CN110090303B - Use of opioid receptor agonists for the treatment of malignant tumors - Google Patents

Abstract

The invention discloses an application of an opiate receptor stimulant in the preparation of a medicine for treating malignant tumors. The present invention is based on the oncosuppressive gene role of TET2 in AML and the activation of TET2 expression by opioid receptor activation (such as the clinically approved antidiarrheal drug loperamide), and finds new uses for leukemia therapy beyond the conventional pharmacological effects of these opioid receptor agonists. Also provides important scientific basis for the 'old medicine new use' of opioid receptor agonists such as loperamide and the like. The application of loperamide in preparing AML treatment medicines provided by the invention obviously improves the defects of conventional AML chemotherapy (high chemotherapeutic dose, long treatment course, large side effect and frequent intolerance of patients), and has less adverse drug reactions and lighter side effects.

Description

Use of opioid receptor agonists for the treatment of malignant tumors

Technical Field

The invention belongs to the technical field of biological medicines. In particular to the application of an opioid receptor agonist in the preparation of a medicament for treating malignant tumors.

Background

Acute Myelogenous Leukemia (AML) is one of the most common lethal hematopoietic malignancies and also the most common Acute leukemia in adults, with complex genetic diversity and widely varying response to therapy. Several AML subtypes, including MLL-fused AML, have poor prognosis even when given chemotherapy. The incidence of AML increases with age, and elderly patients, due to their physical problems, are poorly tolerated by high-intensity chemotherapy, with particularly poor prognosis. By standard chemotherapy, only 30-50% of young AML patients and 5-20% of elderly patients under the age of 60 can survive for more than 5 years. The options for AML treatment are very limited. For the past few decades, the first line treatment regimen has been primarily standard "7 + 3" chemotherapy, i.e., the combination therapy of nucleoside analogs, represented by cytarabine (Ara-C), with anthracyclines, represented by Doxorubicin (DOX). In recent years, it has been gradually recognized that the separate discussion of genetic (genetic) abnormalities, such as genetic mutations, chromosomal remodeling, gene copy number abnormalities, etc., has failed to fully explain the complex pathological manifestations and prognosis of AML; epigenetic regulation is a further fine-tuning on a genetic basis. Recent studies by the research team of the applicant find that the fine adjustment can often play a decisive role in the occurrence and development of leukemia, drug response and the like. On the basis, further research on the action mechanism of epigenetic regulation in leukemia and development of new ways for treating leukemia by intervening in epigenetic regulation are imminent.

The TET (Ten-eleven transition) family protein TET1/2/3 achieves DNA demethylation by hydroxylating 5-methylcytosine (5-methylcytosine,5mC) to 5-hydroxymethylcytosine (5-hydroxymethynychytosine, 5 hmC). Recent studies have found that TET-mediated 5hmC modification plays an important role in many important physiological and pathological processes, such as differentiation and development. The applicant's research team discovered and reported for the first time the important role of TET 1-mediated DNA 5hmC modification in AML.

Similar to TET1, another member of the TET family, TET2, also has the function of catalyzing DNA modification at 5hmC, with AML mutated at the TET2 gene exhibiting low 5hmC levels. In contrast to the oncogene role of TET1, TET2 has been identified as a tumor suppressor gene in a variety of tumors, including leukemia. TET2 mutation exists in various subtypes of AML, and the mutation rate of functional deletion of TET2 gene can reach 7-23% in AML. Furthermore, the TET2 mutation is often accompanied by other disease-causing gene mutations, such as the NPM1 mutation. It was found that TET2 mutant AML generally has a poorer prognosis. In addition to genetic mutations, the level of TET2 expression is also closely related to the development of AML. In vitro experiments, the down regulation of TET2 can obviously promote the proliferation of AML cells; whereas overexpression of TET2 inhibited AML cell expansion. New studies have shown that in Tet 2-induced knockdown hematopoietic stem/precursor cells (HSPCs), restoration of Tet2 expression by withdrawal of the inducer is effective in promoting myeloid differentiation and cell death. Certain drugs, such as vitamin C, in combination with PARP inhibitors kill AML cells by activating the enzymatic activity of TET 2. Furthermore, like TET1, TET2 may also function in a kinase activity independent manner. Given the two modes of action, dependent and independent of the kinase activity of TET family members, remodeling TET2 expression in tumor cells clearly has new implications compared to remodeling TET2 functional activity, and the question of how to treat AML by effectively promoting TET2 expression has not been hitherto thought to be the answer. The above studies suggest that enhancing the expression of TET2 in AML by signal stimulation would be a new concept, a new option, for the treatment of AML.

The role of opioid receptor signaling pathways in tumors has long been controversial. Opioid agonists such as morphine, fentanyl, and the like are frequently used as a potent analgesic adjuvant therapy in the treatment of tumors. It is not clear whether such agents have a direct tumor-inhibiting effect. Research reports that methadone and doxorubicin act synergistically to inhibit tumor growth in acute lymphocytic leukemia (pre-B-ALL). Endorphin treatment can cause apoptosis in an AML cell line HL-60 cultured in vitro, and the mechanism thereof has not been studied in depth. At present, some opioid receptor signal pathway modulators are put into clinical use and are routinely used for the therapeutic purposes of sedation, analgesia, antidiarrheal and the like. Such as the opioid receptor agonist loperamide (loperamide; also known as cloperamide, robumeil, chemical name 4-p-chlorobenzene-4-hydroxy-N, N-dimethyl-alpha, alpha-diphenyl-1-p-piperidyl butanamide, formula C ₂₉ H ₃₃ ClN ₂ O ₂ ) The long-acting antidiarrheal powder is a long-acting antidiarrheal medicine, is mainly used for clinically treating acute and chronic diarrhea caused by various reasons, and is particularly suitable for long-term treatment of chronic diarrhea. Whether the above opioid receptor agonists have epigenetic regulatory mechanisms and can be directly used for AML treatment has not been reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and discloses application of an opioid receptor agonist in preparing a medicament for treating malignant tumors. Based on the oncostatin effect of TET2 in AML and the activation of TET2 expression by opioid receptor activation (such as the clinically approved antidiarrheal loperamide), the use of opioid receptor agonists (such as the opioid receptor agonist) for developing novel therapies for AML targeting the opioid receptor and TET2 pathways and for developing drugs for treating acute myeloid leukemia other than the conventional pharmacological effects of these opioid receptor agonists is disclosed. Further, the opioid receptor agonist includes but is not limited to one or more of loperamide, trimebutine and asimadoline.

The invention has the advantages that:

the invention provides an important research basis for treating malignant tumors, particularly leukemia by taking an opioid receptor signal pathway and TET2 as drug targets; also provides important scientific basis for the 'old medicine new use' of opioid receptor agonists such as loperamide and the like.

Abnormal expression or function of TET2 is a common pathogenesis of various malignant tumors including AML, and how to effectively regulate expression of tumor TET2 has not been reported, thereby achieving the purpose of therapeutic intervention. The invention is the first case of specific molecular targeted therapy aiming at the regulation and control of an opioid receptor signal pathway and TET2 expression, has obvious killing effect on various subtype AML cells, has broad-spectrum AML resistance function, and can also be suitable for other malignant tumors with similar molecular mechanisms.

Traditional AML chemotherapy has high dose, long course of treatment, large side effects, often intolerance of patients, and is especially serious for elderly patients. The loperamide AML treatment regimen provided by the present invention significantly ameliorates the above-mentioned disadvantages of conventional AML chemotherapy. The medicament used in the treatment scheme provided by the invention is loperamide which is commonly used for clinically checking diarrhea, and the medicament has fewer adverse reactions and lighter side effects; in addition, the administration dosage in the animal model is lower (only 0.25 mg/kg; here, the conventional chemotherapy dosage of AML 5+3 course: Ara-C50 mg/kg, DOX 3mg/kg) can be referred and compared, the course of treatment is only 7 days, no obvious toxic or side effect is found in the course of treatment and after the course of treatment is finished, and the animal tolerance is good.

Compared with the previously reported vitamin C capable of activating the enzymatic activity of TET2, the invention can directly activate the expression of TET2 and fundamentally up-regulate TET2, thereby simultaneously strengthening the activity dependence and the independent function of TET2 kinase, and having stronger treatment application prospect.

Drawings

FIGS. 1A-C show the effect of various opioid receptor agonists and antagonists on AML cell activity and cell proliferation.

Wherein, FIG. 1A shows the effect of opioid receptor agonist loperamide (OPA1), trimebutine (OPA2), asimadoline (OPA3), opioid receptor antagonist polyethylene glycol nalenol (OPANT1) or naltrexone (OPANT2) on the activity of AML cell line THP-1.

FIG. 1B is a graph showing the effect of the opioid receptor agonist loperamide (OPA1), trimebutine (OPA2), asimadoline (OPA3), the opioid receptor antagonist polyethylene glycol nalenol (OPANT1) or naltrexone (OPANT2) on proliferation of the AML cell line THP-1.

Figure 1C is a graph of the effect of the opioid receptor agonist loperamide (OPA1) on the activity of various AML cell lines.

FIGS. 2A-B are graphs showing the effect of opioid receptor agonists and antagonists on TET2 expression and cellular 5-hmC modification.

Among them, fig. 2A shows the effect of opioid receptor agonist loperamide (OPA1), trimebutine (OPA2), asimadoline (OPA3), opioid receptor antagonist polyethylene glycol nalenol (OPANT1) or naltrexone (OPANT2) on the expression level of TET2 in AML cell line THP-1.

FIG. 2B is a graph of the effect of the opioid agonist loperamide (OPA1), the opioid antagonist naltrexone (OPANT2) on the level of modification of DNA 5hmC in the AML cell line THP-1.

Figures 3A-B are graphs showing the in vivo therapeutic effect of opioid agonists exemplified by loperamide in a mouse AML model.

Wherein, FIG. 3A shows the in vivo therapeutic effect of loperamide in mouse MLL-AF9(MA9) -AML.

FIG. 3B is the in vivo efficacy of loperamide in mouse AML-ETO9a (AE9a) -AML.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following embodiments.

1. Material

KASUMI-1 cells were purchased from Shanghai stem cell banks of Chinese academy of sciences;

THP-1, MV 4; 11. cells such as NB4 and U937 were purchased from ATCC.

2. Reagent

Loperamide, naltrexone, trimebutine, and asimadoline were purchased from shanghai ceramic Biochemical technologies, ltd;

the polyethylene glycol naloxonol is purchased from seleck;

MTT kit, DMSO, RPMI-1640 medium from Sigma;

the TET2 quantitative PCR primer is synthesized by Hangzhou Youkang biological design;

fetal bovine serum was purchased from Gibco;

Trizol-RNAasso is available from TAKARA;

RNA reverse transcription kit and QPCR kit were purchased from Nanjing Novozam Biotech, Inc.;

the plasmid extraction kit was purchased from Nanjing Novozam Biotech Co.

Example 1 assay of opioid receptor agonists and antagonists for AML cell activity and proliferation.

1.1 cell culture

THP-1 cells, MV 4; 11 cells, NB4 cells, U937 cells and KASSUMI-1 cells were cultured in a medium containing 90% volume fraction of RPMI-1640, 10% fetal bovine serum and 1% of diabase at 37 ℃ and 5% CO ₂ Culturing in an incubator with concentration and saturated humidity.

1.2 cell Activity i.e. MTT assay

And (3) MTT preparation: to 5mg of MTT, 1ml of sterile PBS was added for dissolution to prepare a 12mM MTT solution; storing in a refrigerator at-20 deg.C in dark place.

The cultured cells were inoculated in a 96-well plate in an amount of 100. mu.l per 10000 cells per well, 1. mu.M loperamide (OPA1), trimebutine (OPA2), asimadoline (OPA3), opioid receptor antagonist polyethylene glycol nalenol (OPANT1) or naltrexone (OPANT2), or DMSO control, respectively, and after incubating the cells in an incubator at 37 ℃ for 48 hours, 10. mu.l of 12mM MTT solution was added per well, the incubator at 37 ℃ was incubated for 4 hours, 50. mu.l of DMSO solution per well was added to terminate the MTT reaction, the incubation was carried out for 10 minutes, and the absorbance at 540nm was measured by a microplate reader.

1.3 cell proliferation assay

The cultured cells were plated in a 96-well plate at 100. mu.l per 10000 cells per well, and 1. mu.M loperamide (OPA1), trimebutine (OPA2), acemadoline (OPA3), opioid receptor antagonist polyethylene glycol nalenol (OPANT1) or naltrexone (OPANT2), or DMSO control, respectively, and viable cells were counted at 12hrs, 24hrs, 48hrs, 72hrs, and 96hrs after the plating.

As shown in FIG. 1, various opioid receptor agonists including loperamide (OPA1), trimebutine (OPA2) and asimadoline (OPA3) all showed significant inhibition of cell activity and cell proliferation of the AML cell line THP-1 (FIGS. 1A-B). Among them, loperamide (OPA1) showed the most significant inhibitory effect (FIG. 1A-B). The opioid receptor antagonists, macrogol (OPANT1) and naltrexone (OPANT2), enhanced AML cell activity and cell proliferation (fig. 1A-B).

The opioid receptor agonist loperamide (OPA1) pair comprises THP-1, MV 4; 11, KASUMI1, U937, NB4, which represent different AML subtypes, all showed significant inhibition of cell activity in various AML cell lines (fig. 1C), suggesting that loperamide may have universal efficacy in various AML subtypes.

Example 2 detection of opioid receptor agonists and antagonists for TET2 expression and cellular 5-hmC modification. 2.1 Total RNA extraction from cells

Taking 2x10 cells in logarithmic growth phase ⁶ Repeatedly blowing and beating 500 microliters of Trizol-RNAasso, uniformly mixing, standing on ice for 5min, then adding 100 microliters of chloroform, violently shaking for 15sec, and standing at room temperature for 5 min; centrifuging at 12000g/4 ℃/15 min; taking the supernatant to a new EP tube, adding equal amount of isopropanol to precipitate RNA, mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000g/4 ℃/10 min. Discarding the supernatant, washing the precipitate with 75% anhydrous ethanol, and centrifuging at 7500g/4 deg.C for 5 min; the supernatant was discarded, the EP tube was dried in a fume hood for 10min, then 20. mu.l of DEPC water was used to dissolve the RNA, the RNA concentration was determined, and the sample was stored in a freezer at-80 ℃.

2.2 reverse transcription of RNA and QPCR detection

The system is as follows:

QPCR System:

2.35 hmC immunofluorescent staining

After 48h of the drug or DMSO control treatment, 1x10 was collected ⁶ Each cell was treated with 5After washing once with 00. mu.l PBS, at 2500rpm, centrifuging for 2.5min, discarding the supernatant, adding 80. mu.l PBS to resuspend, dropping on a charged glass slide, and after 20min, blocking with 60. mu.l 4% paraformaldehyde + 0.3% Triton-X100 for 10 min. After 3 washes with 100. mu.l PBS, primary antibody (anti-5 hmC antibody, #39769)1:200) was incubated overnight at 4 ℃. The primary antibody was washed 3 times with 100. mu.l PBS, the secondary antibody was incubated (immunofluorescence 488 rabbit secondary antibody (# A-11008)1:1000), incubated 1h at room temperature in the dark, the secondary antibody was washed 3 times with 100. mu.l PBS, and finally mounted with a DAPI-containing mounting plate (Shanghai san-assist Biotech Co., Ltd., #36308ES11) and observed by fluorescence microscopy.

FIGS. 2A-B show the effect of various opioid receptor agonists and antagonists on TET2 expression and cellular 5-hmC modification.

Wherein, fig. 2A shows: various opioid receptor agonists including loperamide (OPA1), trimebutine (OPA2), and asimadoline (OPA3) significantly elevated TET2 expression in the AML cell line THP-1, while opioid receptor antagonists, polyethylene glycol naloxonol (OPANT1) and naltrexone (OPANT2), inhibited TET2 expression.

FIG. 2B shows: the opioid receptor agonist loperamide (OPA1) significantly enhanced the level of DNA 5hmC modification in the AML cell line THP-1, while the opioid receptor antagonist naltrexone (OPANT2) inhibited the cellular DNA 5hmC modification. The above results suggest: opioid receptor agonists have an enhancing effect on TET 2-mediated DNA 5hmC modification, whereas opioid receptor antagonists counter act.

Example 3 in vivo therapeutic efficacy of loperamide in mouse AML model

3.1 bone marrow transplantation experiments

MLL-AF9 or AML-ETO9a primary AML mouse bone marrow cells (primary donor: B6.SJL (CD45.1) wild mouse bone marrow cells infect MLL-AF9 or AML-ETO9 a; primary receptor: C57BL/6(CD 45.2); primary AML: CD45.1 at the time of collecting samples ⁺ ％>90%) as donor cells, 1 × 10 ⁵ Each donor cell was resuspended in 100. mu.l PBS and transplanted into each second-generation recipient (C57 BL/6; CD45.2) mouse irradiated with a semi-lethal dose (480rads) by tail vein injection.

3.2 drug treatment

Loperamide or DMSO control at 0.25mg/kg was administered 1 week after transplantation, i.p. once a day for 7 days. The symptoms and phenotype of the mice were observed daily. When the mice are dying and meet the human-path sacrifice standard, the mice are sacrificed, and samples of peripheral blood, liver, spleen, bone marrow and the like are collected. The survival time was recorded. Each group of 8-10 weeks old C57BL/6(CD45.2) second generation recipient mice 10, randomly grouped, and half of the male and female.

As shown in fig. 3A-B, opioid agonists, for example loperamide, showed significant in vivo efficacy in the mouse AML model. Loperamide extended the survival time of half of leukemic mice from 21 days (fig. 3A control group) to more than 40 days (fig. 3A dosing group) in the mouse MLL-AF9(MA9) -AML model, i.e., none of the mice in the dosing group died of leukemic morbidity within 40 days after bone marrow transplantation; in a somatic model of AML-ETO9a (AE9a) -AML loperamide extended the survival time of half of leukemic mice from 22 days (fig. 3B control group) to more than 40 days (fig. 3B dosing group), and none of the mice in the dosing group died due to leukemia onset within 40 days after bone marrow transplantation. In conclusion, opioid agonists represented by loperamide showed potent therapeutic effects on various leukemias including MLL-AF9(MA9) -AML and AML-ETO9a (AE9a) -AML.

Sequence listing

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Claims (1)

1. Use of an opioid receptor agonist in the manufacture of a medicament for the treatment of acute myeloid leukemia, the leukemia cells of which are THP-1 cells, which present an opioid receptor signaling pathway; the opioid receptor agonist is one or more of trimebutine and acipimelin.

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