Use of docosahexaenoic acid in the preparation of a medicament for treating anti-nmdar encephalitis

Abstract

This invention discloses the application of docosahexaenoic acid (DHA) in the preparation of drugs for treating N-methyl-D-aspartate receptor (NMDAR) encephalitis. This invention used DHA to intervene in the treatment of mice with anti-NMDAR encephalitis, and found that the treated mice showed recovery in behavior, antibody titers, NMDAR endocytosis, and hippocampal synaptic plasticity, demonstrating that DHA can treat the antibody production mechanism of anti-NMDAR encephalitis. This invention provides a precise therapeutic drug regimen targeting the pathogenesis of anti-NMDAR encephalitis, which can reduce the numerous adverse reactions of existing first-line treatments and has good application prospects.

Description

Technical Field

[0001] This invention relates to the application of docosahexaenoic acid (DHA) in the preparation of drugs for treating NMDAR encephalitis, and belongs to the field of biomedical technology. Background Technology

[0002] Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune encephalitis mediated by autoantibodies targeting NMDAR in the central nervous system. It is the most common type of autoimmune encephalitis, accounting for approximately 80% of all cases. This disease commonly affects young adults aged 18–45, with women accounting for approximately 80% of cases. Some cases are associated with viral infections or ovarian teratomas. Typical clinical manifestations include behavioral and psychiatric abnormalities, seizures, cognitive impairment, motor disorders, and autonomic dysfunction, often leading to misdiagnosis as mental illness or viral encephalitis.

[0003] Currently, first-line treatment for NMDAR encephalitis includes glucocorticoids, plasma exchange, and intravenous immunoglobulin; early resection is necessary for patients with concurrent tumors. Although these treatments significantly improve the prognosis of most patients, the mortality rate for severely ill patients, such as those with refractory status epilepticus, central hypoventilation, or severe altered consciousness, remains 5%–10%. It is noteworthy that there are currently no drug-specific treatments approved by regulatory agencies worldwide that directly target the pathogenesis of NMDAR encephalitis. Existing immunotherapy regimens suffer from delayed response, significant individual variability in efficacy, and numerous potential side effects.

[0004] Fatty acids and their metabolites are key regulators of autoimmune homeostasis, primarily participating in the maintenance of immune balance by modulating inflammatory responses, immune cell function, and exerting neuroprotective effects. Based on chemical structure, fatty acids can be classified into saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, with polyunsaturated fatty acids playing a central role in neuroimmune regulation. Polyunsaturated fatty acids are further divided into two complementary families, ω-3 and ω-6, which require balanced intake. The ω-3 series mainly includes α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), primarily exhibiting anti-inflammatory and neuroprotective effects. Fatty acids with different structures have differential regulatory effects on B cell function. In B cells, fatty acid metabolism precisely regulates their activation, differentiation, and antibody secretion processes, a mechanism particularly prominent in autoimmune diseases such as systemic lupus erythematosus. Studies have shown that eicosapentaenoic acid supplementation can inhibit plasma cell differentiation and autoantibody production, and enhance anti-inflammatory capabilities.

[0005] Docosahexaenoic acid (DHA) is an ω-3 long-chain polyunsaturated fatty acid with important physiological functions. Its chemical structure is characterized by 22 carbon atoms and 6 cis double bonds, systematically named 22:6(ω-3). The molecule's spatial conformation, due to the specific arrangement of its multiple cis double bonds, exhibits a highly curved three-dimensional structure, which gives it unique physicochemical properties in biological membrane systems. When integrated into the phospholipid bilayer of the cell membrane, DHA effectively reduces the orderliness of the membrane system through its inherent spatial conformation, significantly enhancing the fluidity and plasticity of the membrane structure. This property plays a crucial role in maintaining the plasticity of neural synapses and the efficiency of visual signal transduction. Given the limited conversion efficiency of endogenous synthesis pathways in the human body, this substance is defined as a conditionally essential nutrient in nutrition and must be obtained through diet or supplements. However, the role of DHA in the treatment of NMDAR encephalitis has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to address the current lack of therapeutic drugs for NMDAR encephalitis. This invention provides the application of DHA in the preparation of drugs for treating NMDAR encephalitis. Through in vivo animal experiments, this invention demonstrates that the use of DHA holds promise for achieving precise treatment of NMDAR encephalitis through targeted metabolic immunity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the application of docosahexaenoic acid in the preparation of drugs for treating NMDAR encephalitis.

[0009] Preferably, the drug comprises a pharmaceutically effective ingredient and pharmaceutically acceptable excipients, wherein the pharmaceutically effective ingredient is docosahexaenoic acid (DHA).

[0010] Preferably, the pharmaceutically acceptable excipient is one or more of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, and buffer.

[0011] Preferably, the dosage form of the drug is an oral preparation or an injectable preparation.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention provides DHA as a novel drug regimen for treating NMDAR encephalitis. DHA can target the pathogenesis of NMDAR encephalitis and the pathogenic NMDAR antibody, reduce antibody production, and thus reduce the severity of the disease. It is a precision treatment targeting the pathogenesis and can reduce the adverse reactions with many side effects of existing first-line treatments.

[0013] 2) As an essential nutrient for the human body, DHA has a high safety profile at appropriate doses, and its long-term side effects are significantly lower compared to traditional treatments such as potent immunosuppressants. Furthermore, DHA's beneficial effects on cognition, mood, and overall brain health support comprehensive neurological rehabilitation. Attached Figure Description

[0014] Figure 1 Behavioral and memory changes and treatment effects in anti-NMDAR encephalitis mice: (ab) Results of cognitive function assessment using the Y-maze test in the control group, anti-NMDAR encephalitis mice, and the treatment group receiving DHA treatment; On day 33, all anti-NMDAR encephalitis mice showed significant cognitive decline, while the control group did not; DHA treatment restored the cognitive level of NMDAR mice. (a) shows the typical path diagram. Each group contained 14 mice. (cd) Compared to the control group, untreated anti-NMDAR encephalitis mice exhibited anxiety in the elevated cross maze test; this anxiety was significantly alleviated after treatment. (c) shows the typical path diagram. Each group contained 12 mice. (e) Through forced swimming experiments, it was found that compared to the control group, untreated model group mice spent significantly more time immobile in the water, exhibiting depressive symptoms, while the treated mice showed relative relief.

[0015] Figure 2 The total IgG level in the serum of mice with NMDAR encephalitis was significantly reduced. We collected peripheral blood serum samples after euthanizing the mice and performed total IgG ELISA on the serum samples. The results showed that the total IgG level in the model group mice was significantly increased, indicating that the mice produced a large amount of IgG after modeling. However, IgG levels were significantly downregulated after DHA treatment, demonstrating that DHA treatment can reduce the production of pathogenic antibodies in mice.

[0016] Figure 3DHA treatment restored NMDAR levels in mice with anti-NMDAR encephalitis: (a) Density analysis of total cell surface NMDAR (GluN1) clusters, PSD-95, and synaptic NMDAR clusters (defined as NMDAR clusters colocalized with PSD-95) in the CA3 region of the hippocampus; top: CA3 of control mice, middle: anti-NMDAR encephalitis mice, bottom: anti-NMDAR encephalitis mice after DHA treatment; scale bar = 10 µm; for each mouse, we examined 9 square images similar to Figure A (3 images each of CA1, CA3, and DG regions) to quantify the NMDAR cluster density in the three groups of mice, showing (bd) that the total NMDAR clusters and synaptic NMDAR clusters in the hippocampus (CA1, CA3, DG) were significantly downregulated in anti-NMDAR encephalitis mice, while the number of total NMDAR clusters and synaptic NMDAR clusters increased after DHA treatment, demonstrating that DHA treatment can reduce the endocytosis of NMDAR receptors.

[0017] Figure 4 To investigate the effects of long-term potentiation (LTP) in the hippocampus and the excitability of pyramidal neurons in anti-NMDAR mice and after DHA treatment: (ab) Compared with the control group, untreated anti-NMDAR encephalitis mice showed a significant decrease in the slope of excitatory postsynaptic potentials (fEPSPs); anti-NMDAR encephalitis mice treated with DHA showed recovery of fEPSPs; (c) Compared with the control group, untreated anti-NMDAR encephalitis mice showed a significant downregulation of pyramidal neuronal excitability, while mice treated with DHA showed recovery of neuronal excitability; supporting the ameliorative effect of DHA on NMDAR encephalitis-related neurological dysfunction. Detailed Implementation

[0018] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] Example 1 This embodiment first constructs an anti-NMDAR encephalitis mouse model, and the construction method is as follows: 1) Anesthesia: Female C57BL / 6 mice aged 8-10 weeks were randomly divided into a model group and a control group. Mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol at a dose of 300 μL / mouse.

[0022] 2) Initial immunization: 300 μg of GluN1 (359-378) peptide fragments were dissolved in 100 μL of PBS (pH 7.4) and thoroughly emulsified with 800 μg of Mycobacterium tuberculosis in 100 μL of incomplete Freund's adjuvant. Each model group mouse was subcutaneously injected with this emulsion mixture on both sides of its back. Simultaneously, each mouse was intraperitoneally injected with 200 μL of PTX solution diluted in PBS at pH 7.4 with a concentration of 1 μg / mL. The control group received the same dose of emulsion without GluN1 (359-378).

[0023] 3) Second PTX injection: 48 hours after the first immunization, inject 200 μL of PTX solution with a concentration of 1 μg / mL into the peritoneum again.

[0024] 4) Boost immunization: On day 14 after the first immunization, repeat all procedures of the first immunization (including anesthesia, subcutaneous injection of peptide emulsion and intraperitoneal injection of PTX).

[0025] 5) Fourth PTX injection: On day 16 after the first immunization, 200 μL of PTX solution with a concentration of 1 μg / mL was injected intraperitoneally again.

[0026] The establishment of the anti-NMDAR encephalitis mouse model was confirmed through behavioral, immunological, and brain tissue pathological examinations. The anti-NMDAR encephalitis mice exhibited anxiety, cognitive impairment, and depression in behavior, and their serum contained high titers of anti-NMDAR antibodies. The endocytosis of NMDAR in the hippocampus of their brain tissue was reduced.

[0027] After modeling, the mice were treated with DHA for 35 days. The experiment was divided into three groups: sham group, control group (GluN1-PBS), and treatment group (GluN1-DHA). The treatment group received 200 μL of DHA in PBS solution (pH 7.4, drug concentration 40 mg / kg) intraperitoneally daily, while the control group received 200 μL of PBS solution daily until day 35.

[0028] Following treatment, mice were subjected to three behavioral tests—Y-maze, elevated cross maze, and forced swimming—before sacrifice. The Y-maze primarily verified cognitive dysfunction; the elevated cross maze primarily verified anxiety; and forced swimming primarily verified depression. The results are as follows: Figure 1 As shown. Figure 1 ab represents the results of cognitive function assessment using the Y-maze test; on day 33, all anti-NMDAR encephalitis mice showed significant cognitive decline, while control mice did not, and DHA treatment restored the cognitive level of anti-NMDAR encephalitis mice. Figure 1 cd represents the results of assessing anxiety in mice using the elevated cross maze test. The results showed that, compared with the control group, untreated anti-NMDAR encephalitis mice exhibited anxiety in the elevated cross maze test, while the anxiety in the DHA treatment group was significantly alleviated. Figure 1 e represents the results of the forced swimming test to assess depressive mood in mice. The results showed that, compared with the control group, the untreated model group (control group) mice spent significantly longer periods of time remaining still in the water and exhibited depressive mood, while the treated group mice showed relative relief.

[0029] Mice were sacrificed on day 35, and whole blood serum was collected. The total IgG level in the serum was measured, demonstrating a significant decrease in total IgG levels after DHA intervention. The results are as follows: Figure 2 As shown in the figure. Paraffin sections of mouse hippocampal tissue were also collected and stained with NMDAR. Comparison with the model group revealed a significant improvement in NMDAR endocytosis, while PSD-95 protein showed no significant decrease, demonstrating that synaptic integrity was not affected. The results are as follows. Figure 3 As shown.

[0030] This embodiment also performed electrophysiological analysis on mouse hippocampal slices, primarily using EPSP to assess hippocampal synaptic plasticity. The results showed that hippocampal synaptic plasticity significantly decreased after modeling, but essentially returned to normal levels after treatment. Patch-clamp techniques were used to assess the excitability of mouse pyramidal neurons, revealing a significant decrease in pyramidal neuronal excitability after modeling, which was improved after DHA treatment. This demonstrates the ameliorative effect of DHA on neurological functional impairment related to NMDAR encephalitis. The results are as follows: Figure 4 As shown.

[0031] The above results indicate that treatment with DHA against NMDAR encephalitis can alleviate the clinical manifestations and antibody titers in mice with NMDAR encephalitis, thus demonstrating its therapeutic effect against NMDAR encephalitis and its ability to reduce the severity of the disease.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of docosahexaenoic acid in the preparation of drugs for treating NMDAR encephalitis.

2. The application according to claim 1, characterized in that, The drug comprises a pharmacologically active ingredient and pharmaceutically acceptable excipients, wherein the pharmacologically active ingredient is docosahexaenoic acid (DHA).

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

4. The application according to any one of claims 1 to 3, characterized in that, The drug is available in oral or injectable formulations.

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