CN116492325A - Drug development for inhibiting Tet target protein by taking itaconic acid as starting point - Google Patents

Abstract

The invention provides an application of itaconic acid in treating Tet enzyme related diseases. In particular, the invention provides the use of itaconic acid to treat diseases associated with Tet enzyme, particularly cytokine storm-related diseases, by inhibiting Tet enzyme activity. The invention proves that the intraperitoneal injection of the metabolite small molecular itaconic acid can lead the mice to resist LPS-induced endotoxemia, and is particularly characterized by reducing cytokine level, relieving pulmonary edema and liver injury and prolonging the survival time of the mice. Itaconic acid is naturally-occurring, safe and effective, and has application prospect for developing medicines for treating diseases related to Tet enzyme.

Description

Drug development for inhibiting Tet target protein by taking itaconic acid as starting point

Technical Field

The invention relates to the field of biological medicine. In particular to the application of itaconic acid and derivatives thereof in preparing medicaments for treating Tet enzyme related diseases.

Background

Cytokine storm syndrome (also known as cytokine release syndrome) is a common systemic inflammatory response caused by infection with pathogenic bacteria such as viruses and is characterized by numerous cells producing and releasing large amounts of pro-inflammatory cytokines. This uncontrolled inflammatory response can lead to septic shock, multiple organ damage, and even ultimately organ failure. Severe new coronatine pneumonia and the resulting death thereof are typical cytokine storm syndromes.

In addition to the use of antibiotics to inhibit the source of infection in pathogenic bacteria such as viruses and bacteria, the elimination of excessive inflammatory responses including cytokine storm-induced disease-induced tissue damage is also an important component of therapy. Currently common anti-inflammatory drugs are classified as steroids and non-steroids. Wherein, the steroid is generally a hormone medicine (such as adrenal gland hormone) with strong anti-inflammatory effect, but has side effects of water-sodium retention, becoming fatigued, osteoporosis and the like. The nonsteroidal antiinflammatory effect is weak, the side effects are mainly gastrointestinal tract reaction, and the risk of cardiovascular diseases is also caused. Therefore, there is a need to develop safer and more effective anti-inflammatory drugs.

The epigenetic enzyme Tet family has three members (Tet 1/2/3) and participates in the active demethylation process of DNA, and is closely related to the gene transcription regulation. Previous studies have shown that upon infection by pathogenic bacteria such as bacteria, viruses, fungi, etc., host macrophages undergo demethylation of genomic DNA, activating expression of genes associated with inflammation such as NF- κb and the interferon pathway, and the immune response of the organism. Tet2 plays a role in this process.

The Tet protein depends on alpha-ketoglutarate as a cofactor, and small molecules of metabolites which can inhibit the activity of Tet enzyme, which are discovered before, are mostly analogues of alpha-ketoglutarate. They inhibit the enzymatic activity of Tet proteins by competitively binding to their active centers, such as hydroxyglutarate, succinic acid, fumaric acid, etc. However, the metabolite molecules have influence on the activities of a plurality of enzymes which are dependent on alpha-ketoglutarate, and have the problems of non-specific targets, low selectivity on Tet enzyme and the like. The above metabolite molecules accumulate slowly or not at all when the pathogenic bacteria infect macrophages. The development of specific Tet inhibitors is currently blank.

There is therefore a need in the art to provide a medicament which is capable of treating Tet enzyme related diseases, in particular cytokine storm syndrome, but which has no effect on the activity of other alpha-ketoglutarate dependent enzymes.

Disclosure of Invention

The invention aims to provide a medicine which can treat Tet enzyme related diseases and has no influence on the activity of other alpha-ketoglutarate dependent enzymes.

In a first aspect of the invention, there is provided a formulation combination comprising:

(A) A pharmaceutical composition comprising itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an accelerator thereof, as an active ingredient;

(B) Reagents for detecting Tet enzyme.

In another preferred embodiment, the itaconic acid derivative is chemically modified itaconic acid, preferably octyl modified itaconic acid.

In another preferred embodiment, the accelerator is selected from the group consisting of: any one or more of an itaconic acid metabolic precursor, an enzyme required for metabolism to produce itaconic acid, a metabolic enzyme activator for itaconic acid or precursor production, a promoter element that upregulates the level of gene or mRNA or protein expression of an enzyme required for metabolism to produce itaconic acid, and/or an expression vector.

In another preferred embodiment, the itaconic acid metabolic precursor is cis-aconitic acid.

In another preferred embodiment, the enzyme required for the metabolic production of itaconic acid is aconitate decarboxylase Irg1.

In another preferred embodiment, the Tet enzyme comprises: tet1, tet2, tet3, or a combination thereof.

In another preferred embodiment, the Tet enzyme comprises: tet2, tet3, or a combination thereof.

In another preferred embodiment, the reagent for detecting Tet enzyme comprises: reagents for detecting the content of a Tet enzyme, reagents for detecting the activity of a Tet enzyme, reagents for detecting the expression level of a gene encoding a Tet enzyme, or combinations thereof.

In another preferred embodiment, the reagent for detecting a Tet enzyme is a reagent for detecting a Tet enzyme activity.

In another preferred embodiment, the detection of the Tet enzyme content refers to detecting the Tet enzyme content in the cell.

In another preferred embodiment, the cells include macrophages and monocytes.

In another preferred embodiment, the cells are peripheral blood mononuclear cells.

In another preferred embodiment, the detection of the Tet enzyme activity means detection of the content of a Tet enzyme catalytic product.

In another preferred embodiment, the detection of Tet enzyme activity refers to the detection of the 5-hydroxymethylcytosine (5 hmC) content of DNA.

In another preferred embodiment, said detecting Tet enzyme activity comprises detecting Tet enzyme activity in vitro and detecting Tet enzyme activity in a tissue.

In another preferred embodiment, the active ingredient is 0.1-99% by weight of the total weight of the medicament.

In another preferred embodiment, the Tet enzyme is Tet2 and the pharmaceutical composition further comprises an additional agent for the prevention and/or treatment of a cytokine storm-related disease.

In another preferred embodiment, the anti-inflammatory component comprises: steroidal anti-inflammatory drugs (e.g., hydrocortisone, prednisone, dexamethasone), non-steroidal anti-inflammatory drugs (e.g., aspirin, acetaminophen, indomethacin, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib), or combinations thereof.

In another preferred embodiment, the pharmaceutical composition is formulated as an oral or non-oral formulation.

In another preferred embodiment, the pharmaceutical composition is selected from the group consisting of: injection, inhalant, tincture, powder, granule, capsule, oral liquid, tablet, pill, suspension, emulsion, buccal tablet, or dripping pill.

In another preferred embodiment, the pharmaceutical composition is administered orally or by injection.

In another preferred embodiment, the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs for preventing and/or treating cytokine storm-related diseases.

In another preferred embodiment, the pharmaceutical composition is administered to a human or non-human mammal (e.g., a rodent).

In another preferred embodiment, the combination of formulations is used to carry out a method for preventing and/or treating a disease in which the Tet enzyme is increased or the Tet enzyme activity is modulated.

In another preferred embodiment, the method for preventing and/or treating Tet enzyme increase or Tet enzyme activity up-regulation of disease comprises the steps of;

(i) Obtaining a sample to be tested from a subject;

(ii) Detecting the content of the Tet enzyme or the activity of the Tet enzyme in the sample to be detected by using the reagent (B) for detecting the Tet enzyme;

(iii) If the test sample is detected to be positive for the Tet enzyme, the pharmaceutical composition (A) is applied to the test subject to prevent and/or treat the Tet enzyme increase or the diseases related to the Tet enzyme activity up-regulation.

In another preferred embodiment, the Tet enzyme positivity comprises: the content of the Tet enzyme increases, the activity of the Tet enzyme increases, and the expression level of the gene encoding the Tet enzyme increases.

In another preferred embodiment, the increase in Tet enzyme means that the ratio of the Tet enzyme content (E1) to the physiological content (E0) (i.e.E1/E0) is 1.2 or more, preferably 1.5 or more, more preferably 2.0 or more

In another preferred embodiment, the Tet enzyme content refers to the Tet enzyme content in monocytes or macrophages.

In another preferred embodiment, the Tet enzyme content refers to the Tet enzyme content in peripheral blood mononuclear cells.

In another preferred embodiment, the increase in Tet enzyme activity means that the ratio of the level of formation of the Tet enzyme catalytic product (L1) to the level of formation of the catalytic product in physiological conditions (L0) (i.e.L1/L0) is 1.2 or more, preferably 1.5 or more, more preferably 2.0 or more.

In another preferred embodiment, the Tet enzyme catalytic product comprises 5-hydroxymethylcytosine (5 hmC). In another preferred embodiment, the Tet enzyme is Tet1 and the disease is a fertilized egg and embryo dysplasia related disease.

In another preferred embodiment, the Tet enzyme is Tet2 and the disease is a cytokine storm-related disease.

In another preferred embodiment, the cytokine storm-related disease is a disease in which abnormal increase of cytokines in a short period of time causes decrease of organ function.

In another preferred embodiment, the cytokines include NF-. Kappa.B and cytokines and chemokines that regulate the JAK-STAT signaling pathway.

In another preferred embodiment, the cytokines include, but are not limited to: IL1 beta, IL6, IL12, IFN alpha, IFN beta, IFN gamma, CXCL9, CXCL10, CXCL11, CXCL5.

In another preferred embodiment, the cytokine storm-related disease comprises: infectious diseases such as bacteria or viruses, autoimmune diseases, or combinations thereof.

In another preferred embodiment, the infectious disease includes bacterial infection and viral infection.

In another preferred embodiment, the infectious disease comprises: endotoxemia, acute pneumonia, acute hepatitis, acute meningitis, acute keratitis, or combinations thereof.

In another preferred embodiment, the autoimmune disease comprises: multiple sclerosis.

In another preferred embodiment, the Tet enzyme is Tet3 and the disorder is a neurological disorder.

In a second aspect of the invention there is provided the use of a combination of formulations as described in the first aspect of the invention for the preparation of a kit for the treatment of diseases in which Tet enzyme is increased or activity is upregulated.

In another preferred embodiment, the kit wherein the reagent for detecting Tet enzyme and the pharmaceutical composition are each independently packaged.

In another preferred embodiment, the kit further comprises a label and/or instructions, wherein the instructions note that the kit is useful for treating diseases in which Tet enzyme is increased or activity is upregulated.

In a third aspect of the invention, there is provided the use of itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an enhancer thereof, for the manufacture of a formulation or pharmaceutical composition for the treatment of a Tet enzyme-related disease.

In a fourth aspect of the invention, there is provided a method of non-therapeutically inhibiting Tet enzyme activity in vitro comprising the steps of:

releasing the Tet enzyme from itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or a promoter thereof, thereby inhibiting the activity of the Tet enzyme.

In another preferred embodiment, the Tet enzyme is a free Tet enzyme, or an intracellular Tet enzyme.

In another preferred embodiment, the cells are monocytes or macrophages.

In a fifth aspect of the invention, there is provided a method of treating and/or preventing a Tet enzyme-related disorder comprising the steps of:

administering to a subject in need thereof a therapeutically effective amount of itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an enhancer thereof, to treat and/or prevent a Tet enzyme-related disease.

In another preferred embodiment, the method comprises the steps of:

(i) Obtaining a sample to be tested from a subject;

(ii) Detecting the content of the Tet enzyme or the activity of the Tet enzyme in the sample to be detected by using the reagent (B) for detecting the Tet enzyme;

(iii) If it is detected that there is an increase in the Tet enzyme or an increase in the Tet enzyme activity in the sample to be tested, the pharmaceutical composition (A) is administered to the subject, thereby treating and/or preventing the Tet enzyme-related disease.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 shows the results of experiments for specific inhibition of Tet2 by itaconic acid.

FIG. 1a shows an in vitro enzyme activity flow chart for detecting Tet based on an in vitro enzyme activity system of immobilized substrate DNA and 5hmC fluorescence detection.

FIG. 1b shows the determination of the effect of itaconic acid and of the indicated metabolites on Tet2 enzyme activity in vitro.

FIG. 1c shows the determination of genomic 5hmC levels of itaconic acid and OI inhibited macrophages by LC-MS.

Figure 1d shows that LPS stimulated macrophages to produce high levels of itaconic acid.

FIG. 1e shows that accumulation of itaconic acid results in inhibition of Tet2 activity.

FIG. 1f shows that itaconic acid accumulation results in other enzymes that do not affect alpha-ketoglutarate dependence, such as histone demethylase KDM.

FIG. 2 shows the results of experiments on itaconic acid to inhibit Tet2 and prevent expression of inflammatory related genes such as NF- κB and the interferon pathway.

FIG. 2a shows that about 72% of the genes in macrophages that are induced by LPS are inhibited by either itaconic acid or Tet2 gene knockout.

FIG. 2B shows that NF- κB and interferon pathway inflammation related genes are up-regulated by Tet2 induction and can be significantly down-regulated by itaconic acid mediated inhibition of Tet2 activity.

Figure 3 shows itaconic acid protects mice against endotoxemia by inhibiting Tet 2.

Figure 3a shows a schematic representation of a mouse model of endotoxemia induced by intraperitoneal injection of itaconic acid and LPS.

FIG. 3b shows that itaconic acid reduces LPS-induced increases in pro-inflammatory cytokines and chemokines in Tet2 wild-type chimeric mice, but does not reduce Tet2 ^HxD Increased proinflammatory cytokines and chemokines in chimeric mice.

FIG. 3c shows that itaconic acid reduces LPS-induced liver damage in Tet2 wild type chimeric mice, but does not reduce Tet2 ^HxD Liver injury in chimeric mice.

FIG. 3d, FIG. 3e, FIG. 3f show that itaconic acid can alleviate LPS-induced pulmonary edema and lung injury in Tet2 wild type chimeric mice, but cannot alleviate Tet2 ^HxD Pulmonary edema and lung injury in chimeric mice. Fig. 3d shows the wet/dry weight ratio of lung tissue in mice, fig. 3e shows HE staining and fig. 3f shows clinical scoring of lung lesions.

FIG. 3g is a Kaplan-Meier survival curve showing that itaconic acid protects the LPS-induced mortality of Tet2 wild type chimeric mice, but does not protect Tet2 ^HxD Chimeric mice.

FIG. 4 shows the in vitro determination of the effect of itaconic acid on Tet3 enzyme activity.

The display values are the average of three replicates and s.d. P values were compared multiple times using t-test or two-way anova. * P < 0.05, p < 0.01, p < 0.001, p < 0.0001, n.s. no significant.

Detailed Description

The inventor of the present invention has developed the use of itaconic acid in preparing medicines for treating diseases related to Tet enzyme, especially diseases related to cytokine storm for the first time through extensive and intensive research. Specifically, the inventors have demonstrated that intraperitoneal injection of the small metabolite molecule with unmodified, naturally occurring, safe and effective itaconic acid can protect mice against LPS-induced endotoxemia, specifically by reducing cytokine levels, alleviating pulmonary edema and liver injury, and extending survival of mice. On this basis, the present invention has been completed.

In the invention, itaconic acid is the first specific inhibitor of the Tet family enzyme, especially specific inhibition of Tet2, which only inhibits the activity of the Tet enzyme, but does not inhibit other enzymes which are activated by alpha-ketoglutarate similar to the Tet enzyme, through in vitro enzyme activity, nuclear magnetic resonance analysis, cell experiments and in vivo animal experiments. Therefore, the method of the invention can prevent the interference to other normal enzyme functions while treating the cytokine storm.

Meanwhile, the invention defines that the Tet enzyme is a main functional protein target for mediating the anti-inflammatory effect of itaconic acid. The in vivo experiments of the invention prove that the Yikang medicineAcid is used for treating Tet2 ^HxD The inflammatory reaction of mice (i.e., the catalytic inactivation of Tet2 enzyme) did not lead to a significant decrease in the inflammatory index. But it has a significant inflammation-inhibiting effect on wild-type (tet2+/+) mice. Accordingly, the invention provides the combined use of the reagent for detecting the Tet2 activity and the itaconic acid so as to realize the accurate treatment of the inflammation.

Therefore, the invention develops a Tet2 enzyme activity specific inhibitor aiming at the Tet targeting protein by utilizing a safe natural metabolite molecule, namely itaconic acid as an inhibitor pharmacophore starting point, and prevents the expression of inflammation related genes such as NF- κB, interferon pathway and the like and the immune response of organisms, and the Tet2 enzyme activity specific inhibitor is used as an original medicament for safely and effectively treating diseases caused by excessive inflammatory reactions including cytokine storm.

Terminology

As used herein, the terms "pharmaceutical composition of the present invention", "medicament of the present invention" are used interchangeably and refer to a formulation or pharmaceutical composition as defined in the first aspect of the present invention containing itaconic acid, or a pharmaceutically acceptable salt or derivative thereof, or an enhancer thereof, as an active ingredient.

As used herein, the terms "detection reagent", "detection reagent of the present invention", "Tet enzyme detection reagent" and "Tet enzyme detection reagent of the present invention" are used interchangeably and refer to the reagent for detecting Tet enzyme as defined in the first aspect of the present invention.

As used herein, the terms "formulation combination", "drug and detection reagent combination", "formulation combination of the invention", "drug and detection reagent combination of the invention" are used interchangeably and refer to the formulation combination of the first aspect of the invention.

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the terms "subject," "subject in need thereof" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

Itaconic acid

Itaconic acid is named as methylene succinic acid and itaconic acid, is unsaturated binary organic acid and has a molecular formula of C ₅ H ₆ O ₄ As shown in the following formula 1:

itaconic acid is metabolized in vivo by decarboxylation of aconitic acid (aconate). Immune response gene 1 (Immune-response gene 1, irg 1) is transcribed up-regulated in LPS activated macrophages, the gene encoding protein is aconitate decarboxylase (Aconitate decarboxylase 1, ACOD1), and the metabolite is Itaconic acid (ITA).

In resting macrophages, irg1 is not expressed and itaconic acid is barely detectable; irg1 is rapidly induced to express in immune cells such as macrophages during pathogen infection and inflammatory response, and allows intracellular concentrations of itaconic acid up to several millimoles within hours after infection. Chemically modified derivatives of itaconic acid, such as 4-octyl-itaconic acid, are useful as anti-inflammatory and delay the survival period of endotoxemia mice.

The itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an accelerator thereof, has been found to inhibit the activity of a Tet enzyme, and has no influence on the activity of an alpha-ketoglutarate-dependent enzyme other than a Tet enzyme, and has good safety.

Tet enzyme

The epigenetic enzyme Tet family has three members (Tet 1/2/3) and participates in the active demethylation process of DNA, and is closely related to the gene transcription regulation. Previous studies have shown that upon infection by pathogenic bacteria such as bacteria, viruses, fungi, etc., host macrophages undergo demethylation of genomic DNA, activating expression of genes associated with inflammation such as NF- κb and the interferon pathway, and the immune response of the organism.

All the Tet proteins of the Tet family (Tet 1/2/3) contain a catalytically functional carbon-terminal domain, belong to the Cupin-like dioxygenase superfamily and exhibit alpha-ketoglutarate (alpha-KG) and ferrous ion-dependent dioxygenase activity. The Tet protein oxidizes 5mC to 5hmC through these domains and requires alpha ketoglutarate as a co-substrate for enzymatic activity.

Studies have shown that the loss of function of the DNA demethylase Tet2 has anti-inflammatory effects in cells and in animals, and inhibitors targeting Tet2 are found: itaconic acid. The method provides basis and foundation for developing brand new anti-inflammatory drugs for treating excessive inflammatory reaction (including cytokine storm syndrome) by taking Tet2 as target enzyme and itaconic acid as inhibitor pharmacophore starting point.

The preferred targeting cells of the invention are inflammatory cells such as macrophages. Tet2 knockdown resulted in about 90% reduction of 5hmC in macrophages, and itaconic acid failed to further reduce 5hmC content in Tet 2-deficient macrophages. In addition, the LPS-induced endotoxemia phenotype of Tet2 inactivated mutant mice was completely consistent with mice treated with itaconic acid. Itaconic acid treatment no longer exerted anti-inflammatory protection on Tet2 inactivated mutant mice (fig. 3). The above results demonstrate that in cells or mice, the primary functional target of itaconic acid anti-inflammatory is Tet2.

In the present invention, the inventors have used Tet2 knockout mice and macrophages cultured in vitro, and found that Tet2 is the most predominant DNA demethylase (90% in ratio) in macrophages. The inventor also discovers that the LPS-induced mouse endotoxemia can be obviously relieved, the plasma cytokine level is reduced, pulmonary edema and liver injury are relieved, and the survival period of the mouse is prolonged through Tet2 gene mutation or Tet enzyme activity inhibition. Thus, itaconic acid as a Tet2 inhibitor will help avoid excessive immune responses and resulting tissue damage in the body upon infection by pathogenic bacteria.

In the detection of competitive binding of itaconic acid to alpha-ketoglutarate to Tet2 protease, it was confirmed by Nuclear Magnetic Resonance (NMR) that itaconic acid binds to Tet2 similarly to alpha-ketoglutarate. In vitro and cell level experiments, the ability of itaconic acid to inhibit the activity of Tet2 enzyme was also verified, wherein the IC50 of itaconic acid to inhibit the activity of Tet2 enzyme in the in vitro experiments was 171 μm; the itaconic acid in the cell experiment result can inhibit the activity of Tet (mainly Tet 2) enzyme in macrophages to the extent of 90%.

The present invention also demonstrates the binding and neutralizing activity of itaconic acid on Tet3 enzyme. The Tet family enzymes (i.e., tet1, tet2 and Tet 3) have the same active center and all depend on the combination with alpha-ketoglutarate to generate enzyme activity, and the itaconic acid medicament of the invention acts by competitively combining with alpha-ketoglutarate and inhibiting the enzyme activity. Therefore, the present invention can exert an effect of inhibiting the Tet family enzyme.

It is noted that itaconic acid or its derivatives (e.g., octyl itaconic acid) are most preferred for preparing the pharmaceutical composition of the present invention or the formulation combination of the present invention because itaconic acid and its derivatives have no effect on the activity of alpha-ketoglutarate dependent enzymes other than Tet enzyme and have good safety. However, analogues of alpha-ketoglutaric acid, such as hydroxyglutaric acid, succinic acid, fumaric acid, and the like, can also be used to prepare the formulation combinations of the invention. Those skilled in the art will be able to select according to the actual circumstances.

Cytokine and cytokine storm

As used herein, the term "cytokine" is a low molecular weight soluble protein that is an immunogen, mitogen or other stimulatory agent that induces the production of a variety of cells, and has a variety of functions, including modulating innate and adaptive immunity, hematopoiesis, cell growth, and damaged tissue repair. Cytokines can be classified into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. Cytokines are produced primarily by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.).

As used herein, the term "cytokine storm" refers to the phenomenon that causes rapid and massive production of various cytokines such as TNF-alpha, IL-1, IL-6, IL-12, IFN-alpha, IFN-beta, IFN-gamma, MCP-1, IL-8, etc., in body fluids after the body is infected with microorganisms, and is an important cause of acute respiratory distress syndrome and multiple organ failure. The immune system works routinely to clear infection, but extreme immune attacks are "cytokine storms" if the immune system is activated to a limited extent or is out of control. Immune cells communicate with each other through cytokines, which are small molecules released into the blood by cells, which can wash immune cells to the infected site, phagocytize damaged cells, and even penetrate the vessel wall. Cytokines can also trigger inflammation, causing swelling, heating, and pain in the destroyed body. Cytokine storms also trigger the release of nitric oxide in large amounts. These factors can reduce blood pressure to dangerous levels, causing severe patients to die from severe septic shock.

The pharmaceutical composition can effectively inhibit cytokine storm by inhibiting the activity of Tet2 enzyme, and cytokines which can be inhibited by the pharmaceutical composition comprise FKB and cytokines and chemokines regulated by JAK-STAT signal paths. In particular, cytokines that the pharmaceutical compositions of the present invention are capable of inhibiting include, but are not limited to: IL1 beta, IL6, IL12, IFN alpha, IFN beta, IFN gamma, CXCL9, CXCL10, CXCL11, CXCL5.

Pharmaceutical composition

The pharmaceutical compositions provided by the invention preferably contain 0.1 to 99wt% of the first active ingredient, the remainder being the second active ingredient, a pharmaceutically acceptable carrier, a diluent or solution or a salt solution.

The first active ingredient of the present invention can be directly used for treating or preventing cancer metastasis. In addition, it may also be used in combination with other therapeutic agents, i.e. the second active ingredient.

The second active ingredient may be any pharmaceutical ingredient capable of preventing and/or treating cancer or cancer metastasis, including but not limited to chemotherapeutic agents, endocrine therapeutic agents, targeted therapeutic agents, and the like.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.

The compounds and pharmaceutical compositions provided herein may be in a variety of forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, and may be presented in a suitable solid or liquid carrier or diluent and in a suitable sterilizing device for injection or infusion.

The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The dosage unit of the formulation generally comprises from 0.05 to 1000mg of the active compound of the invention, preferably from 1mg to 500mg of the active compound of the invention.

The pharmaceutical compositions of the present invention may be used clinically in mammals, including humans and animals, by oral, nasal, dermal, pulmonary or gastrointestinal routes of administration. Most preferably orally. Most preferably, the daily dosage is 0.01-400mg/kg body weight, and the medicine is administered once or in divided doses of 0.01-200mg/kg body weight. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Typically starting from a small dose, the dose is gradually increased until the most suitable dose is found.

The agents or inhibitors of the invention may be administered by a variety of different means, for example, by injection, spraying, nasal drops, eye drops, permeation, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped by other materials and introduced into the body.

Typically, the active ingredient of the present invention or pharmaceutical compositions containing it may be administered in unit dosage form by the enteral or parenteral route, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, ocular, pulmonary and respiratory routes, skin, vaginal, rectal and the like.

The dosage form may be a liquid, solid or semi-solid dosage form. The liquid preparation can be solution (including true solution and colloid solution), emulsion (including O/W type, W/O type and multiple emulsion), suspension, injection (including water injection, powder injection and transfusion), eye drop, nasal drop, lotion, liniment, etc.; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like.

The active ingredients of the invention can be prepared into common preparations, slow-release preparations, controlled-release preparations, targeted preparations and various microparticle administration systems.

For the preparation of the active ingredient according to the invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrating agent can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.

In order to make the administration unit into a capsule, the active ingredient of the present invention may be mixed with a diluent, a glidant, and the mixture may be directly placed in a hard capsule or a soft capsule. Or mixing the effective components with diluent, binder, and disintegrating agent, granulating or micropill, and making into hard capsule or soft capsule. The various diluents, binders, wetting agents, disintegrants and glidants used in the preparation of the tablets of the invention may also be used in the preparation of the capsules of the invention.

For the preparation of the active ingredients according to the invention, water, ethanol, isopropanol, propylene glycol or mixtures thereof may be used as solvents and appropriate amounts of solubilizers, cosolvents, pH regulators, osmotically adjusted agents as are customary in the art may be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the PH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide and the like; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.

In addition, colorants, preservatives, fragrances, flavoring agents, or other additives may also be added to the pharmaceutical formulation, if desired.

The active ingredients or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.

When the active ingredient of the present invention has a synergistic effect with other therapeutic agents, its dosage should be adjusted according to the actual situation.

Formulation combinations and kits

The present invention provides a formulation combination comprising:

(A) A pharmaceutical composition comprising itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an accelerator thereof, as an active ingredient;

(B) Reagents for detecting Tet enzyme.

The formulation combinations of the invention are useful for specifically treating diseases in which Tet enzyme is elevated.

In particular, the invention may comprise reagents for detecting the Tet1 enzyme and is suitable for detecting and treating diseases positive for the Tet1 enzyme, preferably diseases associated with fertilized egg dysplasia.

Alternatively, the invention may comprise reagents for detecting the Tet3 enzyme and is suitable for detecting and treating Tet3 enzyme-positive diseases, preferably neurological diseases.

In a preferred embodiment, the invention may comprise reagents for detecting a Tet2 enzyme and is suitable for detecting and treating a Tet2 enzyme positive disease, preferably a cytokine storm related disease.

Alternatively, the invention may comprise a combination of reagents for detecting any two or more of the above enzymes. The present invention is also applicable to cases where any two or more of the above-mentioned diseases are accompanied by morbidity.

The preparation combination of the invention, or a kit prepared by using the preparation combination, can be used for implementing a method for preventing and/or treating Tet enzyme increase or Tet enzyme activity up-regulation diseases. The method mainly comprises the following steps:

(i) Obtaining a sample to be tested from a subject;

(ii) Detecting the content of the Tet enzyme or the activity of the Tet enzyme in the sample to be detected by using the reagent (B) for detecting the Tet enzyme;

(iii) If the test sample is detected to be positive for the Tet enzyme, the pharmaceutical composition (A) is applied to the test subject to prevent and/or treat the Tet enzyme increase or the diseases related to the Tet enzyme activity up-regulation.

The Tet enzyme positives include, but are not limited to: the content of the Tet enzyme increases, the activity of the Tet enzyme increases, and the expression level of the gene encoding the Tet enzyme increases.

Reagents for detecting Tet enzyme of the present invention include, but are not limited to: reagents for detecting the content of a Tet enzyme, reagents for detecting the activity of a Tet enzyme, reagents for detecting the expression level of a gene encoding a Tet enzyme, or combinations thereof. The person skilled in the art is able to select a corresponding detection method based on the detection target.

The main advantages of the invention include:

the invention provides an application of itaconic acid in treating Tet enzyme related diseases, which has the following advantages:

1) Itaconic acid is a natural metabolite small molecule, and is safe and effective.

2) Itaconic acid has specific targets in macrophages, has no influence on the activity of alpha-ketoglutarate dependent enzymes except Tet enzyme, and has small side effect.

3) The invention discovers that the Tet2 gene knockout mice still develop normally, so that the Tet2 serving as a drug target has high safety.

4) The invention discovers that itaconic acid is combined with the common active center of the Tet family enzyme, has inhibition effect on the Tet family enzyme and has wide application range.

4) Itaconic acid specifically inhibits the Tet family enzyme and the effect thereof in the anti-inflammatory disease process, and provides a pharmacophore (pharmacophore) taking itaconic acid as a starting point for developing a novel efficient Tet inhibitor.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 specific inhibition of Tet2 by itaconic acid

1.1ITA in vitro inhibiting Tet2 Activity level

In this example, the in vitro inhibition of Tet by itaconic acid was examined. Conversion of 5mC to 5hmC (characterizing Tet enzyme activity levels) was detected by an in vitro enzyme activity system based on immobilized substrate DNA and 5hmC fluorescence detection (fig. 1 a).

Results: ITA can inhibit mTET2 ^CD And its half inhibitory concentration (IC 50) is 171. Mu.M (FIG. 1 b), the IC50 is similar to L-type 2-hydroxyglutarate (L-2-HG, 115. Mu.M) and is significantly higher than D-2-HG (1,057. Mu.M), succinic acid>5 mM) and fumaric acid>5mM)。

1.2 exogenous ITA inhibits the action of Tet family enzymes in cells

The inventors treated Tet2 with octyl modified ITA (OI) or unmodified ITA ^+/+ And Tet2 ^-/- Mouse Bone Marrow Derived Macrophages (BMDMs) to determine whether ITA can inhibit the action of Tet family enzymes in cells.

Results: at Tet2 ^+/+ Exogenously added OI or ITA in BMDMs of (A) can reduce overall 5hmC by up to 95%, but at Tet2 ^-/- Little effect in cells and detectionTo Tet2 ^-/- Basal levels of 5hmC in cells relative to Tet2 ^+/+ The cells were reduced by about 92% (fig. 1C).

The results of this example show that Tet2 is a key Tet enzyme that controls 5hmC production in macrophages, and that exogenous ITA can almost completely inhibit Tet2.

1.3 inhibition of Tet enzyme by ITA produced by inflammatory response

In this example, LPS was used to induce cells to produce inflammatory responses, and the inhibition of Tet enzyme by ITA produced by inflammatory responses was observed.

An increase in Irg1 mRNA levels was observed after 30 minutes of treatment of RAW264.7 macrophages with LPS and protein accumulation began after 2 hours. Correspondingly, it was detected that endogenous ITA of the cells rapidly accumulated to millimolar levels, up to 3.75mM, within 2-4 hours after LPS stimulation. And accumulation of ITA can be completely eliminated after deletion of the Irg1 gene (FIG. 1 d). By LC-MS/MS (fig. 1 e), it was found that overall 5hmC was reduced in Irg1-WT RAW264.7 cells under LPS stimulation, but remained at relatively high levels throughout Irg 1-deficient (Irg 1-KO) cells. In addition, LPS-induced ITA had no effect on overall histone methylation of RAW264.7 cells (fig. 1 f).

In this example, IRG1 expression levels increased after LPS stimulation, the product ITA also accumulated rapidly, and a concomitant decrease in 5hmC was observed, which characterizes Tet enzyme activity levels. The results indicate that ITA produced by IRG1 in the inflammatory response can selectively inhibit Tet enzyme. Furthermore, ITA did not affect other ketoglutarate-dependent enzymes, such as histone demethylase KDM, demonstrating that ITA has specificity for the Tet enzyme.

Example 2 itaconic acid inhibits Tet2 and inhibits NF-. Kappa.B and expression of inflammatory-related genes such as the Interferon pathway

2.1 transcriptome analysis of ITA inhibition of Tet enzyme action

In this example, to further investigate the inhibition of Tet enzyme by ITA, transcriptome changes caused by OI treatment (inhibiting Tet2 catalytic activity) or Tet2 deletion (Tet 2-KO) in LPS-stimulated RAW264.7 cells were compared.

Results: in Tet2-WT cells 1846 genes were found to be down-regulated following OI treatment, of which 712 genes belong to genes that can be up-regulated by LPS activation and up-regulated at least 2-fold. Of these 712 genes, 509 (71.5%) were also down-regulated by the Tet2 deletion (fig. 2 a). Many of these genes are involved in innate immune and inflammatory responses, including cytokine-mediated signaling pathways such as TNF, toll-like receptors and NF- κb.

This example demonstrates that a significant portion of the genes associated with the LPS-induced, innate immune and inflammatory responses are regulated by ITA and Tet 2.

2.2 mechanisms by which ITA affects gene expression by inhibiting the catalytic Activity of Tet2

Tet2 has both a function of relying on its catalytic activity and a function of independent activity. In this example, the mechanism by which ITA affects gene expression by inhibiting the catalytic activity of Tet2 was verified.

The inventors constructed a point mutant mouse of Tet2H1795R knock-in (KI) (H1795 in mouse Tet2 corresponds to the high frequency mutation site Tet2H 1881 in human acute myeloid leukemia, which is critical for the catalytic activity of Tet 2). Then Tet2 is separated ^+/+ And BMDM (bone marrow derived macrophage) of Tet2H1795R (Tet 2-ki), treated with LPS alone or in combination with OI, respectively, followed by gene expression profiling.

Results: of the 1463 genes induced by LPS and downregulated by OI treatment, 607 (41.5%) were also downregulated in Tet2-KI cells. The KEGG signaling pathway of these 607 genes was analyzed and found to be similar to Tet2-KO cells, in which genes involved in innate immune and inflammatory responses were also enriched.

The results of this example demonstrate that ITA also has a certain inhibitory effect on the catalytic activity of Tet2 and is capable of down-regulating innate immunity and inflammatory response-related genes expressed based on the catalytic activity of Tet 2.

EXAMPLE 3 protection of mice against endotoxemia by Tet2 inhibition with itaconic acid

In this example, the functional significance of the Irg1/ITA/Tet2 axis was verified.

The present inventors have derived from catalytically inactive Tet2 by transplantation ^HxD Knock-in (KI) mutant or wild-type (WT) mouse bone marrow cells, Chimeric mice were developed. Tet2 ^HxD KI mutant mice contain double substitution mutations of H1295Y and D1297A in mouse Tet2, corresponding to human Tet 2H 1382 and H1384, respectively, and are able to disrupt binding to the essential cofactor fe2+.

Two transplanted mice (wild type bone marrow transplanted and mutant transplanted mice) were advanced 12 hours to receive 50mg/Kg of ITA for intraperitoneal injection, followed by intraperitoneal injection of 25mg/Kg of LPS for endotoxin model modeling (FIG. 3 a). Four hours after molding, collecting the abdominal leucocytes and plasma of the mice, detecting the expression of cytokines in serum by a commercial ELISA kit, detecting the activities of glutamic pyruvic transaminase ALT and glutamic oxaloacetic transaminase AST (liver injury markers), collecting the measured dry-wet weight ratio (lung edema index) of lung tissues, and performing HE histochemical staining (lung injury index).

Results: tet2 compared to WT chimeric mice ^HxD LPS-induced inflammatory responses were inhibited in chimeric mice, including reduced levels of pro-inflammatory cytokines and chemokines (e.g., il-6, TNF- α, cxcl 9) in serum (fig. 3 b), reduced liver injury (fig. 3 c), pulmonary edema (fig. 3 d), and lung injury (fig. 3 e-f) and prolonged survival (fig. 3 g). Furthermore, inhibition of the above-described inflammatory response in terms of molecules, tissues and general due to Tet2 catalytic inactivation was also observed after injection of LPS into ITA-pretreated WT chimeric mice (fig. 3 b-g).

The results of this example provide direct in vivo evidence that Tet2 is the primary functional target of ITA. Play a critical physiological role in immunomodulation against and the Irg1/ITA/Tet2 axis.

Example 4 itaconic acid inhibits Tet3 enzyme

In this example, flag tagged Tet3 was overexpressed in HEK293T cells and cells were treated with corresponding concentrations of octyl modified itaconic acid or itaconic acid. The relative content of the Tet3 enzyme metabolite 5hmC in the cells was detected by liquid chromatography-mass spectrometry.

Results: as shown in fig. 4, both itaconic acid and octyl modified itaconic acid treated groups showed a significant decrease in 5hmC compared to the control group. This example demonstrates that both itaconic acid and octyl modified itaconic acid are effective in inhibiting 5hmC production in cells over-expressing Tet3 enzyme, suggesting that they have significant inhibition of Tet3 enzyme.

Discussion of the invention

On the other hand, in the past studies on itaconic acid, although there is a report on the medical function of itaconic acid, the mechanism is always unknown, so that itaconic acid cannot be utilized in a targeted manner, and the therapeutic effect is maximized. Aiming at the problem, the animal experiment result shows that the intraperitoneal injection of itaconic acid can effectively relieve LPS-induced endotoxemia, and is particularly characterized by reducing the level of plasma cytokines, avoiding pulmonary edema and liver injury and prolonging the survival period of mice; in mice lacking Tet2 enzyme activity, itaconic acid does not have the above-described anti-inflammatory and body-protecting effects. The results strongly demonstrate that itaconic acid has an effect of alleviating endotoxemia by inhibiting the activity of Tet2 enzyme.

Accordingly, the invention provides the combined use of the reagent for detecting the Tet2 activity and the itaconic acid so as to realize the accurate treatment of the inflammation.

On the other hand, the catalytic activity of Tet enzyme depends on activation of α -ketoglutarate, and in fact, the variety of enzymes in the body that depend on activation of α -ketoglutarate is large, and other enzymes are often affected when inhibitors that competitively bind to their active centers are used. However, itaconic acid of the present invention is the first specific inhibitor of Tet family enzymes, especially specific inhibition Tet2, which only inhibits Tet enzyme activity, but not other enzymes that rely on alpha-ketoglutarate for activation as the Tet enzyme.

In particular, the invention has been experimentally demonstrated that LPS-induced ITA has no effect on overall histone methylation of macrophages. This demonstrates that ITA has no effect on other enzymes that are dependent on alpha-ketoglutarate, such as histone demethylase KDM, demonstrating that ITA has specificity for the Tet enzyme. Therefore, the method of the invention can prevent the interference to other normal enzyme functions while treating the cytokine storm.

Further, since the Tet family enzymes (i.e., tet1, tet2 and Tet 3) have the same active center, all rely on the combination with alpha-ketoglutarate to exert the enzymatic activity, and the itaconic acid medicament of the present invention acts by competitively combining with alpha-ketoglutarate to inhibit the enzymatic activity, the present invention can exert the effect of inhibiting Tet family enzymes in a broad spectrum. Furthermore, itaconic acid has been demonstrated in the examples to also have binding and neutralizing activity against Tet3 enzyme, and to inhibit the production of 5hmC, a metabolite thereof. Therefore, the invention has wide application prospect in treating Tet family enzyme related diseases.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A combination of formulations, wherein the combination of formulations comprises:

(A) A pharmaceutical composition comprising itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an accelerator thereof, as an active ingredient;

(B) Reagents for detecting Tet enzyme.

2. The combination of claim 1, wherein the Tet enzyme comprises: tet2, tet3, or a combination thereof.

3. The combination of claim 1, wherein the reagent for detecting Tet enzyme is a reagent for detecting Tet enzyme activity.

4. A combination of preparations as claimed in claim 3, wherein the detection of the Tet enzyme activity is the detection of the content of the Tet enzyme catalytic product, preferably the detection of the 5-hydroxymethylcytosine (5 hmC) content of DNA.

5. The combination of agents according to claim 1, for use in the practice of a method for preventing and/or treating a disease in which Tet enzyme is increased or Tet enzyme activity is modulated.

6. The combination of claim 5, wherein the Tet enzyme is Tet2 and the disorder is a cytokine storm-related disorder.

7. The combination of claim 6, wherein the cytokine storm-related disease comprises: bacterial or viral infectious diseases, autoimmune diseases, or combinations thereof.

8. Use of a combination of formulations according to any one of claims 1 to 7 for the preparation of a kit for the treatment of diseases in which Tet enzyme is increased or up-regulated in activity.

9. Use of itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or an accelerator thereof, for the preparation of a formulation or pharmaceutical composition for the treatment of a Tet enzyme-related disorder.

10. A method of non-therapeutically inhibiting Tet enzyme activity in vitro comprising the steps of:

releasing the Tet enzyme from itaconic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, or a promoter thereof, thereby inhibiting the activity of the Tet enzyme.