Use of small molecule compound LZ-09 in the preparation of a drug for treating and / or preventing multiple sclerosis

Abstract

The present application relates to the technical field of medicine, especially relates to application of a small molecule compound LZ-09 in preparation of a medicine for treating and / or preventing multiple sclerosis. The present application adopts H2O2 damage OLN-93 cells as a nerve cell oxidative stress model, and the result shows that LZ-09 treatment significantly weakens the cell viability decrease caused by H2O2. The classic EAE model is made by using MOG35-55 to induce C57BL / 6 mice, and it is found that after LZ-09 intervention, the mouse neurological deficit symptom score is significantly reduced, and the disease progression is delayed, and spinal cord tissue HE and LFB staining find that the inflammation infiltration and demyelination are reduced. Therefore, the compound can effectively alleviate the symptoms of multiple sclerosis, can be used as a medicine for treating multiple sclerosis, and has wide development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of a small molecule compound, LZ-09, in the preparation of medicaments for the treatment and / or prevention of multiple sclerosis. Background Technology

[0002] Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelinating lesions in the white matter of the central nervous system. The most commonly affected sites are the periventricular white matter, optic nerve, spinal cord, brainstem, and cerebellum, exhibiting temporal multiple involvement (DIT) and spatial multiple involvement (DIS). Its etiology remains unclear, but it may be related to multiple factors including genetics, environment, and viral infection. MS commonly affects adolescents, with a higher incidence in females. Globally, approximately two million people suffer from this disease, and its incidence in my country is increasing annually. The experimentally allergic encephalomyelitis (EAE) model is an autoimmune disease primarily mediated by specifically sensitized CD4+ T cells, characterized by perivascular mononuclear cell infiltration and demyelination in the central nervous system. It is a classic experimental animal model of MS. The EAE animal model is an important approach for studying the pathological process and pathogenesis of MS and is of great significance in clinical neuroimmunological research.

[0003] Chinese patent CN103800412A, published on May 21, 2014, discloses a novel use of *Panax japonicus* extract as a drug for multiple sclerosis. The extract is obtained by first extracting *Panax japonicus* with an ethanol-water solution, then with water-saturated n-butanol, and finally by acetone precipitation. This invention has significant application value for the prevention and / or treatment of multiple sclerosis. Chinese patent CN104072389A, published on October 1, 2014, discloses a compound for preparing a drug to treat multiple sclerosis. This compound, when used in the preparation of a drug to treat MS, has better anti-inflammatory effects; reduces or minimizes side effects on the liver and kidneys; does not produce teratogenicity; and is a potential therapeutic agent for neuropathies (including MS).

[0004] Currently, there is no definitive cure for multiple sclerosis (MS), therefore early treatment is advocated: acute phase treatment focuses on alleviating symptoms and improving disability as quickly as possible; remission phase treatment, also known as disease-modifying therapy (DMT), focuses on reducing relapse rates, decreasing the number of brain and spinal cord lesions, slowing disease progression, and improving patients' quality of life. Currently, DMT drugs approved for marketing in China include oral teriflunomide, fingolimod, sinimod, and injectable recombinant human beta-1b interferon. However, these drugs are expensive, have significant side effects, and limited efficacy. Even with standardized medication, some patients still cannot control disease progression, leading to numerous problems in clinical application. Therefore, finding a low-cost drug with no side effects is a key research focus for MS treatment.

[0005] Currently, there are no reports on the application of the small molecule compound LZ-09 in the present invention in the preparation of drugs for the treatment and / or prevention of multiple sclerosis. Summary of the Invention

[0006] The first objective of this invention is to address the shortcomings of the prior art by providing the use of the small molecule compound LZ-09 in the preparation of medicaments for the treatment and / or prevention of multiple sclerosis.

[0007] A second objective of this invention is to provide a formulation.

[0008] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0009] The use of the small molecule compound LZ-09, as shown in formula (I), in the preparation of drugs for the treatment and / or prevention of microglia-mediated diseases, wherein the small molecule compound LZ-09 is 3-(3-(2,4-difluorophenyl-)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)but-2-yl)-6-iodobenzo[d][1,2,3]triazine-4(3H)-one

[0010]

[0011] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0012] An agent for the prevention and / or treatment of microglia-mediated neuroinflammatory diseases, said agent comprising the small molecule compound LZ-09 of claim 1 or at least one pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0013] As a preferred example, the formulation includes one or more of the following: tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres, injections, liposomes, or aerosols.

[0014] The advantages of this invention are:

[0015] 1. This invention discloses the application of the small molecule compound LZ-09 in the preparation of drugs for the treatment and / or prevention of multiple sclerosis, which is of great significance for the development and application of drugs for multiple sclerosis.

[0016] 2. This invention uses H2O2-damaged OLN-93 cells as a neuronal oxidative stress model. Different concentrations of LZ-09 were co-cultured with H2O2-damaged OLN-93 cells, and cell proliferation and the neuroprotective effect of LZ-09 were observed. The results showed that LZ-09 treatment significantly reduced the decrease in cell viability induced by H2O2. This invention also used MOG35-55 to induce a classic EAE model in C57BL / 6 mice to evaluate the preventive and therapeutic effects of LZ-09 on the EAE model. The results showed that EAE mice exhibited symptoms such as weight loss, limb incoordination, and even disability. HE and LFB staining of spinal cord tissue revealed inflammatory infiltration and demyelination. After treatment with LZ-09, the scores of neurological deficit symptoms in mice were reduced, and disease progression was slowed. Simultaneously, HE and LFB staining of spinal cord tissue showed a decrease in inflammatory infiltration and demyelination. These results indicate that LZ-09 can effectively alleviate the symptoms of multiple sclerosis and can be used as a drug for the treatment of multiple sclerosis, with broad prospects for development and application. Attached Figure Description

[0017] Figure 1 The effect of LZ-09 on the survival rate of OLN-93 cells;

[0018] Figure 2 The effect of different concentrations of H2O2 on the survival rate of OLN-93 cells (compared with the control group, *P<0.05, **P<0.01);

[0019] Figure 3 The effect of different concentrations of LZ-09 on the survival rate of OLN-93 cells damaged by H2O2 (**P<0.01 compared with the control group);

[0020] Figure 4 Comparison of neurological deficit scores among different groups of mice (compared with the EAE model group *P<0.05);

[0021] Figure 5 Images of hematoxylin-eosin stained spinal cord tissue from each group of mice (×200);

[0022] Figure 6 Images of Laugh fast blue staining of spinal cord tissue from each group of mice (×20). Detailed Implementation

[0023] As a preferred example, the small molecule compound LZ-09 used in the following embodiments for treating and / or preventing microglia-mediated diseases is (E)-3-(3,4-dihydroxyphenyl)-N-(4-fluorophenylethyl)acrylamide (Formula I).

[0024]

[0025] As will be understood by those skilled in the art, 3-(3-(2,4-difluorophenyl-)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)but-2-yl)-6-iodobenzo[d][1,2,3]triazine-4(3H)-one is a commercially purchased and screened compound product.

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0027] Example 1

[0028] 1. Drug preparation

[0029] LZ-09 powder was formulated into a 1 g / L stock solution using a pharmaceutically acceptable carrier.

[0030] 2. Cell Culture

[0031] OLN-93 cells were cultured normally in DMEM high-glucose medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator, with the culture medium changed every 2–3 days. Depending on cell growth, appropriate amounts of cell culture were used for passage.

[0032] 3. Cell grouping and drug administration

[0033] OLN-93 cells were divided into a control group and an experimental group. The experimental group was given LZ-09 at concentrations of 1, 10, and 50 μg / ml, respectively, while the control group was given an equal amount of DMSO. The cells were then cultured.

[0034] 4. CCK-8 assay for cell viability

[0035] OLN-93 cells were evenly seeded into 96-well plates, and grouped and treated with drugs according to the above method. After culturing for 24 hours, CCK-8 solution was added as required by the instructions, and the cells were cultured for another 4 hours. Then, the optical density (OD) value of each well was detected at 450 nm using a microplate reader, and the cell viability of each group was calculated.

[0036] 5. CCK8 assay to detect the effect of LZ-09 on the survival rate of H2O2-damaged cells

[0037] Cells were seeded using the same method as in step 4, and cultured for another 24 hours. The supernatant was discarded, and H2O2 at final concentrations of 50, 100, and 150 μg / ml was added. After 12 hours of treatment, the absorbance was measured at 450 nm using the method described above, and cell viability was calculated. The final damaging concentration of H2O2 was determined to be 100 μg / ml, which was used as the control group. The experimental groups were treated with LZ-09 at concentrations of 1, 10, and 50 μg / ml, respectively, for 2 hours. Then, H2O2 at a final concentration of 100 μg / ml was added, and treatment continued for another 12 hours. The absorbance was measured at 450 nm using the method described above, and cell viability was calculated.

[0038] 6. Experimental Results

[0039] CCK-8 assay showed that, compared with the control group, there was no significant change in cell viability of OLN-93 cells after treatment with different concentrations of LZ-09, and the difference was not statistically significant (P>0.05), indicating that LZ-09 has no effect on oligodendrocyte viability. Figure 1 After treatment with H2O2, it was found that its damage to cells showed a certain concentration dependence, and the degree of damage increased with the increase of hydrogen peroxide concentration, reaching a damage rate of about 55% at 100 μg / ml (after 12 h of H2O2 treatment). Therefore, a concentration of 100 μg / ml of H2O2 was selected for the next efficacy experiment. Figure 2 Treatment with different concentrations of LZ-09 (1, 10, 50 μg / ml) significantly reduced the decrease in cell viability induced by H2O2. Figure 3 ).

[0040] Example 2

[0041] 1. Modeling methods

[0042] The EAE model was induced using currently accepted methods. MOG33-35 powder was dissolved in phosphate-buffered saline (PBS) to a concentration of 2.5 mg / mL. -1 It was mixed with an equal volume of CFA and Mycobacterium tuberculosis (H37RA) to maintain a Mycobacterium tuberculosis concentration of 4 mg·mL⁻¹. -1A milky white, water-in-oil antigen emulsion was prepared using an electric homogenizer. The antigen emulsion was injected subcutaneously at four points on both sides of the lumbar spine of mice, with a total injection volume of 200 μL per mouse. The day of immunization was recorded as day 0. On day 0 and day 2, pertussis toxin (PTX) 500 ng per mouse was injected intraperitoneally.

[0043] 2. Animal grouping, medication administration, and neurological function scoring

[0044] Eight to ten-week-old SPF-grade female C57BL / 6 mice were given free access to food for one week before the experiment. They were randomly divided into a normal control group, an EAE group, and an LZ-09 intervention group, with nine mice in each group. Except for the control group, the other groups underwent modeling as described above. The LZ-09 intervention group received intraperitoneal injections of 20 mg / kg LZ-09 twice daily, starting from the day of EAE modeling. The normal control group and EAE group received an equal volume of physiological saline instead of LZ-09. Administering the medication at the same time each day and recording neurological function scores were conducted. The neurological function scoring criteria were as follows: 0 points, no clinical symptoms; 1 point, tail dragging, mild hind limb weakness; 2 points, moderate hind limb weakness; 3 points, severe bilateral hind limb weakness; 4 points, quadriplegia; 5 points, convulsions, near death, or death. If the mouse's symptoms fell between two scoring criteria, the score was calculated as ±0.5. A neurological function score ≥1 point was considered indicative of disease.

[0045] 3. Histological staining

[0046] Mice in each group were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg, 2.5 mL / kg) at the peak of disease incidence after immunization (day 21). Spinal cord tissue was harvested from mice on ice and placed in sterile EP tubes, flash-frozen in liquid nitrogen, and stored at -80°C. Spinal cord tissue samples from the EAE model group and the LZ-09 intervention group were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were prepared at 25 μm intervals and subjected to hematoxylin-eosin (HE) staining and Lloyd's blue (LFB) staining. Inflammatory infiltration and demyelinating lesions of the spinal cord were observed under a light microscope.

[0047] 4. Experimental Results

[0048] (1) Effects of LZ-09 on neurological function scores in EAE model mice

[0049] Neurological function score results ( Figure 1The results showed that the normal control group had normal activity and no clinical symptoms, while the EAE model group began to show symptoms from day 9 after modeling. Mice exhibited lethargy, reduced eating and activity, and gradually developed neurological deficits, including tail dragging or complete tail paralysis, unsteady gait, unilateral or bilateral hind limb paralysis, forelimb paralysis, and even total paralysis. Compared with the EAE model group, the LZ-09 intervention group had significantly lower average clinical scores on days 12–21 of administration than the EAE group (P<0.05), suggesting that LZ-09 can effectively reduce the clinical scores of EAE mice.

[0050] (2) Effects of LZ-09 on inflammatory infiltration and demyelination of spinal cord tissue in EAE mice

[0051] HE and LFB staining results showed that the spinal cord tissue of mice in the EAE model group had significant inflammatory cell infiltration, with a large number of mononuclear cell aggregates, obvious demyelination lesions, loss of myelin sheath, and loose structural density. In contrast, the LZ-09 intervention group showed a significant reduction in the number and area of ​​mononuclear cell infiltration and aggregation in the spinal cord tissue, with tightly connected spinal cord structures and no obvious demyelination lesions. Figure 2 , Figure 3 .

[0052] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of the small molecule compound LZ-09 (Formula I) in the preparation of medicaments for the treatment and / or prevention of multiple sclerosis, characterized in that, The small molecule compound is 3-(3-(2,4-difluorophenyl-)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)but-2-yl)-6-iodobenzo[d][1,2,3]triazine-4(3H)-one. Formula (I)

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