Application of pyruvate kinase M2 activator in the preparation of drugs for treating psoriasis

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a pyruvate kinase M2 activator in the preparation of a drug for treating psoriasis. The TEPP-46 activator increases the activity of PKM2 pyruvate kinase by inducing the formation of PKM2 tetramerization and inhibiting the formation and nuclear entry of the dimer form, thereby blocking the signal pathway necessary for aerobic glycolysis. The present invention also provides a pharmaceutical composition for treating psoriasis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a pyruvate kinase M2 activator in preparing a medicine for treating psoriasis. Background Art

[0002] Psoriasis is a chronic, immune-mediated inflammatory skin disease that can occur at any age and seriously affects the patient's quality of life. Its pathogenesis has not yet been fully elucidated. IL-23 and Th17 cell responses are considered to be important driving factors of psoriasis. The main characteristics of psoriasis are abnormal differentiation and proliferation of keratinocytes, which are manifested as shortened epidermal transit time and cell cycle. Plaque psoriasis is the most common variant, accounting for more than 80% of psoriasis cases. It is clinically characterized by erythematous scales or plaques, which often occur on the extensor surface, but can also affect the intertrigeminal area, palms, soles and nails. Psoriasis is currently incurable. For patients with mild psoriasis, topical medications are still the main treatment. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a use of a pyruvate kinase M2 activator in the preparation of a medicament for treating psoriasis.

[0004] To achieve the above purpose, the solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides an application of a pyruvate kinase M2 activator in the preparation of a drug for treating psoriasis, wherein the pyruvate kinase M2 (PKM2) activator is a thieno[3,2-b]pyrrolo[3,2-b]pyridazinone (TEPP-46) pyruvate kinase M2 (PKM2) activator.

[0006] Preferably, the TEPP-46 activator increases the PKM2 pyruvate kinase activity by inducing the formation of PKM2 tetramerization and inhibiting the formation and nuclear entry of the dimer form, thereby blocking the signaling pathway necessary for aerobic glycolysis, which is a key step in T cell activation and effector function.

[0007] Preferably, the TEPP-46 activator affects T cell activation and function by impairing the glycolytic rate and capacity of T cells.

[0008] Preferably, the TEPP-46 activator limits the generation of Th17 cells and the development of T cell-mediated autoimmunity.

[0009] In a second aspect, the present invention also provides a pharmaceutical composition for treating psoriasis, comprising the pyruvate kinase M2 activator as described above.

[0010] Preferably, the pyruvate kinase M2 activator is a thieno[3,2-b]pyrrolo[3,2-b]pyridazinone (TEPP-46) pyruvate kinase M2 (PKM2) activator.

[0011] Preferably, the therapeutically effective dosage of the TEPP-46 activator is not less than 0.2 wt%, preferably 0.2 wt%-10 wt%.

[0012] Preferably, the TEPP-46 activator is in the form of gel, and the solvent used for the TEPP-46 gel is Carbomer 940.

[0013] Preferably, the TEPP-46 activator is administered to psoriasis patients in the form of a gel by applying the gel.

[0014] Preferably, the preparation method of TEPP-46 gel comprises: dissolving carbomer 940 in deionized water, standing overnight, and adjusting the pH value of the carbomer gel to 5-7 with NaOH on the next day to prepare the carbomer gel; then dissolving TEPP-46 in DMSO solvent, adding the added amount of TEPP-46 to the carbomer gel to prepare the TEPP-46 gel, and stirring until completely dissolved.

[0015] Preferably, the mass concentration of the carbomer gel is 0.3-0.7wt%, preferably 0.5wt%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The experimental model diagram used in the mouse animal experiment in the embodiment of the present invention; in the figure, A: mouse experimental model diagram; B: PASI scores of 5 groups of mice (n=5). Ordinary one-way ANOVA was used to compare the means of independent samples; *** indicates P<0.001.

[0017] Figure 2 This is a chart of psoriasis mice treated with 0.2% TEPP-46 gel in the embodiment of the present invention; in the figure, A: typical skin lesions on the back and ears of 3 groups of mice; B: statistics of ear thickness of 3 groups of mice (n=5); C: HE staining of the back and ear skin of 3 groups of mice, scale: 100μm; D: left is the epidermal thickness of the back skin (n=5); right is the epidermal thickness of the ear skin (n=5); E: body weight of 3 groups of mice (n=5); F: spleen index of 3 groups of mice (n=5). Ordinary one-way ANOVA was used for comparison between independent sample means. ns means no statistical difference, * means P<0.05, ** means P<0.01, *** means P<0.001, **** means P<0.0001. DETAILED DESCRIPTION

[0018] The present invention provides an application of a pyruvate kinase M2 activator in preparing a drug for treating psoriasis. The pyruvate kinase M2 (PKM2) activator is a thieno[3,2-b]pyrrole[3,2-b]pyridazinone (TEPP-46) pyruvate kinase M2 (PKM2) activator.

[0019] Pyruvate kinase (PK) is the rate-limiting enzyme in the terminal step of glycolysis. It can catalyze the reaction of its substrate phosphoenol pyruvate (PEP) with adenosine diphosphate (ADP) to produce pyruvate and adenosine triphosphate (ATP), and plays an important role in aerobic glycolysis. Different isoforms of pyruvate kinase are expressed in different tissues. There are four isomeric tissue-specific forms, including R-type pyruvate kinase (PKR), L-type pyruvate kinase (PKL), M1-type pyruvate kinase (PKM1) and M2-type pyruvate kinase (PKM2). The PKR isoform is mainly present in red blood cells, but its biological activity is still unknown. The PKL isoform is mainly present in the liver and kidney. PKM1 and PKM2 subtypes are generated by two different splicing modes of PKM gene pre-mRNA, and both contain sequences encoded by exon 9 and exon 10, respectively. PKM1 is mainly distributed in myocardium, skeletal muscle, and brain tissue, while PKM2 is expressed in all proliferating cells, especially in tumors and embryonic tissues. It can be seen that among the four subtypes, PKM2 is most closely related to the occurrence and development of tumors. At present, the research on the correlation between PKM2 and tumors mainly focuses on the abnormal expression of PKM2 and its abnormal kinase activity.

[0020] As early as 1966, Halprin et al. used biochemical methods to measure 22 sugar metabolism enzymes in the epidermis of psoriasis patients, including lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase, aldolase, malate dehydrogenase, pyruvate kinase, hexokinase, phosphofructokinase, hexose phosphate isomerase, etc., and found that the activities of these enzymes were higher than those of the adjacent normal-looking skin. Halprin believed that the increase in the proliferation ability of psoriatic epidermal cells was not due to the increase in the concentration of one or several key enzymes, but was the result of the increase in the concentration of all these enzymes in the entire metabolic reaction.

[0021] The pathogenesis of psoriasis is complex, and overactive adaptive immune responses play an important role in it, including adaptive immune disorders centered on Th1 / Th17, and the secretion of effector cytokines mainly including TNF-α, IL-23, and IL-17. Among them, IL-17 is a key molecule in the pathogenesis of psoriasis, and drugs targeting IL-17 have also achieved good therapeutic effects in clinical practice. IL-17 is mainly produced by TH17 / CD8+T, innate immune lymphocytes ILC3 (innate lymphoid cells), mast cells, neutrophils, etc. TH17 is the main source of IL-17. Initial CD4+T cells recognize antigenic peptides presented by antigen-presenting cells through TCR receptors (the first signal), and under the joint action of costimulatory molecules (the second signal), in a certain cytokine environment, such as IL-23, IL-6, TGF-β, and IL-1β, CD4+T cells will differentiate into TH17 cells. The IL-23 / IL-17 axis plays an important role in psoriasis. DC cells secrete IL-23 when stimulated by self-antigens (such as LL37) or other microorganisms (such as fungi). IL-23 binds to its receptor and promotes the phosphorylation of the JAKs pathway, thereby promoting the transcription of ROR-γt and STAT3, causing CD4+T cells to differentiate into TH17 and secrete IL-17A, TNF-α, and IL-22, thus forming a positive feedback loop.

[0022] In the process of TH17 differentiation, in addition to a specific cytokine environment, energy metabolism also needs to be readjusted, such as increased glycolysis, glutamine metabolism, and fatty acid metabolism. Therefore, regulating energy metabolism has become a new target for the treatment of autoimmune diseases.

[0023] The phenomenon of glycolytic metabolism in the TH17 differentiation process is also known as the Warburg effect. Dr. Warburg initially observed this phenomenon in most cancer cells, and it occurs even when oxygen concentration is sufficient. Pyruvate kinase 2 (PKM2), as the rate-limiting enzyme of glycolysis, is involved in the pathogenesis of many tumors. In addition, PKM2 can also exert biological functions by regulating many transcription factors, such as hypoxia-inducible factor HIF-α and phosphorylation of STAT3. PKM2 expression is upregulated in most cancer cells and also in psoriasis lesions. In addition, recent studies have found that PKM2 can regulate TH1 / TH17 differentiation.

[0024] PKM2 exists in two forms in cells, namely, a tetramer with high activity (pyruvate kinase activity) and a dimer with low activity. PKM2 in its tetramer form exhibits high pyruvate kinase catalytic activity, catalyzing the reaction of phosphoenolpyruvate and ADP to generate pyruvate and ATP, and the generated pyruvate is more likely to enter the tricarboxylic acid cycle pathway; while the dimer form is mainly involved in carcinogenic events. Compared with the tetramer, the dimer has lower pyruvate kinase activity, and the pyruvate generated by its catalysis is more likely to generate lactate, causing tumor cells to choose the aerobic glycolysis pathway; in addition, the low-activity dimer often blocks the glycolysis pathway due to its slow catalytic rate, causing the intermediate metabolites of glycolysis to enter the glycolysis bypass pathway (such as the pentose phosphate pathway) to synthesize the nutrients required for tumor cell proliferation. In addition to pyruvate kinase activity, the dimeric PKM2 also has protein kinase activity, enters the cell nucleus to regulate the phosphorylation and subsequent acetylation modification of target proteins, and promotes the transcription of proto-oncogenes and the occurrence and development of tumors. Studies have shown that after PKM2 enters the nucleus, it will upregulate the expression of Cyclin D1 and oncogene transcription factor c-Myc, promoting tumor cell proliferation. Therefore, selecting PKM2 activators to stabilize the PKM2 tetramer structure and inhibit the PKM2 dimer form can be used as an effective strategy for anticancer drug research.

[0025] The small molecule activator of PKM2, thieno[3,2-b]pyrrole[3,2-d]pyridazinones (TEPP-46), provided by the present invention, induces the formation of PKM2 tetramers by stabilizing the interaction between PKM2 subunits, inhibits the formation and nuclear entry of PKM2 in the form of dimers, increases the activity of PKM2 pyruvate kinase, and blocks the signal pathway necessary for aerobic glycolysis. Aerobic glycolysis is a key step in T cell activation and effector function. TEPP-46 severely impairs the glycolysis rate and capacity of T cells, thereby affecting T cell activation and function. The embodiments of the present invention confirm that TEPP-46 severely limits the generation of Th17 cells and the development of T cell-mediated autoimmunity in a dose-dependent manner, and clinically proves the potential application of TEPP-46 in psoriasis pharmaceuticals.

[0026] In the embodiment of the present invention, the TEPP-46 activator is administered in the form of a gel, and the solvent used in the TEPP-46 gel is Carbomer 940. Carbomer 940 is a polyacrylic acid cross-linked polymer that can form a high-viscosity gel at a very low concentration. It has a variety of uses in medicaments and can be used as a thickener, suspending agent, gel matrix, bioadhesive material, and skeleton material for controlled-release agents. Carbomer has a simple process and good stability, and is commonly used in topical preparations, and is widely used in emulsions, creams, and gels.

[0027] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0028] The present invention provides for the first time a TEPP46 carbomer gel having a TEPP46 content of not less than 0.2wt%, which is proved to be effective in treating and / or improving psoriasis through animal experiments. The present invention uses a PKM2 activator TEPP-46 at a dosage of not less than 0.2wt% to treat psoriasis mice through animal experiments. After treatment with the 0.2wt% TEPP46 carbomer gel, the symptoms are alleviated and effectively relieved, indicating the potential application of the PKM2 activator TEPP-46 in psoriasis pharmaceuticals.

[0029] Example:

[0030] 1. Animal experiment materials:

[0031] TEPP46 was purchased from Selleck, and the solvent was Carbomer 940.

[0032] BALB / c mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and routinely raised in the SPF animal room of Zhongshan Hospital Affiliated to Fudan University.

[0033] 2. Animal experimental model:

[0034] The mouse animal experimental model used in this example is listed in Table 1. Figure 1 shown.

[0035] Table 1

[0036]

[0037] 3. Preparation of TEPP-46 gel:

[0038] 0.5g of Carbomer 940 was dissolved in 99.5g of deionized water, and allowed to stand overnight. The next day, NaOH was used to adjust the pH of the Carbomer gel to 5-7 to prepare a 0.5% Carbomer gel. TEPP-46 was dissolved in DMSO, and the added amount of TEPP-46 was added to Carbomer to prepare low, medium and high concentration TEPP-46 gels, and stirred until completely dissolved.

[0039] 4. Mouse animal experiment:

[0040] Dosage: Apply on skin.

[0041] After shaving the back of all mice, the model group mice were applied with a total of 62.5 mg of 5% imiquimod cream and carbomer on the back skin and unilateral ear once a day, the treatment group mice were applied with a total of 62.5 mg of 5% imiquimod cream and carbomer gel containing low, medium and high concentrations of TEPP-46 on the back skin and unilateral ear daily, and the blank group mice were applied with an equal amount of vaseline for 6 consecutive days. The health of the mice was observed daily, and the weight, PASI score and ear thickness of the mice were recorded. The mice were killed 24 hours after the last application of the drug, and the back and ear skin of the mice were taken for histochemical HE staining; the spleen of the mice was taken and weighed.

[0042] 5. Result analysis:

[0043] First, the five groups of mice were scored using the PISA score, such as Figure 1 As shown, the results suggest that high concentration of TEPP46 gel (ie, 0.2%) has statistical significance in treating psoriasis model mice. Therefore, the therapeutically effective dosage of the TEPP-46 activator provided by the present invention is not less than 0.2wt%, such as 0.2wt%-10wt%.

[0044] Compared with the control group, the inflammatory lesions on the back and ears of the group treated with 0.2% TEPP-46 gel were reduced. After treatment with 0.2% TEPP-46 gel, the thickness of the mouse ears and the PASI score were reduced, and the difference was statistically significant. Figure 2 As shown, HE staining of mouse skin showed that after treatment with 0.2% TEPP-46 gel, epidermal thickness was significantly reduced, hyperkeratosis and follicular plugs were improved, and epidermal thickness decreased after treatment. There was no difference in weight change among the three groups of mice during treatment, and there was no statistical difference in spleen index between the treatment group and the control group, indicating that the drug treatment did not cause immunosuppression in mice. In summary, 0.2% TEPP-46 gel effectively improved psoriasis-like inflammatory lesions induced by imiquimod, and PKM2 is expected to become a new target for the treatment of psoriasis, suggesting the potential of TEPP-46 in the treatment of psoriasis topical preparations.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating psoriasis, characterized in that: including pyruvate kinase M2 activators; The pyruvate kinase M2 activator is TEPP-46; The therapeutically effective dosage of the TEPP-46 activator is not less than 0.2 wt %; The TEPP-46 activator is in the form of gel, and the solvent used in the TEPP-46 gel is Carbomer 940; The TEPP-46 activator is administered to psoriasis patients in the form of a gel by applying the gel; The preparation method of TEPP-46 gel comprises: dissolving carbomer 940 in deionized water, standing overnight, and adjusting the pH value of the carbomer gel to 5-7 with NaOH on the next day to prepare the carbomer gel; then dissolving TEPP-46 in DMSO solvent, adding an added amount of TEPP-46 to the carbomer gel to prepare the TEPP-46 gel, and stirring until completely dissolved; the mass concentration of the carbomer gel is 0.3-0.7wt%.

2. The pharmaceutical composition according to claim 1, characterized in that The therapeutically effective dosage of the TEPP-46 activator is 0.2 wt%-10 wt%.

3. The pharmaceutical composition according to claim 1, characterized in that The mass concentration of the carbomer gel is 0.5wt%.

4. Use of a pyruvate kinase M2 activator in the preparation of a drug for treating psoriasis, characterized in that: The pyruvate kinase M2 activator is TEPP-46; The TEPP-46 activator increases PKM2 pyruvate kinase activity by inducing the formation of PKM2 tetramerization and inhibiting the formation and nuclear entry of the dimer form, thereby blocking the signaling pathway necessary for aerobic glycolysis, which is a key step in T cell activation and effector function; The TEPP-46 activator affects T cell activation and function by impairing the glycolysis rate and capacity of T cells; The TEPP-46 activator limits the generation of Th17 cells and the development of T cell-mediated autoimmunity; The TEPP-46 activator is administered in the form of a gel.

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