉素损伤心肌细胞ROS的影响对比图，其中，Control：空白对照组；**：与模型组相比，P<0.05；
图6为实施例5中NMN对阿霉素H9c2心肌细胞ATP水平的影响对比图；其中，C：空白对照组；**：与模型组相比，P<0.05；
图7为实施例6中NMN对阿霉素H9c2心肌细胞线粒体膜电位影响的荧光图像示例，原始放大倍数＝400×，标尺＝100μm；其中，C：空白对照组；DO：模型组；DO+DE：阳性对照组；DO+NMN组对比模型组，绿色荧光减弱；
图8为实施例6中NMN对阿霉素H9c2心肌细胞线粒体膜电位的影响对比图；其中，C：空白对照组；**：与模型组相比，P<0.05；
图9为实施例7中NMN对阿霉素H9c2心肌细胞凋亡影响的荧光图像示例，原始放大倍数＝400×，标尺＝100μm)；其中，C：空白对照组；DO：模型组；DO+DE：阳性对照组；DO+NMN组对比模型组，绿色荧光减弱；
图10为实施例7中NMN对阿霉素H9c2心肌细胞凋亡的影响对比图；其中，C：空白对照组；**：与模型组相比，P<0.05。
具体实施方式
为了使本发明目的、技术方案更加清楚明白，下面结合附图，对本发明作进一步详细说明。下述实施例中所述实验方法，如无特殊说明，均为常规方法；实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行；所述试剂和材料，如无特殊说明，均可从商业途径获得。
1、下列实施例采用的材料如下：
本发明所述蒽环类抗生素优选包括阿霉素、柔红霉素、阿克拉霉素、伊达比星、表柔比星或米托蒽醌中的一种，在本发明实施例中以阿霉素和表柔比星为例进行相应实验。
H9c2细胞株为大鼠心肌细胞，购买于美国ATCC细胞库。H9c2细胞在心肌细胞完全培养基溶液中培养，培养温度为37℃，在含有95％空气和5％二氧化碳(CO 2)的培养箱中培养。H9c2细胞在T75瓶内长至80％以上时，吸出培养基，用PBS清洗1-3次，加入胰酶消化1.5min左右，消化中尽可能使胰酶接触到瓶壁所有细胞，加入完全培养基溶液终止消化，900r离心3分钟，收集细胞沉淀，用含10％小牛血清的DMEM稀释至细胞密度为10 6，混匀加入96孔板中，每孔100μL细胞悬液，培养第二天用于实验。
心肌细胞完全培养基溶液：体积比为89％DMEM+10％FBS(FBS:胎牛血清，购自公司大连美仑生物技术有限公司；货号PWL001)+1％双抗(双抗：青霉素/链霉素溶液，购自公司大连美仑生物技术有限公司；货号MA0110)。
右雷佐生(DEXRA)：购自公司湖北威德利化学科技有限公司；货号Y694。
烟酰胺单核苷酸(NMN)：化学式C 11H 15N 2O 8P，CAS号1094-61-7。
DCFH-DA活性氧ROS荧光探针：购自公司大连美仑生物技术有限公司；货号MB4682；用量1ml；孵育时间15～60min；荧光波段：激发波长504nm，发射波长529nm。
CCK-8试剂盒，购自公司大连美仑生物技术有限公司；货号MA0218-L-10000T。
LDH检测试剂盒，购自公司北京索莱宝科技有限公司；货号BC0685。
TUNEL细胞凋亡检测试剂盒，购自公司上海碧云天生物技术有限公司；货号C1088。
ATP检测试剂盒，购自公司上海碧云天生物技术有限公司；货号，S0026B。
脂质氧化(MDA)检测试剂盒，购自公司上海碧云天生物技术有限公司；货号S0131S。
线粒体膜电位检测试剂盒(JC-1)，购自公司北京索莱宝科技有限公司；货号M8650-100T。
2、实验过程
将H9c2细胞分别暴露于阿霉素(DOX)为0.5/1/2/10/20μM浓度下，筛选出阿霉素最佳浓度为5μM用于后续实验。H9c2细胞分别暴露于5μM Dox+20μM Dextra和5μM Dox+20μMNMN下共同处理1天。用DMEM处理的细胞作空白对照，以右雷佐生处理的细胞做阳性对照。
3、药物及分组
实验细胞主要分成4组(n＝4)，空白对照组(control)、心肌损伤模型组(DOX)、阳性对照组(DOX+DEX)、DOX+NMN组(DOX+NMN)。
在检测前24h，心肌损伤模型组给予阿霉素5μM，阳性对照组给予右雷佐生20μM，NMN处理组给予NMN 20μM，其余两组分别为5μMDOX+20μMDexra以及5μMDOX+20μMNMN。
实施例1、NMN对阿霉素及其类似物导致H9c2心肌细胞损伤的保护疗效
H9c2心肌细胞用培养基稀释为10 6/ml的细胞悬液，并接种于96孔板中，每孔加入100μL细胞悬液，培养24h后，进行药物处理。分为空白对照组(纯DMEM)、模型组1(5μM DOX)、，模型组2(5μM EPI)、阳性对照组(20μM Dexra)、5μM DOX+20μMDexra组、5μM DOX+NMN组(分别给予NMN 10/20/30/50/100μM)、5μM EPI+NMN组(给予NMN 20μM)。培养24h后，以CCK-8细胞活力检测进行测定，使用酶标仪测定450nm处的吸光度，计算心肌细胞活性。通过倒置光学显微镜观察细胞状态。
结果如图1所示，相对于control组细胞活性106.7％，5μM DOX模型组细胞活性为46.0％，5μM的EPI模型组细胞活性为80.53％。阳性对照组细胞活性为106.8％，而合并使用10μM、20μM、30μM、50μM、100μM的NMN后，细胞活性分别为52.4％、65.5％、66.1％、67.40％、49.8％、39.0％。模型组1、2存活率明显低于空白对照组，NMN浓度在20μM、30μM明显提高细胞存活率。后续实验采用20μM NMN。合并使用NMN后，EPI+NMN组(5μM EPI+20μM NMN)细胞活性为100.30％。NMN对两种典型的蒽环类药物均具有保护作用，对心脏毒性较弱的表柔比星保护效果更佳，并显著提高细胞存活率。
结果如图2所示，相对于control组，DOX模型组细胞数量明显减少，漂浮的死亡细胞增多，细胞形态呈球形反映为应激状态。DO+NMN组细胞，对比模型组1，数量明显增多，与阳性对照组差别不大，提示20μM NMN在促细胞存活能力上即可与右雷佐生接近。
实施例2、NMN降低阿霉素H9c2心肌细胞乳酸脱氢酶(LDH)漏出的影响
每组(n＝4)每个样本取10 6个细胞，充分裂解，丙酮酸法检测细胞内LDH的含量，对照组细胞内LDH为100％，各用药组与心肌损伤模型组细胞内LDH与正常对照组的差值为LDH释放率，在反应体系中产生1μmol丙酮酸为1单位，计算每单位/mg蛋白。培养24h后，用LDH检测试剂盒进行测定。结果显示(图3)不同用药组的LDH漏出率较模型组均明显降低。
实施例3、NMN抑制阿霉素H9c2心肌细胞脂质过氧化水平的影响
氧自由基攻击生物膜中的多不饱和脂肪酸，引发脂质过氧化作用，并因此产生脂质过氧化物丙二醛(MDA)。MDA可与硫代巴比妥酸(TBA)缩合，形成红色产物，在532nm处有最大吸收峰。与实施例2采用相同的处理方式，培养24h后，按照MDA检测试剂盒说明书进行操作。结果显示(图4)，心肌损伤模型组MDA含量与对照组相比显著增高，P<0.05。 DOX+NMN组较模型组均明显降低，证实NMN可抑制活性氧诱导的脂质过氧化。
实施例4、NMN对阿霉素H9c2心肌细胞活性氧(ROS)水平的影响
利用荧光探针DCFH-DA自由穿过细胞膜，进入细胞内后，可以被细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜，从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。检测DCF的荧光就可以知道细胞内活性氧的水平。与实施例2采用相同的处理方式，培养24h后，各组装载DCFH-DA探针，利用激光共聚焦显微镜拍摄，再利用ImageJ图像处理软件处理图像(图5)。
结果显示(图5)，模型组ROS含量与对照组相比显著增高。DOX+NMN组较模型组均明显降低，证实NMN可抑制活性氧自由基产生。
实施例5、NMN对阿霉素H9c2心肌细胞ATP水平的影响
ATP作为最重要的能量分子，在细胞的各种生理、病理过程中起着重要作用。ATP水平的改变，会影响细胞的功能。通常细胞在凋亡、坏死或处于一些毒性状态下，ATP水平会下降。与实施例2采用相同的处理方式，培养24h后，按照ATP检测试剂盒说明书进行操作。
参照图6，心肌损伤模型组ATP含量与对照组相比显著降低。DOX+NMN组较模型组均明显提高，证实NMN可增加ATP产生，维护了线粒体功能。
实施例6、NMN对阿霉素H9c2心肌细胞线粒体膜电位的影响
与实施例2采用相同的处理方式，培养24h后，按照线粒体膜电位检测试剂盒(JC-1)说明书进行操作。取细胞进行JC-1染色缓冲液(1×)重悬后，利用激光共聚焦显微镜拍摄(图7)，利用ImageJ图像处理软件处理图像(图8)。红色荧光为聚集态JC-1探针，表明线粒体电位正常。绿色为单体JC-1探针表明线粒体膜电位异常，提示潜在的线粒体损伤。模型组绿色荧光强度与对照组相比显著提高，P<0.05。DOX+NMN组较模型组均明显降低，证实NMN可改善线粒体膜去极化，改善了线粒体功能。
实施例7、NMN对阿霉素H9c2心肌细胞凋亡的影响
与实施例2采用相同的处理方式，培养24h后，进行TUNEL染色，主要将细胞损伤DNA显示为绿色荧光，蓝色荧光为Dapi标记的细胞核(图9)。染色后经处理，利用激光共聚焦显微镜摄，利用图像分析软件Image-Pro plus 5.0(Media Cybemetrics Inc,USA)计数TUNEL阳性细胞数，TUNEL阳性细胞数/视野表示(图10)。
根据图10显示，模型组荧光强度对照组相比显著增强，DOX+NMN组较模型组荧光强度均明显降低，证实NMN可改善阿霉素诱导的细胞凋亡。
当然，以上仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记 载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 烟酰胺单核苷酸在抗肿瘤药物心肌损伤中的保护应用。
  2. 烟酰胺单核苷酸在蒽环类抗肿瘤药心肌毒性预防和/或治疗中的应用。
  3. 根据权利要求1或2所述的应用，其特征在于，所述抗肿瘤药包括阿霉素、柔红霉素、阿克拉霉素、伊达比星、表柔比星或米托蒽醌中的至少一种。
  4. 根据权利要求3所述的应用，其特征在于，用于制备抑制抗肿瘤药所致的心肌损伤的药物、药物混合物、药物组合物。
  5. 根据权利要求4所述的应用，其特征在于，所述药物、药物混合物或药物组合物中，包括烟酰胺单核苷酸。
  6. 一种药物、药物混合物或药物组合物，其特征在于，其活性成分包括烟酰胺单核苷酸，
    所述药物、药物混合物或药物组合物具有下述1)-5)中至少一种功能：
    1)预防和/或治疗蒽环类肿瘤化疗药的心肌毒性；
    2)缓解蒽环类化疗药引起的心肌细胞乳酸脱氢酶异常；
    3)降低蒽环类化疗药引起的活性氧自由基升高；
    4)缓解蒽环类化疗药引起的心肌细胞脂质过氧化；
    5)缓解蒽环类化疗药引起的线粒体膜去极化及细胞凋亡。
  7. 根据权利要求6所述的药物、药物混合物或药物组合物，其特征在于，所述药物、药物混合物或药物组合物为药学上可接受的任意剂型，包括片剂、胶囊剂、注射剂、颗粒剂、混悬剂和溶液剂中的至少一种。
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CN113662955A (zh) * 2021-09-16 2021-11-19 复旦大学附属中山医院 烟酰胺腺嘌呤二核苷酸在治疗阿霉素诱导的心脏毒性中的应用
CN113750242A (zh) * 2021-10-28 2021-12-07 复旦大学附属中山医院 Nmn联合cd38抑制剂在制备预防或治疗阿霉素所致心脏毒性药物中的应用
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