CN113786490A - Application of preparation capable of regulating and controlling macrophage epigenetic inheritance in preparation of medicine for treating hemophagocytic syndrome - Google Patents

Abstract

The invention relates to application of a preparation capable of regulating and controlling macrophage epigenetic inheritance in preparation of a medicament for treating hemophagocytic syndrome, belonging to the technical field of biology. The preparation capable of regulating and controlling macrophage epigenetic inheritance is applied to preparing the medicine for treating hemophagocytic syndrome, and the preparation capable of regulating and controlling macrophage epigenetic inheritance is a DNA methyltransferase inhibitor, a histone acetylase inhibitor or a gene medicine for down-regulating the expression of DNA methyltransferase. Can reduce the expression level of inflammatory factors, increase blood cell count, relieve hepatosplenomegaly, and reduce the ratio and number of activated macrophages in spleen cells. The hemophagocytic syndrome is primary hemophagocytic syndrome or secondary hemophagocytic syndrome.

Description

Application of preparation capable of regulating and controlling macrophage epigenetic inheritance in preparation of medicine for treating hemophagocytic syndrome

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a preparation capable of regulating and controlling macrophage epigenetic inheritance in preparation of a medicine for treating hemophagocytic syndrome.

Background

Hemophagocytic syndrome (HLH) is a group of clinical syndromes caused by excessive activated proliferation of inflammatory cells including lymphocytes and monocytes and production of a large amount of inflammatory factors due to genetic or acquired immunodeficiency. The clinical manifestations are persistent fever, hepatosplenomegaly, pancytopenia and hemophagia in bone marrow, spleen, liver or lymph nodes. Once HLH occurs, the disease condition is serious and the disease death rate is high. The current clinical treatment means include induction treatment for relieving the storm of the inflammatory factor (such as using immunosuppressive drug cyclosporin, chemotherapeutic drug etoposide, hormone treatment, etc.) and treatment aiming at the cause of disease (such as actively controlling the primary disease or bone marrow transplantation). However, HLH patients are critical and rapidly progressed, the overall remission rate of the current induction treatment means is still limited, and the side effects of the medicines are large. A significant proportion of patients do not have the opportunity to receive treatment for the cause of the disease, and the overall prognosis is poor (Blood 135(16) (2020) (1332) -. Therefore, there is a great need to design and develop new therapeutic strategies to improve HLH therapeutic efficacy.

The malignant positive feedback cycle of "inflammatory factors (from pathogens or abnormal immune cells) -macrophage activation-activation of inflammatory pathways-high expression of inflammatory factors-more macrophage activation" is a common pathophysiological feature of HLH due to different causes. In view of the pathophysiological characteristics of mutual promotion between inflammatory factor storm and activated macrophage and promotion of development of HLH, researchers have used monoclonal antibodies against inflammatory factors, such as interleukin 6(IL-6) and interferon gamma (IFN-y) on the market, to treat HLH in recent years, and have achieved certain therapeutic effects (Blood advances 1(12 (2017) (779-791)). However, the monoclonal antibody can only target specific inflammatory factors, has a single action, and is difficult to effectively control the inflammatory factor storm in which multiple factors cooperatively participate. The JAK/STAT pathway is a common pathway of intracellular signal transduction of various cytokines, mediates various biological reactions such as cell proliferation, differentiation, migration, apoptosis and immunoregulation and is a common pathway of main pathogenic inflammatory factors such as IFN-y, IL-6 and the like in an HLH state (Blood 127(13), (2016) 1666-75). The selective JAK1/2 inhibitor lucagotinib for treating HLH can effectively inhibit the activation of a JAK/STAT pathway and reduce the expression of various inflammatory factors, and has better curative effect than an antibody on an animal level (Blood 134(2) (2019) 147-. However, the clinical therapeutic effect of HLH remains to be examined, and long-term administration increases the risk of lymphoma (Blood 132(7) (2018) 694-706). In addition, most of HLH patients are critically ill patients, and the oral administration compliance of the luccotinib is poor and the price is high, so that the clinical application is possibly limited to a certain extent.

Epigenetics is the regulation and control of gene expression through DNA methylation, histone acetylation, non-coding RNA, etc. under the premise that DNA sequence is not changed, so that the gene expression is heritably changed, and finally, phenotype is changed. Studies have shown that epigenetic regulation plays an important role in the maintenance of tissue homeostasis by macrophages, the response to different stimuli, and the development of innate immune memory. Macrophage activation is often accompanied by epigenetic aberrant changes such as changes in DNA methylation and histone acetylation. When macrophages are activated, the expression of DNA methyltransferase 1(DNA methyltransferase1, DNMT1) and the expression of DNMT3 are up-regulated and down-regulated, and the methylation degree of a peroxisome proliferator-activated receptor alpha (PPAR-alpha) transcription region is further increased to promote the activation of an inflammation pathway and the expression of inflammatory factors (JCI inertia 1(19) (2016) e 87748). These studies suggest that the activation state of the inflammatory pathway and the expression of various inflammatory factors can be effectively regulated by regulating epigenetic abnormality of activated macrophages, and a new treatment concept is expected to be provided for HLH which is mainly characterized by continuous activation of macrophages and storm of inflammatory factors.

Decitabine (DAC) is a water-soluble cytosine analogue, and phosphorylated DAC participates in DNA synthesis, can be combined with DNMT to inhibit DNMT activity, so that the effect of DNA demethylation is achieved. As a DNMT inhibitor, DAC is currently used clinically mainly for the treatment of myelodysplastic syndrome and partial myeloid leukemia. Relevant studies in sepsis and obese mouse models have shown that DAC can control macrophage activation status and reduce inflammatory factor secretion by inhibiting DNMT1 (JCI insight 1(19) (2016) e 87748; Frontiers in immunology 11(2020) 1360.). Therefore, the DAC is adopted to regulate and activate the epigenetic abnormality of the macrophage, so that the activation state of an inflammation channel and the expression of various inflammatory factors can be effectively regulated and controlled, and the inflammation storm can be effectively inhibited, so that the treatment effect of the HLH can be improved.

In the prior art, DAC or azacitidine with demethylation effect similar to DAC is reported to be used for controlling acute myelogenous leukemia or myelodysplastic syndrome combined with HLH in some cases, and certain curative effect is achieved. However, it is not clarified whether DAC or azacitidine can exert a therapeutic effect by controlling HLH by controlling the primary pathogenesis, or whether DAC or azacitidine itself can act on a certain link of HLH (Leuk Lymphoma.2011 Feb; 52(2): 341-3; Intern Med.2018 Oct 15; 57(20): 2995-.

In light of the above background, the present invention employs DAC-regulated epigenetic abnormalities that activate macrophages for therapeutic study of HLH.

Disclosure of Invention

The invention aims to provide an application of a preparation capable of regulating macrophage epigenetics in preparing a medicament for treating hemophagocytic syndrome, in particular to a DNA methyltransferase inhibitor, a histone acetylase inhibitor or a gene medicament for down-regulating the expression of DNA methyltransferase, and specifically relates to a preparation which takes epigenetic abnormality of activated macrophages as a target point, and improves the treatment effect of macrophage syndrome HLH by inhibiting the activation of the activated macrophages and reducing the release of subsequent inflammatory factors. Solves the technical problems of limited HLH induction treatment remission rate and large side effect of medicaments in the prior art.

According to the purpose of the invention, the application of the preparation capable of regulating and controlling macrophage epigenetic inheritance in preparation of the medicine for treating hemophagocytic syndrome is provided.

Preferably, the agent capable of regulating macrophage epigenetics is a DNA methyltransferase inhibitor, a histone acetylase inhibitor or a genetic drug which down-regulates DNA methyltransferase expression.

Preferably, the DNA methyltransferase inhibitor is decitabine or azacitidine; the histone acetylase inhibitor is entinostat; the gene medicine for down-regulating the expression of the DNA methyltransferase is LncRNA, miRNA, siRNA, antisense RNA, RNA aptamer or antisense DNA.

Preferably, the agent capable of modulating macrophage epigenetics is used for reducing the expression level of inflammatory factors;

preferably, the inflammatory factors are IFN-gamma, TNF-alpha, IL-6 and IL-1 beta.

Preferably, the preparation capable of modulating macrophage epigenetics is used for increasing blood cell count, alleviating hepatosplenomegaly and reducing the ratio and number of activated macrophages in spleen cells.

Preferably, the hemophagocytic syndrome is primary hemophagocytic syndrome or secondary hemophagocytic syndrome.

Preferably, the primary hemophagocytic syndrome is a gene deletion-induced hemophagocytic syndrome; the secondary hemophagocytic syndrome is infection, rheumatic disease, neoplastic disease or hemophagocytic syndrome induced by immunotherapy.

Preferably, the immunotherapy is CART therapy or mab therapy.

Preferably, the agent capable of modulating macrophage epigenetics is a free agent or is loaded by a drug delivery system;

preferably, the drug delivery system is a nanoparticle, a liposome, a micelle, a dendrimer or a cell membrane vesicle, and the preparation is loaded in the drug delivery system by physical embedding, adsorption or chemical bonding.

Preferably, the mode of administration of the medicament is subcutaneous, intravenous or intramuscular.

Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) the invention discloses a preparation capable of regulating and controlling macrophage epigenetic inheritance, which can reduce the proportion and the number of spleen activated macrophages in a hemophagocytic syndrome model animal, reduce the expression level of inflammatory factors, relieve symptoms related to inflammatory factor storm such as hepatosplenomegaly and reduction of blood cell count, and prolong the survival time of a hemophagocytic syndrome model mouse.

(2) The medicine provided by the invention is used for regulating and controlling macrophage activation and controlling systemic inflammatory factor storm to treat hemophagocytic syndrome through epigenetic medicine.

(3) In the invention, preferably, the DNA methyltransferase inhibitor is decitabine, and in vitro experiments prove that low-dose decitabine can inhibit the activation of macrophages by regulating and controlling DNA methyltransferase.

(4) The preferable decitabine has demethylation effect at low dose, and has low side effect and good safety in clinical use. The therapeutic target is activated macrophage, is suitable for all types of hemophagocytic syndromes, and has good universality.

Drawings

FIG. 1 is a graph showing the results of DAC modulating macrophage epigenetic abnormalities in vitro, thereby inhibiting macrophage activation.

FIG. 2 is a graph showing the results of DAC reduction of the expression levels of various inflammatory factors in the circulation of HLH model mice induced by CPG stimulation.

FIG. 3 is a graph showing the results of DAC-improved CPG-stimulated reduction of blood cells and hepatosplenomegaly, and reduction of the number of spleen cells and the ratio and number of activated macrophages in mice of the HLH model.

FIG. 4 is a graph showing the results of DAC decreasing the expression levels of various inflammatory factors in the circulation and extending the survival time of HLH model mice induced by polyinosinic acid in combination with LPS.

Figure 5 is a graph of the effect of different modes of administration.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the use of the present invention in the preparation of a medicament for the treatment of HLH is described in detail below with reference to the specific drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Various modifications and changes may be made by those skilled in the art within the scope of the present invention based on the description herein, and such modifications and changes are also encompassed within the scope of the present invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

In the present invention, besides unmethylated cytosine guanine dinucleotide (CPG) -induced HLH model and polyinosinic acid-LPS-induced HLH model, but not limited thereto, primary HLH model of key gene deletion and other secondary HLH models include CPG-IL 18-induced HLH model, LPS-mouse-induced HLH model for aging model, and the like are included.

In the present invention, the disease model is HLH, including HLH of different etiology, including but not limited to primary HLH due to deletion of key genes, secondary HLH induced by severe bacterial, viral or fungal infections, rheumatic diseases or neoplastic diseases, and HLH occurring during immunotherapy, such as CAR-T therapy and certain monoclonal antibody therapies.

According to the invention, decitabine DAC is preferably used as a therapeutic drug, non-methylated cytosine guanine dinucleotide (CPG) or polyinosinic acid is combined with LPS to induce and form an HLH animal model, and in vitro and in vivo experiments prove that DAC can inhibit activation of macrophages by inhibiting DNMT1, inhibit inflammatory factor storm, relieve related symptoms and prolong the survival time of a model mouse, and can be used for treating HLH.

The treatment for HLH in the present invention is preferably achieved by the following technical scheme:

(1) the RAW264.7 cell line stimulated with IFN- γ was a model of pro-inflammatory macrophages in vitro, DAC treated at different doses, cells were collected and western blot was used to assess the expression level of DNA methyltransferase I (DNMT1) and the activated phenotype alteration of macrophages (iNOS is a marker for pro-inflammatory macrophages, Arg-1 is a marker for anti-inflammatory macrophages).

(2) An HLH animal model is established by using a CPG stimulation method, a certain amount of DAC (0.5mg/kg, five times in total, and is parallel to the administration of CPG) is administered in a subcutaneous administration mode, blood is taken for measuring a blood routine, cytokine change in the blood is determined by Elisa, liver and spleen tissues are taken, weighing is carried out, a single cell suspension of spleen cells is obtained through separation, the number of the spleen cells is obtained through cell counting, the ratio of activated cells in the spleen cells is evaluated through flow cytometry, and the number of the activated cells is calculated.

(3) HLH animal models were created using polyinosinic acid in combination with LPS, and after a given amount of DAC (2mg/kg) was administered by subcutaneous administration, cytokine changes in blood were measured by Elisa and animal survival time was monitored.

Example 1

RAW264.7 cells were cultured, treated with DAC (control, 500nM,200nM,40nM) at different concentrations for 72h, incubated with 20ng/ml IFN-y for 24h, cells were collected and western blot was used to evaluate the expression levels of DMNT1, iNOS and Arg-1 in the different treatment groups. FIG. 1 is a graph showing the results of DAC modulating macrophage epigenetic abnormalities in vitro, thereby inhibiting macrophage activation. The low dose of DAC in FIG. 1 down-regulated DNMT1(A-B) and inhibited macrophage activation (A-D). western bolt (a) and corresponding semi-quantitative results (B-D). (B) Relative expression level of DNMT 1; (C) relative expression level of iNOS; (D) relative expression level of Arg-1. The results show that the low-concentration (40nM) DAC treatment group can obviously inhibit the expression of DNMT1 and simultaneously can reduce the labeled iNOS of activated macrophages, which indicates that the low-concentration DAC can regulate and control the apparent abnormality of the activated macrophages and inhibit the activation of the macrophages, and is consistent with the demethylation effect of low-dose DAC reported in the previous research.

Example 2

Mouse models of HLH induced by intraperitoneal injection of CPG (50. mu.g/animal, once every other day, five times total) DAC-treated groups and control groups were given DAC (0.5mg/kg) dissolved in PBS and an equal amount of PBS (once every other day, five times total, and CPG in parallel), and blood was taken after the end of administration and the level of inflammatory factors in serum was measured by Elisa. The results are shown in fig. 2, DAC can significantly reduce the expression level of various inflammatory factors in the circulation of CPG-stimulated HLH model mice compared to the PBS group. (A) IFN-gamma; (B) TNF-alpha; (C) IL-6; (D) IL-1 beta. FIG. 1 illustrates that DAC inhibits the occurrence of inflammatory factor storm by inhibiting macrophage activation.

Example 3

Establishing a CPG-induced HLH mouse model, taking blood after the administration in the embodiment 2 to detect the blood routine, taking out the spleen of the mouse liver, weighing the blood, preparing a single cell suspension of spleen cells, counting, analyzing the ratio of activated macrophages in the spleen cells by flow cytometry, and calculating the number of the activated macrophages in the spleen according to the ratio. As shown in FIG. 3, DAC relieved the symptoms associated with the inflammatory factor storm in CPG-stimulated HLH model mice, increased blood cell counts (A-C), relieved hepatosplenomegaly (D-F & I), and reduced the ratio and number of activated macrophages (G-H) in the spleen cells. (A) Counting white blood cells; (B) counting the hemoglobin; (C) (ii) platelet count; (D) liver weight; (E) spleen weight; (F) number of spleen cells; (G) activated macrophage fraction in spleen; (H) activated macrophage number in spleen; (I) splenic images of model mice were observed externally. The results showed that the blood cell count of DAC-treated group was significantly improved compared to the control group (A-C in FIG. 3), the hepatosplenomegaly of DAC-treated model mice was significantly relieved (D-E & I in FIG. 3), and the ratio and number of activated macrophages in the spleen were significantly lower than those of the control group (F-H in FIG. 3). As shown in FIG. 1 and FIG. 2, DAC treatment can inhibit macrophage activation, and relieve inflammatory factor storm and its related manifestations such as cytopenia, hepatosplenomegaly, etc.

Example 4

An HLH model is established by adopting a polyinosinic acid and LPS method (10 mg/kg of polyinosinic acid is injected into tail vein for 24h, and 5mg/kg of LPS is injected into abdominal cavity), blood is taken after subcutaneous administration according to DAC 2mg/kg, and the expression level of inflammatory factors in the blood is measured by Elisa. As can be seen from FIG. 4, DAC decreased the circulating inflammatory factor expression levels (A-D) and prolonged the survival time (E) of the model mice of HLH induced by polyinosinic acid in combination with LPS. (A) IFN-gamma; (B) TNF-alpha; (C) IL-6; (D) IL-1 β; (E) survival curves of model mice. The results show that DAC-treated groups can significantly reduce circulating inflammatory factor levels (a-D in fig. 4) in HLH model mice and significantly prolong the survival time of mice compared to PBS control group (E in fig. 4).

Example 5

An HLH model is established by adopting a polyinosinic acid and LPS method (after 10mg/kg of polyinosinic acid is injected into tail vein for 24h, LPS is injected into abdominal cavity for 5mg/kg), the survival time of a model mouse is monitored after subcutaneous, intravenous or intramuscular injection according to DAC 2mg/kg, and the result shows that the model mouse can survive for 48 hours after subcutaneous, intravenous and intramuscular injection, and the treatment effects of different administration modes are not different (figure 5).

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The preparation capable of regulating and controlling macrophage epigenetic inheritance is applied to preparing the medicine for treating hemophagocytic syndrome.

2. The use according to claim 1, wherein the agent capable of modulating macrophage epigenetics is a DNA methyltransferase inhibitor, a histone acetylase inhibitor or a genetic drug which down-regulates expression of DNA methyltransferase.

3. The use of claim 2, wherein the DNA methyltransferase inhibitor is decitabine or azacitidine; the histone acetylase inhibitor is entinostat; the gene medicine for down-regulating the expression of the DNA methyltransferase is LncRNA, miRNA, siRNA, antisense RNA, RNA aptamer or antisense DNA.

4. The use of claim 1, wherein the agent that modulates macrophage epigenetics is used to reduce the expression level of an inflammatory factor;

preferably, the inflammatory factors are IFN-gamma, TNF-alpha, IL-6 and IL-1 beta.

5. The use of claim 1, wherein the agent that modulates macrophage epigenetics is used to increase blood cell count, alleviate hepatosplenomegaly, and reduce the ratio and number of activated macrophages in spleen cells.

6. The use of claim 1, wherein the hemophagocytic syndrome is primary hemophagocytic syndrome or secondary hemophagocytic syndrome.

7. The use of claim 6, wherein the primary hemophagocytic syndrome is a gene deletion-induced hemophagocytic syndrome; the secondary hemophagocytic syndrome is infection, rheumatic disease, neoplastic disease or hemophagocytic syndrome induced by immunotherapy.

8. The use of claim 7, wherein the immunotherapy is CART therapy or monoclonal antibody therapy.

9. The use of claim 1, wherein the agent that modulates macrophage epigenetics is a free agent or is loaded by a drug delivery system;

preferably, the drug delivery system is a nanoparticle, a liposome, a micelle, a dendrimer or a cell membrane vesicle, and the preparation is loaded in the drug delivery system by physical embedding, adsorption or chemical bonding.

10. The use of claim 1, wherein said medicament is administered subcutaneously, intravenously, or intramuscularly.

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