A pharmaceutical composition for treating autoimmune uveitis and use thereof

Abstract

The present application provides a kind of anti-autoimmune uveitis pharmaceutical composition and its application, the composition includes the active ingredient capable of improving the local AEA level of retina, the active ingredient is selected from one or more of AEA or its functional analogue, fatty acid amide hydrolase inhibitor or the nucleic acid drug inhibiting its expression and the expression promoter of NAPE-PLD.The present application also provides a detection kit comprising AEA and a plurality of different lipid markers.The present application is based on the analysis of EAU mouse retina lipidomics, for the first time reveals that the cell-specific metabolic imbalance mechanism of AEA synthetase NAPE-PLD is mainly located in Müller cell, and the hydrolytic enzyme FAAH is mainly limited to microglial cell, by supplementing AEA, inhibiting FAAH or promoting the expression of NAPE-PLD, restores the retina AEA homeostasis.Experiments show that intravitreal injection of 500 μM AEA can significantly reduce EAU inflammation.The present application provides a precise, low side effect pharmaceutical composition and detection tool for the prevention and treatment of autoimmune uveitis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to a pharmaceutical composition for treating autoimmune uveitis and its application. Background Technology

[0002] Uveitis is an inflammatory disease of the eye affecting the uvea, retina, retinal vessels, and surrounding tissues, and is one of the core causes of visual impairment in clinical practice. Autoimmune uveitis, in particular, is primarily mediated by an abnormal immune response, which can gradually destroy the normal structure of the retina, impair visual function, and cause irreversible visual damage in severe cases. Experimental autoimmune uveitis (EAU) is currently the most fundamental and commonly used animal model for studying non-infectious uveitis, autoimmune retinitis, and various non-infectious intraocular inflammatory diseases in humans, providing crucial experimental support for exploring the mechanisms of these diseases and developing treatments.

[0003] The pathogenesis of autoimmune uveitis differs fundamentally from common peripheral inflammations such as skin infections and arthritis. Common peripheral inflammations are mostly induced by pathogen invasion or tissue trauma, and are self-limiting, resolving spontaneously once the causative factors are eliminated. However, autoimmune uveitis exhibits significant ocular-specific pathological features: First, the eye possesses a blood-retinal barrier forming a unique immune-immune microenvironment; disruption of this barrier's structure and function is the core initiating factor in uveitis pathogenesis. Second, the intraretinal immune regulation mechanism is highly complex, with Müller cells, microglia, and other glial cells interacting with various immune cells to form a unique ocular immune regulatory network. Third, intraocular inflammation is prone to becoming chronic and recurrent; persistent inflammatory infiltration can cause irreversible damage to photoreceptor cells, ultimately leading to permanent vision loss. Fourth, the unique anatomical structure of the eye greatly hinders drug delivery; systemic administration struggles to achieve effective therapeutic concentrations in the retinal target area, while local administration suffers from rapid intraocular metabolic clearance and short duration of action. In summary, the findings and regulatory mechanisms of peripheral inflammation cannot be directly applied to ocular inflammation. There is an urgent need to develop specific and precise inflammatory intervention strategies targeting the unique immune and metabolic microenvironment of the eye.

[0004] Currently, clinical treatment for uveitis primarily relies on glucocorticoids, immunosuppressants, and biologics, but each therapy has significant limitations. Long-term use of glucocorticoids can easily induce serious complications such as cataracts, glaucoma, intraocular pressure, and secondary infections. Immunosuppressants have systemic toxic side effects such as bone marrow suppression, liver and kidney damage, and elevated blood pressure, and their clinical efficacy varies significantly among individuals. While biologics are effective for some patients, they are costly, and some patients do not respond; long-term use can also increase the risk of severe infections and malignant tumors. Furthermore, existing clinical treatments rely on non-specific broad-spectrum immunosuppression as their core mechanism, failing to achieve precise targeted regulation of intraocular inflammation and failing to address the core challenge of low drug delivery efficiency in the eye. Therefore, identifying novel regulatory targets for local ocular inflammation and developing low-toxicity, highly effective, and specific treatment strategies has significant clinical application value and scientific research significance.

[0005] Lipid metabolism not only participates in cell membrane structure building and the body's energy metabolism, but also profoundly regulates biological processes such as inflammatory signal transduction, immune cell activation, neuroprotection, and tissue damage repair. As a neural tissue with extremely high lipid content, the retina's lipid metabolism homeostasis imbalance is highly likely to be involved in the entire process of the occurrence and development of autoimmune uveitis (EAU). However, the current scientific community still lacks a systematic and clear explanation of the dynamic changes in retinal lipidomics and its core regulatory mechanisms during the peak of EAU inflammation.

[0006] Arachidonic acid ethanolamide (AEA) is an endocannabinoid-related lipid molecule with important biological activities, and its bioactivity in vivo is strictly dependent on the dynamic balance of its synthesis and degradation pathways. N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) is a key functional enzyme regulating AEA synthesis, while fatty acid amide hydrolase (FAAH) is the core enzyme mediating AEA hydrolysis and degradation. Previous studies have confirmed that AEA plays a crucial anti-inflammatory and immunomodulatory role in peripheral inflammation. In peripheral inflammation models such as colitis and arthritis, supplementing AEA or inhibiting FAAH activity can effectively suppress excessive inflammatory activation and reduce tissue inflammatory damage. However, existing research focuses on peripheral tissue inflammation systems and has not yet addressed ocular inflammation, nor has it elucidated the regulatory mechanism of AEA metabolic pathways in the unique intraocular immune-inflammatory microenvironment of uveitis. Due to the presence of the blood-retinal barrier, specific glial cell regulatory networks, and immune exemption characteristics in the eye, the metabolic regulation pattern of AEA in retinal tissue differs significantly from that in peripheral tissues, and the mechanisms of action in peripheral inflammation cannot be directly extrapolated to the eye.

[0007] In summary, there is an urgent need in the field to develop a drug composition that can precisely regulate retinal AEA metabolism, thereby correcting the imbalance between AEA synthesis and degradation mediated by Müller cells and microglia, and targeting abnormal intraocular immune inflammation to achieve precise, efficient, and low-toxicity treatment of autoimmune uveitis. Summary of the Invention

[0008] Based on a systematic analysis of retinal lipidomics during the peak of inflammation in EAU mice, this invention is the first to discover 593 differentially expressed lipids in the retina of EAU mice. Among them, the level of AEA was significantly downregulated, and its metabolism showed a cell type-specific imbalance: the AEA synthase NAPE-PLD was mainly located in Müller cells and its expression was slightly increased during inflammation, while the AEA hydrolase FAAH was mainly limited to microglia and its expression was significantly upregulated during inflammation.

[0009] Based on the above findings, this invention proposes a technical concept to prevent and treat autoimmune uveitis by increasing the local AEA level in the retina. Specifically, it provides an anti-autoimmune uveitis composition based on retinal AEA metabolism, the active ingredient of which is selected from one or more of the following: exogenous AEA or its functional analogues, FAAH inhibitors or nucleic acid drugs that inhibit FAAH expression, and NAPE-PLD expression promoters. This composition can be administered through various local ocular routes such as intravitreal injection, eye drops, and sustained-release implantation for the prevention, relief, or treatment of autoimmune uveitis.

[0010] Furthermore, this invention provides a detection kit containing AEA and various differentially expressed lipid molecules for the detection or assessment of autoimmune uveitis, as well as a method for screening anti-uveitis candidate drugs based on the AEA-FAAH-NAPE-PLD axis. Compared with existing technologies, this invention reveals for the first time the mechanism of AEA metabolic imbalance between Müller cells and microglia in the retina, and provides a therapeutic strategy for precisely regulating retinal AEA levels, which has the advantages of strong targeting and low side effects.

[0011] In a first aspect, the present invention provides a pharmaceutical composition for treating autoimmune uveitis, the composition comprising an active ingredient capable of increasing local AEA levels in the retina; The active ingredient is selected from one or more of the following groups: (a) AEA or its functional analogues; (b) FAAH inhibitors or nucleic acid drugs that inhibit FAAH expression; (c) Expression promoters of NAPE-PLD.

[0012] This invention, through research using an EAU mouse model, found that during the peak of inflammation, the AEA synthase NAPE-PLD in the retina is mainly located in Müller cells and its expression is slightly increased, while the AEA hydrolase FAAH is mainly limited to microglia and its expression is significantly upregulated. This leads to the excessive hydrolysis of AEA produced by Müller cells by the highly expressed FAAH in microglia, resulting in a decrease in local AEA levels in the retina and the failure of endogenous AEA-mediated anti-inflammatory and immune regulatory mechanisms.

[0013] Based on the above mechanism, the composition of the present invention increases the local AEA level in the retina through the following means: for (a) the active ingredient, it directly supplements exogenous AEA or its functional analogues to compensate for the deficiency of endogenous AEA; for (b) the active ingredient, it indirectly increases the AEA level by inhibiting the enzymatic activity of FAAH or downregulating FAAH expression to reduce AEA degradation; for (c) the active ingredient, it enhances the ability of Müller cells to synthesize AEA by promoting the expression of NAPE-PLD. These active ingredients work together through different pathways to restore retinal AEA homeostasis, thereby inhibiting excessive activation of microglia, reducing intraocular immune inflammatory responses, and achieving the effect of preventing and treating autoimmune uveitis.

[0014] Preferably, the AEA functional analogues include AEA derivatives, AEA prodrugs, AEA receptor agonists, or lipid molecules with AEA biological activity.

[0015] Preferably, the lipid molecules with AEA bioactivity are selected from one or more of oleoylethanolamine, palmitoylethanolamine, stearoylethanolamine, linoleoylethanolamine, and docosahexaenoic ethanolamine, mixed in any proportion.

[0016]

[0017] Preferably, the FAAH inhibitor is selected from one or more of small molecule inhibitors, peptide inhibitors, antibodies, siRNA, shRNA, antisense oligonucleotides, or CRISPR interference systems, and is composed of any proportion of these two or more.

[0018] Preferably, the NAPE-PLD expression promoter is a substance that promotes the expression or activity of NAPE-PLD in Müller cells.

[0019] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, sustained-release materials, liposomes, nanoparticles, or hydrogels.

[0020] Preferably, the anti-autoimmune uveitis composition is an intravitreal injection, a subretinal injection, an eye drop, a sustained-release implant, a nanoformulation, a liposome formulation, or a periocular injection.

[0021] In a second aspect, the present invention provides the use of a pharmaceutical composition for treating autoimmune uveitis in the preparation of a medicament for the prevention, relief or treatment of autoimmune uveitis.

[0022] Thirdly, the present invention provides a diagnostic kit for autoimmune uveitis, the kit comprising reagents for detecting lipid biomarker levels in retinal samples of a subject, the lipid biomarkers including AEA, and one or more of the following selected from PG 42:8, PG 42:9, LPC 16:0e, LPC 18:1e, PE 16:1-16:1, SM d42:2, PMe 16:0-16:0, dMePE 16:0-16:0, Hex1Cer d18:1-24:0, and AEA 18:1 in any proportion. The diagnostic kit of the present invention can be used in conjunction with the aforementioned pharmaceutical composition to monitor changes in retinal AEA and related lipid levels after administration of the pharmaceutical composition, thereby assessing the efficacy of the drug. This invention, through retinal lipidomics analysis during the peak of inflammation in EAU mice, revealed 593 differentially expressed lipids in the retina of EAU mice compared to the normal control group. Among these, AEA levels were significantly downregulated, and the aforementioned lipid molecules, including PG, LPC, PE, SM, PMe, dMePE, and Hex1Cer, also showed significant changes. These differentially expressed lipids are involved in pathological processes such as retinal inflammation, cell membrane remodeling, immune signal transduction, and oxidative stress. Therefore, changes in AEA and related lipid molecules can directly or indirectly reflect the inflammatory status, disease progression, and severity of autoimmune uveitis.

[0023] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention, through retinal lipidomics analysis during the peak of inflammation in EAU mice, detected a total of 1115 lipids, identifying 593 differentially expressed lipids. It also demonstrated for the first time that the AEA synthase NAPE-PLD is mainly located in Müller cells, while the AEA hydrolase FAAH is mainly confined to microglia and its expression is significantly upregulated, revealing a novel cell-specific metabolic imbalance mechanism of "Müller cells producing AEA and microglia excessively hydrolyzing AEA." Based on this mechanism, the composition provided by this invention increases local AEA levels in the retina through different pathways such as AEA supplementation, FAAH inhibition, or NAPE-PLD expression, providing diverse drug options for the intervention of autoimmune uveitis.

[0024] 2. The active ingredients of this invention encompass three classes of substances with different mechanisms of action: exogenous AEA and its functional analogs, FAAH inhibitors and nucleic acid drugs, and NAPE-PLD expression promoters. These ingredients can be used alone or in any combination. Furthermore, the compositions can be prepared into various dosage forms such as intravitreal injections, eye drops, sustained-release implants, nanoformulations, liposome formulations, or periorbital injections, exhibiting strong flexibility in formulation development and clinical applicability.

[0025] 3. This invention uses high-throughput lipidomics analysis to screen lipids that are significantly differentially expressed during the peak of EAU inflammation from 1115 lipids, and identifies a group of representative lipid biomarkers. A detection kit developed based on this biomarker combination can achieve highly sensitive detection of these lipids in retinal samples, providing a powerful tool for mechanism research, drug screening, and sample analysis in autoimmune uveitis. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The figure shown is a graph illustrating the differential analysis results of retinal lipidomics during the peak of inflammation in EAU mice provided in the embodiments of this application; wherein Figure 1 A is a statistical graph showing the number of differentially expressed lipids in the retina of EAU mice during the peak of inflammation. Figure 1 B is a volcano map of differentially expressed lipids in the retina of EAU mice (the first 10 differentially expressed lipids are labeled). Figure 2 This paper shows a volcano map of endocannabinoid lipids in the retina of EAU mice (AEA mono-downregulated) provided in an embodiment of this application. Figure 3 This document shows cellular localization diagrams of the AEA synthase NAPE-PLD in the retinas of normal and EAU mice, provided in embodiments of this application. Figure 4 This invention provides cellular localization diagrams of AEA hydrolase FAAH in the retinas of normal and EAU mice, as shown in embodiments of this application. Figure 5 The image shows slit-lamp examination results of ocular inflammation in EAU mice after intravitreal injection of different concentrations of exogenous AEA, as provided in the embodiments of this application. Figure 6The following diagram illustrates the effect of intravitreal injection of different concentrations of exogenous AEA on the retinal histopathology of EAU mice, as provided in this application embodiment; wherein... Figure 6 A represents the clinical scoring results; Figure 6 B is a graph showing the pathology scoring results. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0029] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0030] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0031] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0032] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] To enable those skilled in the art to better understand this application, the following examples will provide a detailed description of a pharmaceutical composition for treating autoimmune uveitis and its application.

[0034] Example Example 1: Lipomics analysis of the retina in EAU mice (1) Establishment of experimental animals and EAU model Six- to eight-week-old female C57BL / 6J mice were randomly divided into a non-immunized control group (Control) and an EAU model group. All mice were housed under SPF conditions with free access to food and water, and were acclimatized for at least 7 days before the experiment.

[0035] The EAU model was established using the IRBP651-670 peptide immunization method: 350 μg / mouse of IRBP651-670 peptide was emulsified with an equal volume of complete Freund's adjuvant containing inactivated Mycobacterium tuberculosis H37Ra, and then subcutaneously injected at multiple sites on the back and tail base of mice. On the day of immunization, 1 μg / mouse of pertussis toxin was injected intraperitoneally to enhance the EAU induction efficiency. Samples were collected on day 14 post-immunization (the peak of EAU inflammation).

[0036] (2) Collection of retinal samples During the peak of EAU inflammation, mice were euthanized and their eyeballs were rapidly enucleated. Microscopic dissection was performed in ice-cold PBS to remove the cornea, lens, vitreous body, sclera, choroid, and RPE layer, and the neuroretina was separated. Both retinas from each mouse were combined into one biological sample, with 5-6 biological replicates per group. The retinas were rapidly washed with pre-cooled PBS, blotted dry, and immediately flash-frozen in liquid nitrogen at -80°C until lipid extraction. To avoid lipid oxidation, both sampling and extraction were performed under low-temperature, light-protected conditions.

[0037] (3) Lipid extraction Retinal lipids were extracted using the MTBE method: each sample was added to pre-cooled methanol containing a mixture of lipid internal standards (SPLASHLipidomix, AEA-d4, OEA-d4, PEA-d4, and internal standards of PC, PE, LPC, SM, Cer, etc.), and BHT was added to a final concentration of 0.01%~0.05% to prevent lipid oxidation; the samples were homogenized thoroughly on ice using a tissue homogenizer; MTBE was added, vortexed, and extracted at 4℃ for 30~60 min; ultrapure water was added to induce layering, and the samples were centrifuged at 12,000~14,000 g for 10~15 min at 4℃; the upper organic phase was collected and dried under nitrogen; the samples were reconstituted with isopropanol / acetonitrile / water and transferred to LC-MS vials.

[0038] (4) LC-MS / MS detection Non-targeted lipidomics detection was performed using the UHPLC-Q Exactive high-resolution mass spectrometry platform. Chromatographic conditions: ACQUITY UPLC CSH C18 column (2.1 × 100 mm, 1.7 μm), column temperature 45–55 °C; mobile phase A: acetonitrile / water (containing 10 mM ammonium formate and 0.1% formic acid); mobile phase B: isopropanol / acetonitrile (containing 10 mM ammonium formate and 0.1% formic acid), gradient elution; flow rate 0.25–0.35 mL / min; injection volume 2–5 μL. Mass spectrometry conditions: ESI positive and negative ion modes were used for acquisition, scan range m / z 200–1500, MS1 resolution 60,000–70,000, MS / MS resolution 15,000–17,500; spray voltage: positive ion +3.5 kV, negative ion -2.8 kV; capillary temperature 300–350 °C. One QC sample (prepared by mixing equal amounts of all experimental samples) is inserted for every 5 to 10 samples to monitor instrument stability.

[0039] Raw mass spectrometry data were processed using LipidSearch software, including peak extraction, retention time correction, lipid annotation, and peak area integration. Lipid features with excessively high missing rates were removed, and lipid abundance was normalized using internal standards. Log2 transformation and Pareto scaling were then performed. Differential lipid screening criteria: fold change ≥ 1.5 or ≤ 0.67 (i.e., |log2FC| ≥ 0.58), and... p <0.05 (or FDR-adjusted) p <0.05).

[0040] The results are as follows Figure 1 As shown in Figure A, a total of 1115 lipids were detected in the retina of EAU mice during the peak of inflammation. Compared with the non-immunized control group (Control), 593 differentially expressed lipids were found in the retina of EAU mice, of which 357 were significantly upregulated and 236 were significantly downregulated.

[0041] A volcano map was constructed based on all detected lipid molecules, such as... Figure 1 As shown in Figure B, the horizontal axis is log2(EAU / Control), and the vertical axis is -log 10(p-value). Red dots represent significantly upregulated lipids, blue dots represent significantly downregulated lipids, and gray dots represent lipids with no significant change. The top 10 differentially regulated lipids by |log2FC| are marked in the figure, including: PG 42:8, PG 42:9, LPC 16:0e, LPC 18:1e, PE 16:1_16:1, SM d42:2, PMe 16:0_16:0, dMePE16:0_16:0, Hex1Cer d18:1_24:0 O, and AEA 18:1. The first 9 are upregulated lipids, and AEA 18:1 is a downregulated lipid.

[0042] Further focusing on lipid categories related to endocannabinoid and N-acylethanolamine metabolism, these lipids were extracted from total lipidomics data, reclassified, and their log2 FC relative to the Control group in the EAU group was calculated. Volcano plots for specific categories were then constructed. Results are as follows: Figure 2 As shown, AEA exhibits monotonically down-regulated behavior in the EAU group (log2FC < 0, and...). p <0.05), while other related NAPE lipids showed no significant changes. These results indicate that AEA levels in the retina specifically decreased significantly during the peak of EAU inflammation, suggesting that abnormal AEA metabolism may be involved in the pathological process of autoimmune uveitis.

[0043] Example 2: Expression and cellular localization of AEA synthase NAPE-PLD in the retina of EAU mice This embodiment aims to detect the expression changes and cellular origin of AEA synthase NAPE-PLD in the retina of normal mice and EAU mice during the peak of inflammation. The experimental groups are shown in Table 1, with 3-5 mice in each group. At least 3 retinal slices were taken from each mouse for quantitative analysis.

[0044] Table 1 Experimental Treatment Groups

[0045] (1) Tissue fixation and sectioning: Mice were anesthetized and perfused with PBS via the heart, followed by perfusion with 4% paraformaldehyde (PFA). Eyeballs were enucleated and post-fixed in 4% PFA for 1–2 h. After washing with PBS, the tissues were dehydrated in 10%, 20%, and 30% sucrose solutions until they settled to the bottom. Then, they were embedded in OCT. Frozen sections were 10–12 μm thick and stored at -20°C or used directly for immunofluorescence staining.

[0046] (2) Immunofluorescence staining Table 2 Antibody Combinations

[0047] After the slides were brought to room temperature, they were washed with PBS, permeabilized with 0.3% Triton X-100 for 10-15 min, blocked with 5% donkey serum or 5% goat serum for 1 h, and then incubated overnight at 4°C with primary antibodies (anti-NAPE-PLD 1:100-1:300, anti-GS 1:300-1:500, anti-Iba1 1:300-1:500). After washing with PBS three times, the slides were incubated with fluorescent secondary antibody at room temperature in the dark for 1 h, counterstained with DAPI (1 μg / mL) for 5-10 min, and finally mounted with anti-fluorescence quencher and imaged under a confocal microscope.

[0048] The results are as follows Figure 3 As shown, immunofluorescence staining results revealed that NAPE-PLD was expressed at low levels in the retinas of normal mice; however, NAPE-PLD expression was slightly increased in the retinas of EAU mice during the peak of inflammation, and it was mainly located in Müller cells. Furthermore, the number of microglia in the retinas of the EAU group was significantly higher than that of the normal group, but only a small number of microglia expressed NAPE-PLD.

[0049] Example 3: Expression and cellular localization of AEA hydrolase FAAH in the retina of EAU mice This embodiment was designed to detect the expression changes of AEA hydrolase FAAH in the retina of normal mice and EAU mice during the peak of inflammation and its cell origin. The experimental groups are shown in Table 3, with 3-5 mice in each group. At least 3 retinal slices were taken from each mouse for quantitative analysis. Table 3 Experimental Groups

[0050] The tissue fixation, sectioning, and immunofluorescence staining procedures were the same as in Example 2, with the immunofluorescence staining antibody groups shown in Table 4: Table 4 Antibody Combinations

[0051] anti-FAAH 1:100~1:300, anti-Iba1 1:300~1:500, anti-CD4 1:100~1:300, DAPI1 μg / mL.

[0052] like Figure 4 As shown, immunofluorescence staining results revealed that FAAH was expressed at low levels in the retinas of normal mice; however, FAAH expression was significantly increased in the retinas of EAU mice during the peak of inflammation, primarily confined to microglia (Iba1 positive). Furthermore, CD4+ was present in the retinas of the EAU group. + T cells infiltrated, but FAAH and CD4 were not co-expressed, indicating that the infiltrating T cells were not the source of FAAH.

[0053] Example 4: Effects of intravitreal injection of exogenous AEA on EAU inflammation This embodiment aims to investigate whether exogenous AEA supplementation can alleviate ocular inflammation caused by end-stage retinitis augerta (EAU) and to functionally validate lipidomics findings. The experimental groups are shown in Table 5, with 4-6 mice per group. If EAU was induced in both eyes, the statistical unit was "per animal" to avoid simply treating both eyes as completely independent samples. Table 5 Experimental Groups

[0054] AEA is a lipid-soluble molecule, easily oxidized and degraded, and should be handled in the dark and at low temperatures. DMSO or ethanol is used as the mother liquor solvent, and the Vehicle group contains the same concentration of DMSO or ethanol as the AEA group.

[0055] Injection was performed on day 9 post-immunization (the rising phase of inflammation), and inflammation was assessed on day 14 post-immunization. Mice were anesthetized and mydriatics were dilated; topical anesthetic was instilled into the ocular surface. Under a microscope, a 33G or 34G microneedle connected to a Hamilton syringe was inserted approximately 0.5–1 mm posterior to the limbus, avoiding the lens. 1 μL of AEA or Vehicle was injected into the vitreous cavity of each eye. After injection, the needle was held in place for several seconds and slowly withdrawn, and antibiotic ointment was applied to the ocular surface. Eyes exhibiting lens damage, vitreous hemorrhage, retinal detachment, or severe regurgitation were excluded.

[0056] Slit-lamp examination was performed on day 14 post-immunization (peak EAU). Observation indicators included corneal opacity, anterior chamber inflammatory cells, anterior chamber flare, iris hyperemia, posterior synechiae, and vitreous opacity. A clinical scoring system of 0-4 points was used, and the scores were completed by two independent observers under blinded conditions. Eyeballs were fixed in 4% PFA, dehydrated, and embedded in paraffin. Sections were prepared along the optic nerve direction and H&E staining was performed to observe pathological changes such as retinal inflammatory cell infiltration, wrinkling, granulomatous lesions, and photoreceptor layer destruction.

[0057] like Figure 5 Slit lamp inspection and Figure 6 Retinal tissue section results showed that, compared with the EAU+Vehicle group, the EAU+AEA medium-dose group (500 μM) had a significantly lower inflammation score, and significantly reduced retinal inflammatory cell infiltration, folding, and photoreceptor damage. The low-dose group (200 μM) and the high-dose group (1000 μM) also showed some improvement, but the 500 μM concentration was the most significant. These results indicate that exogenous AEA supplementation can effectively alleviate ocular inflammation associated with EAU, validating the preventive and therapeutic effects of increasing local retinal AEA levels on autoimmune uveitis.

[0058] In summary, based on a systematic analysis of retinal lipidomics during the peak of inflammation in EAU mice, this invention reveals for the first time the cell-specific imbalance mechanism of retinal AEA metabolism in autoimmune uveitis. The AEA synthase NAPE-PLD is mainly located in Müller cells and its expression is slightly increased, while the AEA hydrolase FAAH is mainly confined to microglia and its expression is significantly upregulated. This leads to excessive hydrolysis of AEA produced by Müller cells by microglia, resulting in a decrease in local AEA levels in the retina. Based on this, this invention provides an anti-autoimmune uveitis composition based on regulating retinal AEA metabolism. Its active ingredient is selected from one or more of exogenous AEA or its functional analogues, FAAH inhibitors or nucleic acid drugs that inhibit FAAH expression, and NAPE-PLD expression promoters. It can be administered via various local ocular routes such as intravitreal injection, eye drops, and sustained-release implantation. Experiments show that intravitreal injection of 500 μM exogenous AEA can significantly reduce the inflammatory response and retinal pathological damage in EAU mice. Furthermore, this invention also provides a detection kit based on differential lipid biomarkers (including AEA and various lipids such as PG 42:8 and LPC 16:0e) and a drug screening method based on the AEA-FAAH-NAPE-PLD axis. Compared with existing technologies, this invention is the first to precisely target the AEA metabolic imbalance between retinal Müller cells and microglia, providing a new strategy with low side effects and high targeting for the prevention and treatment of autoimmune uveitis.

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0061] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0062] The above provides a detailed description of a pharmaceutical composition for treating autoimmune uveitis and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A pharmaceutical composition for treating autoimmune uveitis, characterized in that, The composition contains an active ingredient capable of increasing local AEA levels in the retina; The active ingredient is selected from one or more of the following groups: (a) AEA or its functional analogues; (b) FAAH inhibitors or nucleic acid drugs that inhibit FAAH expression; (c) Expression promoters of NAPE-PLD.

2. The pharmaceutical composition for treating autoimmune uveitis according to claim 1, characterized in that, The AEA functional analogues include AEA derivatives, AEA prodrugs, AEA receptor agonists, or lipid molecules with AEA biological activity.

3. The pharmaceutical composition for treating autoimmune uveitis according to claim 2, characterized in that, The lipid molecules with AEA bioactivity are selected from one or more of oleoylethanolamine, palmitoylethanolamine, stearoylethanolamine, linoleoylethanolamine, and docosahexaenoic ethanolamine, mixed in any proportion.

4. The pharmaceutical composition for treating autoimmune uveitis according to claim 1, characterized in that, The FAAH inhibitor is selected from one or more of small molecule inhibitors, peptide inhibitors, antibodies, siRNA, shRNA, antisense oligonucleotides, or CRISPR interference systems, and is composed of any proportion of these two or more.

5. The pharmaceutical composition for treating autoimmune uveitis according to claim 1, characterized in that, The NAPE-PLD expression promoter is a substance that promotes the expression or activity of NAPE-PLD in Müller cells.

6. The pharmaceutical composition for treating autoimmune uveitis according to claim 1, characterized in that, The pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, sustained-release materials, liposomes, nanoparticles, or hydrogels.

7. The pharmaceutical composition for treating autoimmune uveitis according to claim 1, characterized in that, The anti-autoimmune uveitis composition is an intravitreal injection, subretinal injection, eye drops, sustained-release implant, nanoformulation, liposome formulation, or periocular injection.

8. The use of a pharmaceutical composition for treating autoimmune uveitis as described in any one of claims 1 to 7 in the preparation of a medicament for the prevention, relief or treatment of autoimmune uveitis.

9. A diagnostic kit for autoimmune uveitis, characterized in that, The kit contains reagents for detecting lipid biomarker levels in retinal samples from subjects. The lipid biomarkers include AEA, and one or more of the following selected from PG 42:8, PG 42:9, LPC 16:0e, LPC 18:1e, PE 16:1-16:1, SM d42:2, PMe 16:0-16:0, dMePE 16:0-16:0, Hex1Cer d18:1-24:0, and AEA 18:1 in any proportion. The kit is used to detect changes in retinal AEA and related lipid levels after administration of the pharmaceutical composition according to any one of claims 1 to 7.

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