Use of plasma exosomes in the preparation of a medicament for treating ocular inflammation

Abstract

The application discloses application of plasma exosomes in preparation of a medicine for treating eye inflammation. The eye inflammation is an eye vascular inflammatory disease. The plasma exosomes are healthy human plasma exosomes. The plasma exosomes can effectively inhibit the eye vascular inflammation progression of EAU mice, significantly reduce the eye vascular inflammatory cell and factor infiltration, thereby preventing the retinal function loss and structural regressive changes caused by the eye vascular inflammation. The plasma exosomes are directly derived from the human circulatory system, retain the natural conformation of endogenous immune regulatory molecules, have higher biocompatibility, can avoid the potential immune rejection risk of heterologous exosomes, and have a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of plasma exosome applications, specifically to the application of plasma exosomes in the preparation of drugs for treating ocular inflammation. Background Technology

[0002] In medicine, "ocular inflammation" is not a single disease entity. Based on the different main affected structures and pathological basis, it can be clearly divided into two major categories: ocular vascular inflammatory diseases and ocular neurodegenerative or neuroinflammatory diseases. These two types of diseases are fundamentally different in terms of pathogenesis, target sites, and clinical manifestations. First, in terms of pathological mechanisms, ocular vascular inflammatory diseases include ocular vascular inflammatory diseases associated with systemic immune diseases, specifically including ocular vascular inflammation in autoimmune uveitis, ocular vascular inflammation in autoimmune retinitis (such as Behçet's disease-associated uveoangiitis, Behçet's disease-associated retinal angiitis, etc.), etc. Its main manifestations are immune-mediated vascular endothelial damage and inflammatory cell infiltration. Its core mechanisms include endothelial cell activation, leukocyte adhesion and migration, release of inflammatory factors, and blood-retinal barrier disruption. On the other hand, ocular neurodegenerative or neuroinflammatory diseases (such as glaucomatous optic nerve damage, hereditary optic neuropathy, age-related macular degeneration, etc.) mainly involve neuronal apoptosis, axonal degeneration, and glial cell activation. Its essence is chronic degenerative changes or metabolic damage to neural structures. Secondly, regarding the pathogenic factors, ocular vascular inflammatory diseases are usually closely related to autoimmune abnormalities, infection-induced immune responses, or systemic vasculitis, such as Behcet's disease (BD) and Vogt-Koyanagi-Harada syndrome (VKH syndrome). Neurodegenerative or neuroinflammatory diseases, on the other hand, are more often associated with genetic factors, metabolic abnormalities, ischemia / hypoxia, or mechanical injury (such as increased intraocular pressure). Thirdly, regarding the main target cells, ocular vascular inflammation primarily affects vascular endothelial cells, pericytes, and circulating immune cells, with the lesion core located in the vascular wall and its surrounding inflammatory microenvironment. Neurological diseases, however, mainly affect retinal ganglion cells, photoreceptor cells, and glial cells, representing lesions of the nerve tissue itself. Finally, in terms of pathogenesis and clinical manifestations, ocular vascular inflammatory diseases mainly manifest as vascular leakage, hemorrhage, vascular occlusion and inflammatory exudation. Clinically, retinal vasculitis, macular edema and acute visual fluctuations can be seen. On the other hand, neurodegenerative or neuroinflammatory diseases manifest as optic nerve atrophy, visual field defects and progressive visual loss, which are usually irreversible chronic progressions.

[0003] The traditional treatment for autoimmune uveitis is to suppress active inflammation in the eye and prevent potential vision impairment. Early, continuous, and systemic use of corticosteroids remains the most important and effective treatment for autoimmune uveitis (Chee SP, Jap A, Bacsal K: Prognostic factors of Vogt-Koyanagi-Harada disease in Singapore. Am J Ophthalmol 2009, 147(1).). Treatment should continue for at least 6 months to 1 year, followed by gradual tapering of the hormones. Due to the risk of relapse, it is not recommended to end systemic treatment within the first 3 months. For patients who are intolerant or resistant to glucocorticoids, immunosuppressants such as cyclosporine, azathioprine, methotrexate, and cyclophosphamide are used to control ocular inflammation. However, these methods have varying degrees of side effects.

[0004] Exosomes are membrane-bound vesicles ranging in size from 30 nm to 150 nm, secreted by different types of cells under various normal or pathological conditions. Exosomes play a crucial role in intercellular communication by transferring cargo such as proteins, lipids, and nucleic acids from donor cells to recipient cells. Because exosomes are present in most bodily fluids (such as blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal fluid, and peritoneal fluid) and play an important role in carrying bioactive molecules from cells, they have attracted considerable interest, and their value in the preparation of drugs for treating diseases is being explored.

[0005] Exosomes carry a variety of biomolecular cargoes. Since exosomes are constitutively secreted by most cell types, their cargoes reflect their host diversity (Yoon YJ, Kim OY, Gho YS. Extracellular vesicles as emerging intercellular communicasomes. BMB Rep. 2014;47(10):531–539. Armstrong D, Wildman DE. Extracellular vesicles and the promise of continuous liquid biopsies. J Pathol Transl Med. 2018;52(1):1-8.). After secretion, exosome cargoes can be transferred to neighboring cells and even shuttle long distances to different cell types, playing a crucial role in intercellular communication. From intercellular communication to physiological and pathological processes, exosomes perform beneficial and / or harmful functions, making them promising candidates for the development of drugs to treat various diseases. However, further research is needed to determine which specific types of exosomes will be effective for which specific diseases. Summary of the Invention

[0006] The inventors of this invention unexpectedly discovered during their research that patients with Behcet's disease have excessive vesicle secretion, and the increase in exosomes can aggravate the disease in vivo and in vitro models. Abnormal exosome composition may be a key factor in its pathogenesis. Therefore, it was conceived that exosomes could be used to prepare drugs for treating Behcet's disease-related diseases. Based on this technical concept, this invention provides an application of plasma exosomes in the preparation of drugs for treating ocular inflammation.

[0007] The use of plasma exosomes in the preparation of a drug for treating ocular inflammation, wherein the ocular inflammation is an inflammatory disease of ocular vascular inflammatory disease.

[0008] Preferably, the plasma exosomes are plasma exosomes from healthy individuals. More preferably, the plasma exosomes are peripheral blood plasma exosomes from healthy individuals. The term "healthy individual" refers to a person whose health examination results show all indicators within the normal range or who has no disease.

[0009] The preparation of exosomes from healthy human plasma can be achieved through multiple centrifugations, for example, by the following preparation method, including the following steps:

[0010] (1) After anticoagulating the peripheral blood of healthy individuals with an anticoagulant, centrifuge at 2℃-5℃ and 800g-1200g for 8min-12min, and collect the orange-yellow supernatant, which is the peripheral blood plasma of healthy individuals;

[0011] (2) Dilute peripheral blood plasma from healthy individuals with PBS buffer at pH 7.4, centrifuge at 2℃-5℃ and 300g-500g for 8min-12min, and collect the supernatant to obtain the first supernatant;

[0012] Centrifuge the first supernatant at 2℃-5℃ and 1800g-2200g for 8min-12min, and collect the supernatant to obtain the second supernatant;

[0013] Centrifuge the second supernatant at 2℃-5℃ and 10000g-15000g for 25min-30min, and collect the supernatant to obtain the third supernatant;

[0014] The third supernatant was centrifuged at 2℃-5℃ and 100000g-110000g for 60min-70min. The supernatant was discarded, and the liquid at the bottom of the centrifugation was collected to obtain crude peripheral blood plasma exosomes from healthy individuals.

[0015] The crude peripheral blood plasma exosomes from healthy individuals were resuspended in an appropriate amount of PBS buffer at pH 7.4, centrifuged at 100,000-110,000g for 60-70 minutes at 2℃-5℃, the supernatant was discarded, and the liquid at the bottom of the centrifugation was collected to obtain peripheral blood plasma exosomes from healthy individuals.

[0016] Preferably, the ocular vascular inflammatory disease includes ocular vascular inflammatory diseases associated with systemic immune diseases.

[0017] More preferably, the ocular vascular inflammatory disease includes one or two of the following: ocular vascular inflammation of autoimmune uveitis, ocular vascular inflammation of autoimmune retinitis, etc.

[0018] Furthermore, the ocular vascular inflammatory diseases include one or more of the following: ocular vascular inflammation of Behçet's disease-associated uveitis, ocular vascular inflammation of Behçet's disease-associated retinitis, ocular vascular inflammation of Vogt-Koyanagi-Harada syndrome-associated uveitis, ocular vascular inflammation of Vogt-Koyanagi-Harada syndrome-associated retinitis, diabetic retinopathy, and retinopathy of wet age-related macular degeneration.

[0019] The drug can be formulated for intravenous injection. The active ingredient in the formulation is the plasma exosome, and commonly used excipients in the pharmaceutical industry can be added.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Previous studies in this invention have demonstrated that peripheral blood plasma exosomes from healthy individuals can effectively inhibit the progression of ocular vascular inflammation in EAU mice. Preliminary clinical trials have also confirmed that peripheral blood plasma exosomes from healthy individuals can effectively inhibit the progression of ocular vascular inflammation, significantly reduce the infiltration of inflammatory cells and factors in ocular vasculitis, thereby preventing retinal dysfunction and structural degenerative changes caused by ocular vascular inflammation. Future research will further explore the underlying mechanisms of action to advance the application of plasma exosomes in the treatment of ocular vascular inflammation.

[0022] Existing technologies have widely reported the role of exosomes derived from stem cells or exogenous organisms (such as plants and bacteria) in inflammation regulation. However, most current technologies rely on non-human or in vitro modified exosomes. In contrast, this invention is the first to use natural exosomes directly isolated from peripheral blood of healthy individuals for inflammation regulation, especially ocular inflammation. Its advantages lie in its direct origin from the human circulatory system, preserving the natural conformation of endogenous immune regulatory molecules (such as HLA-G and CD47), resulting in higher biocompatibility and avoiding the potential immune rejection risk of heterologous exosomes. Moreover, it eliminates the need for complex stem cell expansion or gene editing steps, simplifying the process, reducing safety risks, eliminating significant toxic side effects, and demonstrating significant therapeutic efficacy with promising application prospects. Attached Figure Description

[0023] Figure 1 A simplified diagram illustrating the experimental procedure for assessing uveitis inflammation;

[0024] Figure 2 Comparison of fundus photographs of EAU mice in different experimental groups;

[0025] Figure 3 Comparison of clinical scores of fundus inflammation in EAU mice from different experimental groups;

[0026] Figure 4 H&E staining images of fundus pathological sections from healthy mice and EAU mice in each experimental group after 5 consecutive days of injection.

[0027] In this context, IRBP represents photoreceptor-binding retinoid peptide; Day 0 represents the day of modeling; Day 6 represents the 6th day after modeling; (Day 7-11) represents the 7th to 11th days after modeling; (Day 11-17) represents the 11th to 17th days after modeling; post immunization represents the period after modeling; Day 11 represents the 11th day after modeling; Day 14 represents the 14th day after modeling; Day 17 represents the 17th day after modeling; PBS represents the PBS buffer injection group; NC-Exos represents the group injected with peripheral blood plasma exosomes from healthy individuals; BD-Exos represents the group injected with peripheral blood plasma exosomes from Behçet's disease patients; days postimmunization represents the number of days after modeling; EAU score represents the clinical score of fundus inflammation in EAU mice; and Naive represents healthy mice. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can better understand the present invention, but should not be construed as limiting the technical solutions of the present invention.

[0029] Example 1

[0030] (1) Peripheral blood anticoagulated with EDTA from healthy volunteers (EDTA-K2 was prepared into an 18 mg / mL solution using physiological saline or buffer, and then the solution was mixed with peripheral blood at a weight ratio of 1:9 to obtain EDTA-anticoagulated peripheral blood from healthy individuals) was centrifuged at 1000g for 10 minutes at 4°C. The resulting orange-yellow supernatant was the peripheral blood plasma from healthy individuals, which was then aliquoted and stored at -80°C. When needed, it was thawed slowly on ice before use.

[0031] (2) Methods for isolating plasma exosomes (ultracentrifugation) include:

[0032] i. Dilute 5 mL of peripheral blood plasma from healthy individuals to 10 mL with PBS buffer at pH 7.4, then centrifuge at 4°C and 300 g for 10 minutes using a benchtop centrifuge. Collect the supernatant to obtain the first supernatant.

[0033] ii. Take the first supernatant and centrifuge it at 2000g for 10 minutes in a benchtop centrifuge at 4℃. Take the centrifuged supernatant to obtain the second supernatant.

[0034] iii. Using an ultracentrifuge, after pre-cooling at 4°C, pour the second supernatant into an ultracentrifuge tube, centrifuge at 10000g for 30 minutes, and take the centrifuged supernatant to obtain the third supernatant.

[0035] iv. Take the third supernatant and centrifuge at 100,000g for 70 minutes at 4°C. Discard the supernatant and retain 200μL of liquid at the bottom of the centrifuge tube to obtain crude peripheral blood plasma exosomes from healthy individuals. Discard the remaining liquid.

[0036] v. Resuspend the crude peripheral blood plasma exosomes from healthy individuals in an appropriate amount of PBS buffer at pH 7.4, and centrifuge again at 100,000g for 70 minutes at 4°C. Discard the supernatant to obtain 100 μg of peripheral blood plasma exosomes from healthy individuals at the bottom of the ultracentrifuge tube (under the same operating method, the amount of exosomes obtained from different donors will vary due to individual differences, generally ranging from 50 μg to 200 μg).

[0037] vi. Resuspend 100 μg of healthy human peripheral blood plasma exosomes at the bottom of the ultracentrifuge tube using 200 μL of pH 7.4 PBS buffer to obtain healthy human peripheral blood plasma exosomes resuspended in PBS buffer. Aliquot and store at 4°C for one week or -80°C for three months.

[0038] (3) Healthy human peripheral blood plasma exosomes resuspended in PBS buffer were intravenously infused into volunteers with uveitis (with vascular inflammation in the fundus). The dosage was 100 μg of healthy human peripheral blood plasma exosomes per injection, once a day for 5 consecutive days.

[0039] (4) After injection, observe the patient's routine condition and follow up for a long period of time. It was found that with the increase of injection frequency, the inflammation of fundus blood vessels in patients with uveitis gradually decreased. Five days after injection, the inflammation of fundus blood vessels was significantly reduced, and the condition was close to that of healthy people.

[0040] Comparative Example 1

[0041] In Example 1, the peripheral blood of healthy volunteers anticoagulated with EDTA was replaced with peripheral blood of Behcet's disease patients anticoagulated with EDTA. The remaining procedures were the same as in Example 1, and 200 μg of peripheral blood plasma exosomes from Behcet's disease patients were obtained.

[0042] Behçet's disease peripheral blood plasma exosomes from the bottom of an ultracentrifuge tube were resuspended in 400 μL of PBS buffer (pH 7.4) to obtain Behçet's disease peripheral blood plasma exosomes resuspended in PBS buffer. These were then aliquoted and stored at 4°C for one week or at -80°C for three months.

[0043] Peripheral blood plasma exosomes resuspended in PBS buffer were intravenously infused into volunteers with uveitis (with fundus vascular inflammation). The dosage was 100 μg of peripheral blood plasma exosomes from Behçet's disease patients once daily for 5 consecutive days. Patients' routine conditions were observed after injection, and they were followed up long-term. It was observed that with increasing injection frequency, fundus vascular inflammation in uveitis patients further aggravated, and the inflammation significantly worsened after 5 days of injection.

[0044] To investigate the role of peripheral blood plasma exosomes in ocular vascular inflammatory diseases such as Behçet's disease-associated uveitis and Vogt-Koyanagi-Harada syndrome-associated uveitis, as well as ocular vascular inflammation associated with other autoimmune diseases like Behçet's disease-associated retinitis and Vogt-Koyanagi-Harada syndrome-associated retinitis, this study constructed an experimental autoimmune uveitis (EAU) model to simulate fundus vascular inflammation in autoimmune uveitis / retinitis. The EAU model is a classic model for studying autoimmune diseases, with primary lesions located in the choroid and retina. Its fundus lesions can be quantitatively analyzed using clinical scoring, exhibiting good reproducibility and stability, and can be used for inflammation assessment in uveitis and retinitis and for exploring autoimmune mechanisms.

[0045] C57BL / 6j mice were injected subcutaneously at multiple sites with a mixture of interphotoreceptor retinoid binding protein fragment (IRBP) 1-20 peptides (250 μg per mouse) and a complete Freund's adjuvant (CFA) emulsion (200 μL per mouse). Pertussis toxin (PTX) solution (1 μg PTX powder dissolved in 200 μL of pH 7.4 PBS buffer to prepare a 5 μg / mL PTX solution, 100 μL per mouse) was also injected intraperitoneally to induce ocular vascular inflammation in the uveitis / retinitis complex, thus establishing an EAU mouse model. The specific method is as follows: Interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) fragment was dissolved in PBS buffer at pH 7.4, resulting in a dosage of 250 μg IRBP1-20 per mouse. Subsequently, the antigen solution containing 250 μg IRBP1-20 was thoroughly mixed with an equal volume of Complete Freund's adjuvant (CFA), and a stable emulsion (i.e., IRBP1-20 / CFA emulsion) was prepared using ultrasonic emulsification or repeated injection. The final total volume of emulsion injected into each mouse was 200 μL, containing 150 μg IRBP1-20 and approximately 100 μL CFA. The emulsification endpoint was determined by the emulsion not rapidly spreading after being dropped onto water. The prepared emulsion was placed on ice and used as soon as possible.

[0046] During immunization, mice were fixed in place and injected subcutaneously at multiple points on the base of the tail and the lateral thighs of both sides, with each mouse receiving a total of 200 μL of IRBP1-20 / CFA emulsion. To enhance the immune response, pertussis toxin solution was injected intraperitoneally on the day of immunization and on day 1 post-immunization, with each mouse receiving 0.5 μg of PTX per injection, for a total of two injections.

[0047] On day 7 of successful EAU modeling, each mouse was injected daily via the retroorbital vein with either 100 μg of pH 7.4 PBS buffer (PBS group), 100 μg of peripheral blood plasma exosomes from healthy individuals in Example 1 (NC-Exos group), or 100 μg of peripheral blood plasma exosomes from Behçet's disease patients in Comparative Example 1 (BD-Exos group). This injection was repeated for 5 consecutive days. On days 11, 14, and 17, fundus photography and inflammation scoring were performed on each experimental group of EAU mice. It is recommended that the classic clinical fundus scoring method for experimental autoimmune diseases be used for EAU inflammation scoring, referring to the clinical fundus scoring system established and applied to mouse EAU models by Caspi / Chen et al. The scoring criteria include the degree of vitreous opacity, optic disc edema, retinal vascular inflammatory exudation, vascular white sheathing changes, retinal folds or linear lesions, and focal retinal inflammatory infiltration. The scoring system was semi-quantitative, ranging from 0 to 4 points: 0 points indicated no significant inflammation; 0.5-1 points indicated mild vitreous opacity, a small number of inflammatory cells, or mild vascular changes; 2 points indicated moderate vitreous opacity, definite vasculitis, optic disc edema, or focal retinal inflammation; 3 points indicated severe vitreous opacity, widespread vasculitic exudation, significant retinal folds, or multifocal inflammation; and 4 points indicated extremely severe inflammation, characterized by severe vitreous opacity, widespread retinal structural destruction, retinal detachment, or significant atrophic changes. Results are shown below. Figure 2 , Figure 3 and Figure 4 . Figure 2 In the images, the white areas represent inflammatory cell infiltration, and the blood vessels in the three sets of photos show significant differences in thickness: the peripheral blood plasma exosome group from healthy individuals can effectively inhibit inflammatory cell infiltration and edema in the ocular blood vessels. Figure 2 and Figure 4 The results showed that EAU mice injected with PBS exhibited mild to moderate vasculitis and linear lesions in their fundus; EAU mice injected with peripheral blood plasma exosomes from healthy individuals showed very mild fundus inflammation, with only small peripheral lesions or even no disease changes, closely resembling the fundus vascular state of healthy mice; EAU mice injected with peripheral blood plasma exosomes from Behçet's disease patients showed moderate to severe vasculitis and linear lesions in their fundus; statistical analysis was performed on the fundus scores of the three groups of EAU mice (see...). Figure 3 The study found that peripheral blood plasma exosomes in patients with Behcet's disease significantly aggravated vascular damage in EAU uveitis / retinitis, while peripheral blood plasma exosomes in healthy individuals significantly reduced vascular inflammation in EAU uveitis / retinitis.

[0048] The above studies confirmed that peripheral blood plasma exosomes from healthy individuals can effectively inhibit the progression of ocular vascular inflammation in EAU mice. This indicates that peripheral blood plasma exosomes from healthy individuals can effectively inhibit ocular vascular inflammation associated with Behçet's disease-related uveitis, Behçet's disease-related retinitis, Vogt-Koyanagi-Harada syndrome-related uveitis, Vogt-Koyanagi-Harada syndrome-related retinitis, diabetic retinopathy, and wet age-related macular degeneration. Preliminary clinical evidence also confirms that peripheral blood plasma exosomes from healthy individuals can effectively inhibit the progression of ocular vascular inflammation, significantly reduce the infiltration of inflammatory cells and factors in ocular vasculitis, and thus prevent retinal dysfunction and structural degeneration caused by ocular vascular inflammation. Future research will further explore the underlying mechanisms of action and advance the application of plasma exosomes in the treatment of ocular vascular inflammation.

Claims

1. The use of plasma exosomes in the preparation of a drug for treating ocular inflammation, wherein the ocular inflammation is an inflammatory disease of ocular vascular inflammatory disease.

2. The application according to claim 1, characterized in that, The plasma exosomes are plasma exosomes from healthy individuals.

3. The application according to claim 2, characterized in that, The plasma exosomes are peripheral blood plasma exosomes from healthy individuals.

4. The application according to claim 1, characterized in that, The ocular vascular inflammatory diseases mentioned include ocular vascular inflammatory diseases associated with systemic immune diseases.

5. The application according to claim 1, characterized in that, The ocular vascular inflammatory diseases include one or both of the ocular vascular inflammations of autoimmune uveitis and autoimmune retinitis.

6. The application according to claim 1, 4, or 5, characterized in that, The ocular vascular inflammatory diseases include one or more of the following: ocular vascular inflammation of Behçet's disease-associated uveitis, ocular vascular inflammation of Behçet's disease-associated retinitis, ocular vascular inflammation of Vogt-Koyanagi-Harada syndrome-associated uveitis, ocular vascular inflammation of Vogt-Koyanagi-Harada syndrome-associated retinitis, diabetic retinopathy, and retinopathy of wet age-related macular degeneration.

7. The application according to claim 1, characterized in that, The drug is a formulation for intravenous injection.

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