WO2020224055A1 - 阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途 - Google Patents

阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途 Download PDF

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WO2020224055A1
WO2020224055A1 PCT/CN2019/097239 CN2019097239W WO2020224055A1 WO 2020224055 A1 WO2020224055 A1 WO 2020224055A1 CN 2019097239 W CN2019097239 W CN 2019097239W WO 2020224055 A1 WO2020224055 A1 WO 2020224055A1
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opioid receptor
aml
loperamide
tet2
receptor agonist
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蒋晞
赵焕焕
韩菲
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浙江大学
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

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  • the invention is in the field of biomedicine technology. Specifically, it relates to the use of opioid receptor agonists in the manufacture of drugs for treating malignant tumors.
  • AML Acute myeloid leukemia
  • MLL-fused AML Acute myeloid leukemia
  • the first-line treatment plan is mainly the standard "7+3" chemotherapy, that is, nucleoside analogs represented by cytarabine (Ara-C) and doxorubicin (DOX) Representative combination therapy of anthracyclines.
  • genetic abnormalities such as gene mutations, chromosome remodeling, and gene copy number abnormalities
  • epigenetic regulation is It is a further fine adjustment based on genetics.
  • the applicant’s research team has found that this type of fine-tuning can often play a decisive role in the occurrence and development of leukemia, drug reactions, etc. On this basis, it is urgent to further study the mechanism of epigenetic regulation in leukemia, and to develop new ways to intervene in epigenetic regulation to treat leukemia.
  • TET Teen-eleven translocation
  • TET2 Similar to TET1, TET2, another member of the TET family, also has the function of catalyzing DNA 5hmC modification. AML with mutations in the TET2 gene presents a low 5hmC level. Contrary to the oncogene effect of TET1, TET2 has been identified as a tumor suppressor gene in a variety of tumors including leukemia. Many subtypes of AML have TET2 mutations, and the mutation rate of TET2 gene loss of function can be as high as 7-23% in AML. Moreover, TET2 mutations are often accompanied by other disease-causing gene mutations, such as NPM1 mutations. Studies have found that AML with TET2 mutations usually has a poor prognosis.
  • TET2 expression levels are also closely related to the occurrence and development of AML.
  • down-regulation of TET2 can significantly promote the proliferation of AML cells, while overexpression of TET2 inhibits the proliferation of AML cells.
  • New research shows that in Tet2-induced knockout hematopoietic stem cells/precursor cells (HSPC), the recovery of Tet2 expression by removing the inducer can effectively promote myeloid differentiation and cell death.
  • Certain drugs such as vitamin C combined with PARP inhibitors, can kill AML cells by activating TET2 enzyme activity.
  • TET2 can also function in a kinase activity-independent manner.
  • opioid receptor signaling pathways have long been controversial.
  • Opioid receptor agonists such as morphine and fentanyl are often used as powerful analgesics adjuvant therapy in tumor treatment. It is not clear whether such agents have a direct inhibitory effect on tumors.
  • pre-B-ALL acute lymphocytic leukemia
  • methadone and doxorubicin can act synergistically to inhibit tumor growth.
  • endorphin treatment can cause cell apoptosis, and its mechanism has not been thoroughly studied.
  • opioid receptor signaling pathway modulators have been put into clinical practice and are routinely used for sedation, analgesia, antidiarrhea and other therapeutic purposes.
  • opioid receptor agonist loperamide loperamide; alias clopiramide hydrochloride, Luobaomai, chemical name 4-p-chlorobenzene-4-hydroxy-N,N-dimethyl- ⁇ , ⁇ -diphenyl-1 -Piperidinyl butanamide, molecular formula C 29 H 33 ClN 2 O 2
  • loperamide alias clopiramide hydrochloride, Luobaomai, chemical name 4-p-chlorobenzene-4-hydroxy-N,N-dimethyl- ⁇ , ⁇ -diphenyl-1 -Piperidinyl butanamide, molecular formula C 29 H 33 ClN 2 O 2
  • the above opioid receptor agonists have an epigenetic regulatory mechanism and whether they can be directly used in the treatment of AML has not been reported.
  • the purpose of the present invention is to overcome the shortcomings of the prior art, and discloses the use of an opioid receptor agonist in the manufacture of drugs for treating malignant tumors. It is based on the tumor suppressor gene effect of TET2 in AML, and the activation of the opioid receptor signaling pathway to activate the expression of TET2. It uses opioid receptor agonists (such as the antidiarrheal drug loperamide that has been approved for clinical use) to develop on the one hand New AML therapies targeting opioid receptors and TET2 pathways have been developed. On the other hand, the use of these opioid receptor agonists in drugs for the treatment of acute myeloid leukemia beyond the conventional drug effects has been explored. Further, the opioid receptor agonists include but are not limited to one or more of loperamide, trimebutine, and asimadolin.
  • the present invention provides an important research basis for the treatment of malignant tumors, especially leukemia, using opioid receptor signaling pathways and TET2 as drug targets; it also provides an important basis for "old drugs and new uses" of opioid receptor agonists such as loperamide. Scientific basis.
  • TET2 Abnormal expression or function of TET2 is a common pathogenesis of many malignant tumors including AML. How to effectively regulate the expression of tumor TET2 to achieve the purpose of therapeutic intervention has not been reported.
  • the present invention is the first molecular targeted therapy specifically targeting the opioid receptor signaling pathway and TET2 expression regulation. It has a significant killing effect on multiple subtypes of AML cells, has a broad-spectrum anti-AML function, and may also be applicable to other similar molecules Mechanism of malignancy.
  • the loperamide AML treatment program provided by the present invention significantly improves the above shortcomings of conventional chemotherapy for AML.
  • the drug used in the treatment plan provided by the present invention is loperamide, which is commonly used clinically to stop diarrhea, with fewer adverse reactions and mild side effects; and the dose in animal models is lower (only 0.25 mg/kg; here can be Refer to and compare the conventional chemotherapy dose of AML "5+3" course: Ara-C 50mg/kg, DOX 3mg/kg), the course of treatment is only 7 days, no obvious side effects were found during the course of treatment and after the course of treatment, animal tolerated good.
  • the present invention can directly activate the expression of TET2, fundamentally up-regulate TET2, thereby simultaneously strengthening TET2 kinase activity dependent and independent functions, and has a stronger therapeutic application prospect .
  • Figures 1A-C show the effects of multiple opioid receptor agonists and antagonists on AML cell activity and cell proliferation.
  • Figure 1A shows the opioid receptor agonist loperamide (OPA1), trimebutine (OPA2), asimadolin (OPA3), and opioid receptor antagonist polyethylene glycol nanolol (OPANT1) Or the effect of naltrexone (OPANT2) on the activity of AML cell line THP-1.
  • OPA1 opioid receptor agonist loperamide
  • OPA2 trimebutine
  • OPA3 asimadolin
  • OANT1 opioid receptor antagonist polyethylene glycol nanolol
  • OPANT2 opioid receptor antagonist polyethylene glycol nanolol
  • OPANT2 opioid receptor antagonist polyethylene glycol nanolol
  • Figure 1B shows the opioid receptor agonists loperamide (OPA1), trimebutine (OPA2), asimadolin (OPA3), opioid receptor antagonists polyethylene glycol nanolol (OPANT1) or sodium The effect of trexone (OPANT2) on the proliferation of AML cell line THP-1.
  • OPA1 opioid receptor agonists loperamide
  • OPA2 trimebutine
  • OPA3 opioid receptor antagonists polyethylene glycol nanolol
  • OPANT1 polyethylene glycol nanolol
  • THP-1 The effect of trexone
  • Figure 1C shows the effect of opioid receptor agonist loperamide (OPA1) on cell viability of various AML cell lines.
  • Figure 2A-B shows the effects of opioid receptor agonists and antagonists on TET2 expression and cellular 5-hmC modification.
  • Figure 2A shows the opioid receptor agonist loperamide (OPA1), trimebutine (OPA2), asimadolin (OPA3), and opioid receptor antagonist polyethylene glycol nanolol (OPANT1) Or the effect of naltrexone (OPANT2) on the expression level of TET2 in the AML cell line THP-1.
  • OPA1 opioid receptor agonist loperamide
  • OPA2 trimebutine
  • OPA3 opioid receptor antagonist polyethylene glycol nanolol
  • OPANT1 opioid receptor antagonist polyethylene glycol nanolol
  • OPANT2 opioid receptor antagonist polyethylene glycol nanolol
  • Figure 2B shows the effects of opioid receptor agonist loperamide (OPA1) and opioid receptor antagonist naltrexone (OPANT2) on the level of DNA 5hmC modification in the AML cell line THP-1.
  • OPA1 opioid receptor agonist loperamide
  • OANT2 opioid receptor antagonist naltrexone
  • Figure 3A-B shows the in vivo efficacy of an opioid receptor agonist taking loperamide as an example in a mouse AML model.
  • Figure 3A shows the in vivo efficacy of loperamide in mouse MLL-AF9(MA9)-AML.
  • Figure 3B shows the in vivo efficacy of loperamide in mouse AML-ETO9a(AE9a)-AML.
  • KASUMI-1 cells were purchased from Shanghai Stem Cell Bank of Chinese Academy of Sciences;
  • THP-1, MV4; 11, NB4, U937 and other cells were purchased from ATCC.
  • Loperamide, naltrexone, trimebutine, and aximadolin were purchased from Shanghai Taosu Biochemical Technology Co., Ltd.;
  • Polyethylene glycol nanolol was purchased from Selleck;
  • MTT kit DMSO, RPMI-1640 medium were purchased from Sigma;
  • TET2 quantitative PCR primers were designed and synthesized by Hangzhou Youkang Biotechnology;
  • Fetal bovine serum was purchased from Gibco;
  • Trizol-RNAiso was purchased from TAKARA Company;
  • RNA reverse transcription kits and QPCR kits were purchased from Nanjing Novozan Biotechnology Co., Ltd.;
  • the plasmid extraction kit was purchased from Nanjing Novazan Biotechnology Co., Ltd.
  • Example 1 The detection of opioid receptor agonists and antagonists on the activity and proliferation of AML cells.
  • THP-1 cells MV4; 11 cells, NB4 cells, U937 cells and KASSUMI-1 cells are used with 90% volume fraction of RPMI-1640, 10% fetal bovine serum and 1% double antibody medium at a temperature of 37 Cultivate in an incubator at °C, 5% CO 2 concentration and saturated humidity.
  • MTT preparation Add 1ml sterile PBS to 5mg MTT to dissolve it to prepare 12mM MTT solution; store in the refrigerator at -20°C in the dark.
  • loperamide As shown in Figure 1, a variety of opioid receptor agonists, including loperamide (OPA1), trimebutine (OPA2), and aximadolin (OPA3), all have an effect on the AML cell line THP-1. Cell viability and cell proliferation were significantly inhibited ( Figure 1A-B). Among them, the inhibitory effect of loperamide (OPA1) is the most obvious ( Figure 1A-B).
  • the opioid receptor antagonists polyethylene glycol nanolol (OPANT1) and naltrexone (OPANT2) enhanced AML cell viability and cell proliferation (Figure 1A-B).
  • the opioid receptor agonist loperamide (OPA1) showed significant inhibition of cell activity of various AML cell lines representing different AML subtypes including THP-1, MV4; 11, KASUMI1, U937, NB4 ( Figure 1C), suggesting that loperamide may have a universal effect on multiple subtypes of AML.
  • Example 2 Detection of the effects of opioid receptor agonists and antagonists on TET2 expression and cell 5-hmC modification.
  • RNA Discard the supernatant, wash the pellet with 75% absolute ethanol, centrifuge at 7500g/4°C for 5 min; discard the supernatant, put the EP tube in a fume hood to dry for 10 min, and then dissolve the RNA with 20 ⁇ l DEPC water to detect the RNA concentration, and The samples are stored in a refrigerator at -80°C.
  • the system is as follows:
  • Figures 2A-B show the effects of various opioid receptor agonists and antagonists on TET2 expression and cellular 5-hmC modification.
  • Figure 2A shows: a variety of opioid receptor agonists including loperamide (OPA1), trimebutine (OPA2), and aximadolin (OPA3) can significantly increase the THP- In 1 TET2 expression level, the opioid receptor antagonists polyethylene glycol nanolol (OPANT1) and naltrexone (OPANT2) inhibited TET2 expression.
  • opioid receptor agonists including loperamide (OPA1), trimebutine (OPA2), and aximadolin (OPA3) can significantly increase the THP- In 1 TET2 expression level
  • the opioid receptor antagonists polyethylene glycol nanolol (OPANT1) and naltrexone (OPANT2) inhibited TET2 expression.
  • Figure 2B shows that the opioid receptor agonist loperamide (OPA1) significantly enhanced the DNA 5hmC modification level in the AML cell line THP-1, while the opioid receptor antagonist naltrexone (OPANT2) inhibited the cellular DNA 5hmC modification.
  • OPA1 opioid receptor agonist loperamide
  • OPANT2 opioid receptor antagonist naltrexone
  • MLL-AF9 or AML-ETO9a primary AML mouse bone marrow cells (primary donor: B6.SJL(CD45.1) wild-type mouse bone marrow cells to infect MLL-AF9 or AML-ETO9a; primary recipient: C57BL/6( CD45.2); primary AML: CD45.1 + %>90%) as donor cells at the time of sample collection. Resuspend 1x10 5 donor cells in 100 ⁇ l PBS, and transplant into each mouse with a half-lethal dose by tail vein injection (480rads) irradiated second-generation receptor (C57BL/6; CD45.2) mice.
  • mice One week after transplantation, 0.25 mg/kg loperamide or DMSO control was administered, intraperitoneal injection once a day for a total of 7 days. Observe the symptoms and phenotypes of mice daily. When the mouse is dying and meets the humane death standard, the mouse is executed, and samples of peripheral blood, liver, spleen, bone marrow, etc. are collected. Record survival time. Each group of 8-10 weeks old C57BL/6 (CD45.2) second-generation recipient mice, 10 mice were randomly divided into groups, half male and half female.
  • opioid receptor agonists represented by loperamide have shown strong curative effects on a variety of leukemias including MLL-AF9(MA9)-AML and AML-ETO9a(AE9a)-AML. .

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Abstract

阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途。所述用途基于TET2在AML中的抑癌基因作用，以及阿片受体信号通路活化对TET2表达的激活，利用阿片受体激动剂(如已在临床批准使用的止泻药洛哌丁胺)，发掘出这些阿片受体激动剂常规药物作用以外的白血病治疗新用途。也为洛哌丁胺等阿片受体激动剂"老药新用"提供了重要科学依据。洛哌丁胺制备AML治疗药物的用途显著改善AML常规化疗的缺点（化疗剂量高、疗程长、副反应大，患者经常不耐受），药物不良反应较少、副作用较轻。

Description

阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途 技术领域
本发明生物医药技术领域。具体涉及阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途。
背景技术
急性髓细胞性白血病(Acute myeloid leukemia,AML)是最常见的致死性造血系统恶性肿瘤之一，也是成人最常见的急性白血病，具有复杂的基因多样性，对治疗反应差异较大。多种AML亚型即使给予化疗，仍然预后不佳，其中包括MLL融合的AML等。AML的发生率随年龄增长而增长，老年患者因其体质问题，对高强度化疗难以耐受，预后尤其差。通过标准化疗，仅有30-50％低于60岁的年轻AML病人和5-20％的老年患者可以存活超过5年。AML的可选治疗方案十分有限。过去几十年，一线治疗方案主要是标准“7+3”化疗，即以阿糖胞苷(cytarabine,Ara-C)为代表的核苷类似物与以多柔比星(doxorubicin,DOX)为代表的蒽环类药物的组合治疗。近些年人们逐渐认识到，单独讨论基因(遗传学)异常，如基因突变、染色体重构、基因拷贝数异常等，已无法完全解释AML复杂的病理表现和预后；而表观遗传学调控则是在遗传学基础上的进一步精细调整。申请人的研究团队近年来的研究发现，这类微调往往能在白血病的发生发展、药物反应等方面起到决定性作用。在此基础上，进一步研究表观遗传学调控在白血病中的作用机制，以及开发干预表观遗传调控的治疗白血病的新途径迫在眉睫。
TET(Ten-eleven translocation)家族蛋白TET1/2/3通过将5-甲基胞嘧啶(5-methylcytosine,5mC)羟基化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)，实现DNA去甲基化。近几年的研究发现，TET介导的5hmC修饰在很多重要生理、病理过程，如分化、发育中，发挥重要作用。申请人的研究团队首次发现并报道了TET1介导的DNA 5hmC修饰在AML中的重要作用。
与TET1类似，TET家族中的另一个成员TET2同样具有催化DNA 5hmC修饰的功能，TET2基因突变的AML呈现低5hmC水平。与TET1的癌基因作用相反， TET2在多种肿瘤包括白血病中已明确作为抑癌基因发挥作用。AML多种亚型均存在TET2突变，TET2基因功能缺失突变率在AML中可高达7-23％。而且，TET2突变常伴随其他致病基因突变，如NPM1突变。研究发现，TET2突变的AML通常预后较差。除了基因突变以外，TET2表达水平也与AML发生发展密切相关。体外实验中，TET2下调可明显促进AML细胞增殖；而过表达TET2则抑制AML细胞扩增。新的研究显示，在Tet2诱导敲除的造血干细胞/前体细胞(HSPC)中，通过撤除诱导剂而使Tet2表达恢复，可有效促进髓系分化和细胞死亡。某些药物，如维生素C与PARP抑制剂联合使用，可通过激活TET2酶活性，杀伤AML细胞。此外，与TET1类似，TET2也可以激酶活性非依赖的方式发挥作用。考虑到TET家族成员激酶活性依赖和非依赖这两种作用方式，在肿瘤细胞中重塑TET2表达与重塑TET2功能活性相比，无疑具有新的意义，而“如何通过有效促进TET2表达来治疗AML”这一问题却迄今未有理想答案。上述研究提示，通过信号刺激增强TET2在AML中的表达，将会是AML治疗的一个新思路、新选择。
阿片受体信号通路在肿瘤中的作用长久以来广有争议。阿片受体激动剂如吗啡、芬太尼等在肿瘤治疗中时常作为强效镇痛剂辅助治疗。此类药剂对肿瘤是否有直接抑制作用尚不清楚。有研究报道，在急性淋巴细胞白血病(pre-B-ALL)中，美沙酮与多柔比星可协同作用，抑制肿瘤生长。在体外培养的AML细胞系HL-60中，内啡肽处理可引起细胞凋亡，其机制未得到深入研究。目前，一些阿片受体信号通路调节剂已投入临床，常规用于镇静、镇痛、止泻等治疗目的。如阿片受体激动剂洛哌丁胺(loperamide；别名盐酸氯哌拉米、罗宝迈，化学名4-对氯苯-4-羟基-N,N-二甲基-α,α-二苯-1-对哌啶基丁酰胺，分子式C 29H 33ClN 2O 2)，为长效抗腹泻药，临床上主要治疗各种病因引起的急慢性腹泻，特别适用于慢性腹泻的长期治疗。以上阿片受体激动剂是否具有表观遗传学调控机制、能否直接用于AML治疗未有报道。
发明内容
本发明的目的是克服现有技术的不足，公开了一种阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途。它基于TET2在AML中的抑癌基因作用，以及阿片受体信号通路活化对TET2表达的激活，利用阿片受体激动剂(如已在临床批准使用的止泻药洛哌丁胺)，一方面开发出靶向针对阿片受体和TET2通路的 AML新疗法，另一方面发掘出这些阿片受体激动剂常规药物作用以外的用于治疗急性髓细胞性白血病的药物中的用途。进一步的，所述的阿片受体激动剂包括但不限于洛哌丁胺、曲美布汀、阿西玛多林中的一种或多种。
本发明的优点是：
本发明为以阿片受体信号通路和TET2为药物靶点治疗恶性肿瘤，特别是白血病，提供了重要研究基础；也为洛哌丁胺等阿片受体激动剂“老药新用”提供了重要科学依据。
TET2表达异常或功能异常是包括AML在内的多种恶性肿瘤的一个常见发病机制，如何有效调控肿瘤TET2表达，从而起到治疗干预目的却未有报道。本发明为首例特异性针对阿片受体信号通路和TET2表达调控的分子靶向治疗，对多种亚型AML细胞均有显著杀伤效果，具有广谱抗AML功能，也可能适用于其他具有类似分子机制的恶性肿瘤。
传统AML化疗剂量高、疗程长、副反应大，患者经常不耐受，老年患者尤为严重。本发明提供的洛哌丁胺AML治疗方案显著改善AML常规化疗的上述缺点。本发明提供的治疗方案所用药物为临床常用于止泻的洛哌丁胺，此药不良反应较少、副作用较轻；且动物模型中给药剂量较低(仅为0.25mg/kg；这里可参考和比较AML“5+3”疗程常规化疗剂量：Ara-C 50mg/kg,DOX 3mg/kg)，疗程仅为7天，治疗过程中和疗程结束后均未发现明显毒副作用，动物耐受佳。
本发明与先前报道可激活TET2酶活性的维生素C相比，可直接激活TET2的表达，从根本上上调TET2，从而同时强化TET2激酶活性依赖和非依赖两类功能，具更强的治疗应用前景。
附图说明
图1A-C为多种阿片受体激动剂和拮抗剂对AML细胞活性和细胞增殖的作用。
其中，图1A为阿片受体激动剂洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)、阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)或纳曲酮(OPANT2)对AML细胞系THP-1活性的影响。
图1B为阿片受体激动剂洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)、阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)或纳曲酮(OPANT2)对AML细胞系THP-1增殖的影响。
图1C为阿片受体激动剂洛哌丁胺(OPA1)对多种AML细胞系细胞活性的影响。
图2A-B为阿片受体激动剂和拮抗剂对TET2表达和细胞5-hmC修饰的作用。
其中，图2A为阿片受体激动剂洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)、阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)或纳曲酮(OPANT2)对AML细胞系THP-1中TET2表达水平的影响。
图2B为阿片受体激动剂洛哌丁胺(OPA1)、阿片受体拮抗剂纳曲酮(OPANT2)对AML细胞系THP-1中DNA 5hmC修饰水平的影响。
图3A-B为以洛哌丁胺为例的阿片受体激动剂在小鼠AML模型中表现出的在体疗效。
其中，图3A为洛哌丁胺在小鼠MLL-AF9(MA9)-AML中的在体疗效。
图3B为洛哌丁胺在小鼠AML-ETO9a(AE9a)-AML中的在体疗效。
具体实施方式
以下通过实施例对本发明作进一步的说明，但本发明并不限于以下实施方式。
1、材料
KASUMI-1细胞购自中科院上海干细胞库；
THP-1、MV4；11、NB4、U937等细胞均购自ATCC。
2、试剂
洛哌丁胺、纳曲酮、曲美布汀、阿西玛多林购自上海陶素生化技术有限公司；
聚乙二醇纳诺醇购自Selleck公司；
MTT试剂盒、DMSO、RPMI-1640培养基购自Sigma公司；
TET2定量PCR引物由杭州有康生物设计合成；
胎牛血清购自Gibco；
Trizol-RNAiso购自TAKARA公司；
RNA逆转录试剂盒及QPCR试剂盒均购自南京诺唯赞生物科技有限公司；
质粒抽提试剂盒购自南京诺唯赞生物科技有限公司。
实施例1 阿片受体激动剂和拮抗剂对AML细胞活性及增殖作用的检测。
1.1细胞培养
THP-1细胞、MV4；11细胞、NB4细胞、U937细胞和KASSUMI-1细胞用含有90％体积分数的RPMI-1640、10％的胎牛血清和1％的双抗培养基，在温度为37℃，5％CO 2浓度以及饱和湿度的培养箱中培养。
1.2细胞活性即MTT检测
MTT配制：向5mg MTT中加入1ml无菌PBS进行溶解，制备12mM MTT溶液；避光保存于-20℃冰箱。
取培养细胞以每孔10000个细胞，100μl种于96孔板中，分别给予1μM洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)、阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)或纳曲酮(OPANT2)，或DMSO对照，将细胞放于37℃培养箱孵育48小时后，每孔加入10μl 12mM MTT溶液，37℃培养箱孵育4小时，每孔加入50μl DMSO溶液，终止MTT反应，孵育10min，酶标仪检测540nm吸光度。
1.3细胞增殖检测
取培养细胞以每孔10000个细胞，100μl种于96孔板中，分别给予1μM洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)、阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)或纳曲酮(OPANT2)，或DMSO对照，于给药后12hrs、24hrs、48hrs、72hrs、96hrs分别计数活细胞。
如图1所示，包含洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)在内的多种阿片受体激动剂均对AML细胞系THP-1的细胞活性和细胞增殖有着显著抑制(图1A-B)。其中，洛哌丁胺(OPA1)的抑制作用最为明显(图1A-B)。阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)和纳曲酮(OPANT2)则增强AML细胞活性和细胞增殖(图1A-B)。
阿片受体激动剂洛哌丁胺(OPA1)对包含THP-1,MV4；11,KASUMI1,U937,NB4在内的、代表不同AML亚型的多种AML细胞系细胞活性均表现出显著抑制(图1C)，提示洛哌丁胺对多种亚型AML可能具有普适性疗效。
实施例2 阿片受体激动剂和拮抗剂对TET2表达和细胞5-hmC修饰作用的检测。
2.1细胞总RNA提取
取对数生长期细胞2x10 6，加500微升Trizol-RNAiso反复吹打混匀，冰上 静置5min，然后加100μl氯仿，剧烈振荡15sec，室温静置5min；12000g/4℃/15min离心；取上清液到一新EP管中，并加等量的异丙醇沉淀RNA,混匀后室温静置5min,12000g/4℃/10min离心。弃掉上清，用75％无水乙醇清洗沉淀，7500g/4℃离心5min；弃掉上清，将EP管放入通风厨干燥10min，再用20μl DEPC水溶解RNA，检测RNA浓度，并将样品保存于-80℃冰箱。
2.2 RNA逆转录及QPCR检测
体系如下：
Figure PCTCN2019097239-appb-000001
QPCR体系：
Figure PCTCN2019097239-appb-000002
2.3 5hmC免疫荧光染色
上述药物或DMSO对照处理48h后各收集1x10 6个细胞，分别用500μl PBS洗一遍，2500rpm,离心2.5min，弃上清，各加80μl PBS重悬，滴在带电荷的载玻片上，20min后分别用60μl的4％多聚甲醛+0.3％Triton-X100封闭10min。用100μl PBS洗涤3次，然后孵育一抗(抗5hmC抗体(Active Motif，#39769)1:200)，4℃过夜。用100μl PBS洗涤一抗3次，孵育二抗(免疫荧光488兔二抗(Thermofisher,#A-11008)1:1000)，避光室温孵育1h，用100μl PBS洗涤二抗3次，最后用含有DAPI封片剂(上海翊圣生物科技有限公司，#36308ES11)封片、荧光显微镜观察。
图2A-B显示了多种阿片受体激动剂和拮抗剂对TET2表达和细胞5-hmC修饰的作用。
其中，图2A显示：包含洛哌丁胺(OPA1)、曲美布汀(OPA2)、阿西玛多林(OPA3)在内的多种阿片受体激动剂均可显著提升AML细胞系THP-1中TET2表达水平，阿片受体拮抗剂聚乙二醇纳诺醇(OPANT1)和纳曲酮(OPANT2)则抑制TET2表达。
图2B显示：阿片受体激动剂洛哌丁胺(OPA1)显著增强AML细胞系THP-1中DNA 5hmC修饰水平，阿片受体拮抗剂纳曲酮(OPANT2)则抑制细胞DNA 5hmC修饰。上述结果提示：阿片受体激动剂对TET2介导的DNA 5hmC修饰具有增强效果，阿片受体拮抗剂则起到相反作用。
实施例3 洛哌丁胺在小鼠AML模型中表现出的在体疗效
3.1骨髓移植实验
取MLL-AF9或AML-ETO9a初代AML小鼠骨髓细胞(初代供体：B6.SJL(CD45.1)野生型小鼠骨髓细胞感染MLL-AF9或AML-ETO9a；初代受体：C57BL/6(CD45.2)；初代AML：收取样本时CD45.1 +％>90％)为供体细胞，将1x10 5个供体细胞重悬于100μl PBS，以尾静脉注射移植入每只经过半致死剂量(480rads)照射的二代受体(C57BL/6；CD45.2)小鼠。
3.2药物治疗
移植1周后开始给予0.25mg/kg洛哌丁胺或DMSO对照，每天腹腔注射一次，共给药7天。每日观察小鼠症状、表型。当小鼠濒临死亡、符合人道处死标准时， 处死小鼠，收集外周血、肝、脾、骨髓等样本。记录生存时间。每组8-10周龄C57BL/6(CD45.2)二代受体小鼠10只，随机分组，公母各半。
如图3A-B所示，为以洛哌丁胺为例，阿片受体激动剂在小鼠AML模型中表现出明显的在体疗效。在小鼠MLL-AF9(MA9)-AML模型中，洛哌丁胺将白血病小鼠半数生存时间从21天(图3A对照组)延长至大于40天(图3A给药组)，即在骨髓移植后40天内无一给药组小鼠因白血病发病而导致死亡；小鼠AML-ETO9a(AE9a)-AML的在体疗效模型中，洛哌丁胺将白血病小鼠半数生存时间从22天(图3B对照组)延长至大于40天(图3B给药组)，同样在骨髓移植后40天内无一给药组小鼠因白血病发病而导致死亡。综上所述，以洛哌丁胺为代表的阿片受体激动剂对包含MLL-AF9(MA9)-AML和AML-ETO9a(AE9a)-AML在内的多种白血病均表现出强有力的疗效。

Claims (4)

  1. 阿片受体激动剂在制造用于治疗恶性肿瘤的药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于恶性肿瘤细胞存在阿片受体信号通路。
  3. 阿片受体激动剂在制造用于治疗急性髓细胞性白血病的药物中的用途。
  4. 根据权利要求1或3所述的用途，其特征在于所述的阿片受体激动剂为洛哌丁胺、曲美布汀、阿西玛多林中的一种或多种。
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