Application of milk fat globule epidermal growth factor 8 in preparing drug for treating neuromyelitis optica spectrum disorder

Abstract

An application of milk fat globule epidermal growth factor 8 in preparing a drug for treating neuromyelitis optica spectrum disorder. Milk fat globule epidermal growth factor 8 (MFG-E8) can be used as a drug for treating neuromyelitis optica spectrum disorder, thus broadening the range of application of milk fat globule epidermal growth factor 8 (MFG-E8), and also providing a new option the vast number of NMOSD patients; the present invention can reduce the incidence of neuromyelitis optica in NMOSD patients and improve the quality of life of said patients, the present invention has a significant curative effect, and no obvious toxic side effects have been observed.

Description

Application of human milk fat globule epidermal growth factor 8 in the preparation of drugs for treating neuromyelitis optica spectrum disorders

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 29, 2023, with application number 202311871636.9 and invention name “Application of human milk fat globule epidermal growth factor 8 in the preparation of drugs for the treatment of neuromyelitis optica spectrum diseases”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of human milk fat globule epidermal growth factor 8 in preparing drugs for treating neuromyelitis optica spectrum disorders. Background Art

[0003] Milk fat globule epidermal growth factor VIII (MFG-E8) is a lipophilic glycoprotein originally discovered in mammary epithelial cells and is one of the most important proteins in the milk fat globule membrane (MFGM). Studies have found that human MFG-E8 has three domains: EGF-L, C1, and C2. MFG-E8 can promote the phagocytosis of apoptotic cells by macrophages and plays an important role in angiogenesis, autoimmune diseases, and tumors. Furthermore, MFG-E8 is found on exosomes and can adhere to cell membranes, suggesting its potential for transporting target proteins.

[0004] Studies have reported that MFG-E8 targets chondrocyte senescence and macrophage reprogramming via the NF-κB signaling pathway, preventing osteoarthritis. Other studies have shown that bone marrow MSC-derived MFG-E8 may accelerate wound healing in diabetic mice by promoting angiogenesis, clearing apoptotic cells, infiltrating M2 macrophages, and inhibiting inflammatory cytokines in the wound area. Furthermore, in a rat model of traumatic brain injury, MFG-E8 reduced apoptosis via the integrin β3 / FAK / PI3K / AKT signaling pathway. MFG-E8 alleviated the inflammatory response in a mouse model of subarachnoid hemorrhage by driving microglia toward M2 polarization, providing neuroprotection and improving neurological dysfunction in the model mice. In summary, MFG-E8 plays an important role in inflammatory and traumatic diseases.

[0005] Neuromyelitis optica (NMO) is an acute or subacute demyelinating disorder involving the optic nerve and spinal cord simultaneously or sequentially. First described by Devic (1894), the disease is characterized by acute or subacute onset of monocular or bilateral blindness, preceded or followed by transverse or ascending myelitis days or weeks before onset. Later, it was referred to as Devic's disease or Devic's syndrome. On July 14, 2015, the American Academy of Neurology published the "National Consensus on Diagnostic Criteria for Neuromyelitis Optica Spectrum Disorders (NMSOD)" in its journal Neurology, designating NMO as a unified term for neuromyelitis optica spectrum disorders (NMOSD).

[0006] To date, there is no accurate epidemiological data on NMOSD internationally. According to relevant literature, the current incidence of NMOSD in China is 0.278 cases per 100,000 people per year, and it is estimated that there are approximately 50,000 NMOSD patients in China. On May 11, 2018, the National Health Commission and five other departments jointly developed the "First List of Rare Diseases," which included neuromyelitis optica.

[0007] Currently, there is no effective treatment for NMOSD. For patients in the acute phase of NMOSD, the primary goals are to alleviate acute symptoms, shorten the course of the disease, improve disability, and prevent and treat complications. Treatment options include steroids, plasma exchange, immunoglobulins, and hormones combined with other immunosuppressants. For patients in remission, the primary goals are to reduce relapses and delay disability progression. The most commonly used medications include azathioprine, rituximab, mycophenolate mofetil, methotrexate, mitoxantrone, cyclophosphamide, and cyclosporine A. For patients with other complications, treatment for these complications and rehabilitation training are also recommended.

[0008] Therefore, the development of drugs and innovative treatment strategies for NMOSD has important scientific value and social significance. Summary of the Invention

[0009] In order to solve the problem that there is a lack of drugs with significant therapeutic effects on neuromyelitis optica spectrum disorders (NMSOD) in the prior art, the purpose of the present invention is to provide the use of human milk fat globule epidermal growth factor 8 in the preparation of drugs for treating neuromyelitis optica spectrum disorders.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The first object of the present invention is to provide an application of human milk fat globule epidermal growth factor 8 in the preparation of a drug for treating neuromyelitis optica spectrum disorders.

[0012] Preferably, the neuromyelitis optica spectrum disease includes: neuromyelitis optica, relapsing optic neuritis, longitudinally extending transverse myelitis, multiple sclerosis optica, long-stage transverse myelitis, unilateral or bilateral optic neuritis, optic neuritis or myelitis associated with autoimmune disease, and optic neuritis or myelitis associated with symptomatic or asymptomatic brain lesions.

[0013] The second object of the present invention is to provide the use of human milk fat globule epidermal growth factor 8 in the preparation of a drug for treating neuromyelitis optica.

[0014] Preferably, the drug comprises human milk fat globule epidermal growth factor 8 and a pharmaceutically acceptable carrier.

[0015] Preferably, the carrier includes excipients, diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and stabilizers.

[0016] Preferably, the drug is an oral preparation or an injectable preparation.

[0017] The design principle of the present invention:

[0018] The human milk fat globule epidermal growth factor 8 (MFG-E8) provided by the present invention has a significant therapeutic effect on NMSOD. Experimental results of MFG-E8 in NMOSD cell models and mouse models showed that compared with the model control group, MFG-E8 treatment inhibited astrocyte damage and microglial activation, and improved motor function impairment and related histopathological lesions in the NMOSD mouse model, demonstrating that the MFG-E8 provided by the present invention has a significant therapeutic effect on NMSOD.

[0019] Therefore, compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a new application of human milk fat globule epidermal growth factor 8 (MFG-E8) in the field of medicine, that is, human milk fat globule epidermal growth factor 8 (MFG-E8) can be used as a drug for treating neuromyelitis optica spectrum disorders, broadening the application scope of human milk fat globule epidermal growth factor 8 (MFG-E8). It also provides a new option for the majority of NMOSD patients, can reduce the incidence of neuromyelitis optica in NMOSD patients and improve their quality of life, and has significant therapeutic effects without obvious toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a diagram showing the results of cell immunofluorescence analysis;

[0022] FIG2 is a diagram showing the treatment scheme of MFG-E8 for NMOSD mouse model according to the present invention;

[0023] FIG3 is a diagram showing the results of a fatigue rotating rod test;

[0024] Figure 4 is a diagram showing the results of a gait analysis experiment;

[0025] Figure 5 shows the histopathological results. DETAILED DESCRIPTION

[0026] The following is a further detailed description of the present invention through specific embodiments in the form of examples, but it should not be understood that the scope of the present invention is limited to the following examples.

[0027] Equipment and instruments:

[0028] The human milk fat globule epidermal growth factor 8 (MFG-E8) was purchased from Signalway Antibody (SAB), product number AP70540-1;

[0029] The fatigue tachometer was purchased from Chengdu Taimeng Software Co., Ltd., model ZB-200;

[0030] The gait experiment analysis system was purchased from Shanghai Xinruan Technology Co., Ltd., model XR-FP101.

[0031] Example 1 Cell test

[0032] 1.1 Construction of NMOSD cell model

[0033] 1) Astrocyte isolation and culture

[0034] Poly-D-lysine (PDL) was used to pre-coat culture flasks (purchased from Thermo Fisher Scientific, product number 156367) and astrocyte complete culture medium (45 mL DMEM / F12 + 5 mL FBS + 500 μl NEAA + 500 μl glutamine + 500 μl double antibody + 100 μl sodium pyruvate) was prepared. One-day-old C57 suckling mice (obtained through in-house breeding in the laboratory) were selected, disinfected, decapitated, the skull was peeled, the meninges were removed, the cortex was taken, and 0.05% trypsin was used for digestion. The cells were centrifuged (300 g, 10 minutes), the supernatant was discarded, the culture medium was added again, and the cells were gently pipetted and filtered through a 70 μm filter. The medium was changed every other day for the first time. Thereafter, the medium was changed every 3 days for 14 consecutive days.

[0035] 2) Cell modeling

[0036] Astrocytes were seeded into 24- or 6-well plates. After the cell confluence reached 70-80% and the purity of the astrocytes was identified, the cells were treated with serum IgG (hsAQP4-IgG) (100 ng / mL) from patients positive for aquaporin 4 (AQP4) for 4 hours to establish the model.

[0037] Immunofluorescence staining with GFAP antibody (Santa Cruz Biotechnology, Catalog No. sc-33673) and AQP4 antibody (Santa Cruz Biotechnology, Catalog No. sc-20812) was performed. The model was considered successful when the AQP4 fluorescence intensity was significantly reduced compared with the unmodeled control group.

[0038] 1.2 Therapeutic effect of MFG-E8 on NMOSD cell model

[0039] The experimental group was divided into 3 groups, as follows:

[0040] 1) Negative control group: mouse astrocytes were treated with healthy human serum control IgG (hsCtrl-IgG) (100 ng / mL) and then treated with astrocyte complete culture medium for 4 h;

[0041] 2) NMOSD model control group: mouse astrocytes were treated with hsAQP4-IgG (100 ng / mL) and then treated with astrocyte complete culture medium for 4 h;

[0042] 3) MFG-E8 treatment group: mouse astrocytes were treated with hsAQP4-IgG (100 ng / mL) and then treated with astrocyte complete medium containing MFG-E8 (100 ng / mL) for 4 h;

[0043] After treatment, GFAP and AQP4 immunofluorescence staining was performed as follows: the cell slides fixed with 4% paraformaldehyde were rinsed three times with phosphate buffered saline (PBS) for 5 minutes each time, incubated in blocking solution at room temperature for 1 hour, added with primary antibody (GFAP antibody or AQP4 antibody), incubated in a shaker at 4°C overnight, removed on the next day, rinsed three times with PBS for 5 minutes each time, added with fluorescent secondary antibody, incubated in a shaker at room temperature in the dark for 1 hour, rinsed three times with PBS for 5 minutes each time, and finally mounted with antifade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI). After drying, the slides were observed under a laser confocal microscope (Leica, TCS SP8), photographed, and the fluorescence signal intensity was quantitatively analyzed.

[0044] The experimental results are shown in FIG1 . As shown in FIG1 , MFG-E8 inhibited the overactivation of GFAP-positive astrocytes and restored the expression level of AQP4 in astrocytes.

[0045] Example 2 Animal Experiment

[0046] The MFG-E8 treatment scheme for the NMOSD mouse model of the present invention is shown in Figure 2. The experimental mice were divided into a healthy control group, an NMOSD model group, a hMFG-E8 single-dose group, and a hMFG-E8 two-dose group. The experiment was conducted according to the mouse treatment scheme shown in Figure 2. Figure 2 shows the experimental stages from D(-7) to D10. D(-7) to D0 were the modeling stage. hsCtrl-IgG / hsAQP4-IgG was administered daily from D0 to D10. hMFG-E8 was administered to the hMFG-E8 single-dose group (D2) and the hMFG-E8 two-dose group (D2 and D6). Relevant behavioral assessments and interventions were performed during this period. Samples were collected after anesthesia and perfusion on D10.

[0047] The animal groups and drug administration are as follows:

[0048] The mice were divided into 4 groups, 10 mice per group, and the experiment was carried out according to the mouse treatment plan shown in Figure 2, as follows:

[0049] 1) Healthy control group: On D2 and D6 of the experimental period, the animals were injected with PBS via the tail vein, with 200 μl of PBS injected per animal.

[0050] 2) NMOSD model group: On D2 and D6 of the experimental period, the animals were injected with PBS via the tail vein, with 200 μl of PBS injected per animal.

[0051] 3) hMFG-E8 single-dose group: On D2 of the experimental phase, the animal was given a tail vein injection of hMFG-E8 (20 μg / kg) per injection;

[0052] 4) hMFG-E8 two-time group: On D2 and D6 of the experimental period, the animals were given tail vein injections of drugs, with hMFG-E8 (20 μg / kg) injected into each animal each time.

[0053] 2.1 Construction of a mouse model of neuromyelitis optica spectrum disorder (NMOSD)

[0054] The method is as follows: 8-week-old SPF-grade adult C57 / BL6J female mice (provided by Guangdong Animal Monitoring Institute, weighing 20-25g) were selected. In order to destroy the blood-brain barrier of mice and prevent its self-repair, in the pretreatment stage, on D(-7), four points on the back of the mice were subcutaneously injected with 50ug heat-inactivated H37Ra tuberculin (BD-DIFCO) and 50uL complete Freund's adjuvant mixture (purchased from Sigma-Aldrich) at each point, and 200ng of cough toxin (purchased from Enzo Life Science) were intraperitoneally injected. On D(-4), pertussis toxin was injected intraperitoneally again; on D(-1), pertussis toxin was injected intraperitoneally again; from D0 to D10, purified hsCtrl-IgG / hsAQP4-IgG were continuously injected intraperitoneally, and relevant behavioral assessments and interventions were given during this period. On D10, the mice were anesthetized and perfused for sampling.

[0055] During the modeling period, behavioral evaluations were conducted, including fatigue rotarod test and gait analysis test, to evaluate the modeling effect. The specific experimental process is as follows:

[0056] 1) Fatigue rotating rod test

[0057] Behavioral testing was performed on days 0, 2, 4, 6, 8, and 10 after modeling. The rotarod task was performed using a 6-track fatigue rotator. This device requires mice to maintain balance and walk on a rotating cylinder, starting at 10 revolutions per minute and accelerating uniformly to 40 revolutions per minute over 5 minutes. Each mouse was tested three times at 15-minute intervals, and the time the mouse spent on the device until landing was calculated as the average of the three tests.

[0058] 2) Gait analysis experiment

[0059] Gait analysis was performed using the Catwalk assisted gait analysis system (XR-FP101, SHXINRUAN), which consisted of mice walking freely on a narrow terrain, and the length of the hindlimb stride was calculated as the average of five consecutive steps.

[0060] 3) Behavioral evaluation

[0061] The results of the fatigue rotarod test are shown in Figure 3. The experimental results show that compared with the NMOSD model group, single or double administration of MFG-E8 can significantly prolong the time that NMOSD model mice stay on the rotarod, thereby improving the movement disorders of NMOSD model mice.

[0062] The results of the gait analysis experiment are shown in Figure 4. The experimental results show that compared with the NMOSD model group, single or double administration of MFG-E8 can significantly increase the stride length of NMOSD model mice, thereby improving the movement disorders of NMOSD model mice.

[0063] 2.2 Therapeutic effect of MFG-E8 on NMOSD mouse model

[0064] The mice with successful modeling were sacrificed, and the therapeutic effects of the model group and the stem cell intervention groups with different doses and administration times were evaluated by histopathology, immunohistochemistry / fluorescence and molecular biology methods.

[0065] 2.2.1 Tissue Preparation

[0066] The spinal cord of the mouse was fixed by perfusion with 4% paraformaldehyde, and soaked overnight in 4% paraformaldehyde, which was then replaced with 30% sucrose. After the tissue sank to the bottom, frozen sections were made with a thickness of approximately 40 μm. The sections were immersed in freezing solution and stored in a 4°C refrigerator for later use.

[0067] 2.2.2 Immunofluorescence experiments

[0068] Rinse with PBS three times, 5 minutes each time, incubate with blocking solution at room temperature for 1 hour, add primary antibodies (GFAP antibody, AQP4 antibody, IBA1 antibody (Wako Chemicals, product number 019-19741)), incubate in a shaker at 4°C overnight, take out the next day, rinse with PBS three times, 5 minutes each time, add fluorescent secondary antibody, incubate in a shaker at room temperature in the dark for 1 hour, rinse with PBS three times, 5 minutes each time, and finally seal with antifade mounting medium containing DAPI. After drying, observe under a laser confocal microscope, take pictures, and quantitatively analyze the fluorescence signal intensity.

[0069] 2.2.3 Efficacy evaluation

[0070] The results of the histopathological experiment are shown in Figure 5. The experimental results show that compared with the NMOSD model group, the number of activated (GFAP-positive cells) astrocytes was significantly reduced after single or double administration of MFG-E8, the expression level of astrocyte AQP4 was significantly restored, and the number of microglia representing inflammation (IBA-1-positive cells) was significantly reduced, demonstrating that MFG-E8 can inhibit the pathological changes of astrocytes and suppress inflammation.

[0071] In summary, the present invention has demonstrated through NMOSD cell model and animal model experiments that human milk fat globule epidermal growth factor 8 (MFG-E8) can be used as a drug for the treatment of neuromyelitis optica spectrum disorders, broadening the scope of application of human milk fat globule epidermal growth factor 8 (MFG-E8).

[0072] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Use of human milk fat globule epidermal growth factor 8 in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

2. The application according to claim 1, characterized in that, The neuromyelitis optica spectrum disorders include: neuromyelitis optica, recurrent optic neuritis, longitudinally extensive transverse myelitis, neuromyelitis optica type multiple sclerosis, long-segment transverse myelitis, unilateral or bilateral optic neuritis, optic neuritis or myelitis associated with autoimmune diseases, optic neuritis or myelitis with symptomatic or asymptomatic intracerebral lesions.

3. Use of human milk fat globule epidermal growth factor 8 in the preparation of a medicament for treating neuromyelitis optica.

4. The application according to any one of claims 1 to 3, characterized in that The medicament contains human milk fat globule epidermal growth factor 8 and a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The carrier includes excipients, diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and stabilizers.

6. The application according to any one of claims 1-5, characterized in that, The medicament is an oral preparation or an injection preparation.

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