Pharmaceutical composition for treating allergic conjunctivitis in children, and preparation method and use thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for treating allergic conjunctivitis in children, its preparation method, and its uses. The pharmaceutical composition of this invention comprises hsa-miR-19b mimic and an immune peptide, wherein the immune peptide is a fusion peptide NGF-FGF, a combination of nerve growth factor (NGF) and fibroblast growth factor (FGF). In vitro and in vivo experiments have confirmed that hsa-miR-19b mimic and the fusion peptide NGF-FGF can alleviate the course of allergic conjunctivitis in children and reduce allergic symptoms by inhibiting the expression of inflammatory factors and reducing inflammatory responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for treating allergic conjunctivitis in children, its preparation method, and its uses. Background Technology

[0002] Allergic conjunctivitis in children is an IgE-mediated inflammatory eye disease triggered by allergens. It is a non-infectious ocular surface disease in children and is mainly caused by type I and type IV hypersensitivity reactions. The disease is related to factors such as atopic constitution, genetics, allergens, season, and weather. Mental stress, infection, and cold stimulation can also induce or aggravate symptoms. According to the pathogenesis and clinical manifestations, allergic conjunctivitis can be divided into the following five types: (1) Seasonal allergic conjunctivitis (SAC): related to seasonal allergens such as pollen, often accompanied by allergic rhinitis; (2) Perennial allergic conjunctivitis (PAC): present throughout the year, with mild symptoms, and related to perennial allergens such as dust mites; (3) Giant papillary conjunctivitis (GPC): related to contact lens wear; (4) Vernal keratoconjunctivitis (VKC): more common in children, with corneal complications that can threaten vision; (5) Atopic keratoconjunctivitis (AKC): combined with atopic dermatitis (eczema), involving the eyelids and facial skin. The first three types generally have a good prognosis, while the latter two types (vernal keratoconjunctivitis and atopic keratoconjunctivitis) are usually accompanied by corneal changes and may threaten vision.

[0003] Typical symptoms of allergic conjunctivitis in children include: itchy eyes—the most common symptom, manifested as frequent eye rubbing; red eyes—conjunctival vasodilation, appearing bright red; tearing—increased tear production due to eye discomfort; and a foreign body sensation—a feeling of sand or foreign objects in the eye. Other manifestations include: thick eye discharge upon waking, eyelid edema and swelling, photophobia; infants and young children often exhibit eye rubbing and tearing, while some may primarily present with cough and general malaise; in severe cases, blurred vision may occur. Physical examination may reveal conjunctival hyperemia, edema, and papillary hyperplasia; severe cases may present with Horner-Trantas nodules, corneal shield ulcers, etc.

[0004] Existing treatments generally include health education, allergen avoidance, and symptom relief, such as using artificial tears to dilute and remove allergens and relieve dryness; and rinsing the conjunctival sac with saline solution to remove allergens and secretions. However, these general treatments are not very effective for children with severe or frequent allergic conjunctivitis. For severe or frequent allergic conjunctivitis, immunotherapy can be considered. Immunotherapy has advantages such as convenient administration, high safety, and good compliance, making it particularly suitable for long-term use in children.

[0005] Based on this, the present invention aims to provide an effective immunotherapy composition for allergic conjunctivitis in children, providing effective assurance for clinical treatment. Summary of the Invention

[0006] This invention provides a pharmaceutical composition for treating allergic conjunctivitis in children, a method for preparing the composition, and its use. Specifically, it provides an effective therapeutic composition for allergic conjunctivitis in children, a method for preparing the composition, and its use.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a pharmaceutical composition for treating allergic conjunctivitis in children, characterized in that it comprises hsa-miR-19b mimic and an immune polypeptide.

[0008] Furthermore, the mature miRNA sequence of the hsa-miR-19b mimic is UUGGCAAAUCCAUGCAAAACUGA (SEQ ID NO.1).

[0009] Furthermore, the stem-loop structure sequence of the hsa-miR-19b mimic is CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG (SEQ ID NO.2).

[0010] Furthermore, the immune polypeptide is a fusion polypeptide NGF-FGF, which is a combination of nerve growth factor (NGF) and fibroblast growth factor (FGF).

[0011] Furthermore, the fusion polypeptide NGF-FGF consists of NGF and FGF linked by a linker.

[0012] Furthermore, the connector is selected from (GGGGS)n, (G)n, (EAAAK)n; n=1 or 2 or 3 or 4.

[0013] Furthermore, the amino acid sequence of the nerve growth factor (NGF) is FHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCV (SEQ ID NO.3).

[0014] Furthermore, the amino acid sequence of the fibroblast growth factor (FGF) is MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTL (SEQ ID NO.4).

[0015] Furthermore, the amino acid sequence of the fusion peptide NGF-FGF is FHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVGGGGSVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTL (SEQ ID NO. 5).

[0016] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~200 nM): (10~200) ng / mL.

[0017] Secondly, the present invention provides a method for preparing a pharmaceutical composition for treating allergic conjunctivitis in children, characterized in that hsa-miR-19b mimic is mixed with an immune polypeptide in a certain proportion to obtain the composition.

[0018] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~200 nM): (10~200) ng / mL.

[0019] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~100nM): (50~200)ng / mL.

[0020] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (30~80nM): (75~150)ng / mL.

[0021] Thirdly, the present invention provides a pharmaceutical preparation for treating allergic conjunctivitis in children, characterized in that it comprises hsa-miR-19b mimic, an immune peptide, and a pharmaceutically acceptable carrier and diluent.

[0022] Furthermore, the immune polypeptide is a fusion polypeptide NGF-FGF, which is a combination of nerve growth factor (NGF) and fibroblast growth factor (FGF).

[0023] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~200 nM): (10~200) ng / mL.

[0024] Fourthly, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating conjunctivitis.

[0025] Furthermore, the conjunctivitis is allergic conjunctivitis; even further, the conjunctivitis is childhood allergic conjunctivitis.

[0026] Furthermore, the pharmaceutical composition comprises hsa-miR-19b mimic and an immune peptide.

[0027] Furthermore, the mature miRNA sequence of the hsa-miR-19b mimic is UUGGCAAAUCCAUGCAAAACUGA (SEQ ID NO.1).

[0028] Furthermore, the stem-loop structure sequence of the hsa-miR-19b mimic is CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG (SEQ ID NO.2).

[0029] Furthermore, the immune polypeptide is a fusion polypeptide NGF-FGF, which is a combination of nerve growth factor (NGF) and fibroblast growth factor (FGF).

[0030] Furthermore, the amino acid sequence of the nerve growth factor (NGF) is FHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCV (SEQ ID NO.3).

[0031] Furthermore, the amino acid sequence of the fibroblast growth factor (FGF) is MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTL (SEQ ID NO.4).

[0032] Furthermore, the amino acid sequence of the fusion peptide NGF-FGF is FHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVGGGGSVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTL (SEQ ID NO. 5).

[0033] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~200 nM): (10~200) ng / mL.

[0034] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (10~100nM): (50~200)ng / mL.

[0035] Furthermore, the ratio of hsa-miR-19b mimic to the immune peptide is (30~80nM): (75~150)ng / mL.

[0036] The beneficial effects of this invention are that hsa-miR-19b mimic and the fusion polypeptide NGF-FGF can reduce the inflammatory response and slow down the course of allergic conjunctivitis in children by inhibiting the expression of inflammatory factors and alleviating allergic reaction symptoms. Attached Figure Description

[0037] Figure 1 This is a graph showing the relative content of inflammatory factors in the supernatant of HConEpic human conjunctival epithelial cells.

[0038] Figure 2This image shows the expression of p-ERK1 / 2 and ERK1 / 2 proteins in mouse conjunctival tissue. Lane 1 is the blank group, lane 2 is the model group, lane 3 is the experimental group (Example 1), lane 4 is control group 1 (Comparative Example 1), lane 5 is control group 1 (Comparative Example 2), and lane 6 is control group 1 (Comparative Example 3). Detailed Implementation

[0039] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0040] hsa-miR-19b mimic was purchased from MCE, Cat. No.: HY-R00404. The mature miRNA sequence is UUGGCAAAUCCAUGCAAAACUGA (SEQ ID NO.1), and the stem-loop structure sequence is CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG (SEQ ID NO.2).

[0041] The fusion peptide NGF-FGF was synthesized by Nanjing Genscript Biotech Co., Ltd. The amino acid sequence of the fusion peptide NGF-FGF is as follows: FHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVGGGGSVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTL (SEQ ID NO.5).

[0042] Previous studies have shown that the levels of miR-19b in the serum and tears of children with allergic conjunctivitis are significantly reduced, and the reduction is more significant with the increase of disease severity (Clinical Ophthalmology Journal, Vol. 33, No. 2, 2025), suggesting that miR-19b is a biological marker related to allergic conjunctivitis in children and is of great significance for the diagnosis, treatment and prognosis of allergic conjunctivitis in children.

[0043] Nerve growth factor (NGF) can regulate the function of neurons, lymphocytes, mast cells, fibroblasts and smooth muscle cells, and has the potential to regulate immunity and promote repair.

[0044] Fibroblast growth factor (FGF) has shown clear efficacy in children with allergic conjunctivitis, improving tear film stability and reducing corneal epithelial damage, and is especially suitable for children with corneal epithelial damage or tear film instability.

[0045] Example 1 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising hsa-miR-19b mimic and the fusion peptide NGF-FGF in a ratio of 50 nM: 100 ng / mL.

[0046] Example 2 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising hsa-miR-19b mimic and the fusion peptide NGF-FGF in a ratio of 80 nM: 75 ng / mL.

[0047] Example 3 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising hsa-miR-19b mimic and the fusion peptide NGF-FGF in a ratio of 30 nM: 150 ng / mL.

[0048] Comparative Example 1 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising hsa-miR-19b mimic, fibroblast growth factor (FGF), and nerve growth factor (NGF) in a dosage ratio of 50 nM: 100 ng / mL: 100 ng / mL. The amino acid sequence of the nerve growth factor (NGF) is shown in SEQ ID NO.3; the amino acid sequence of the fibroblast growth factor (FGF) is shown in SEQ ID NO.4.

[0049] Comparative Example 2 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising 80 nM of hsa-miR-19b mimic.

[0050] Comparative Example 3 A pharmaceutical composition for treating allergic conjunctivitis in children, comprising 100 ng / mL of the fusion peptide NGF-FGF.

[0051] Experimental Example 1 HConEpic human conjunctival epithelial cells (purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd., catalog number HTX2257) were cultured in DMEM / F12 medium containing 10% FBS.

[0052] Once the cells were stably passaged and reached 60% confluence, an in vitro model of allergic conjunctivitis was constructed, and experimental groups were formed. The control group received no treatment. The model group, experimental group (Example 1), and control group (Comparative Examples 1-3) were treated with histamine (5 μM) for 24 h. Simultaneously with the addition of histamine, the experimental group received 10 μL of the drug composition from Example 1, and the control groups (Comparative Examples 1-3) received 10 μL of the drug composition from Comparative Examples 1-3, respectively. Cell culture was then incubated for 24 h. The cell culture medium was collected, centrifuged, and the relative levels of inflammatory factors in the cell supernatant were detected using an ELISA kit. Results are shown below. Figure 1 .

[0053] The data from this experiment show that the pharmaceutical composition with hsa-miR-19b mimic and the fusion peptide NGF-FGF as active ingredients can significantly reduce histamine-induced pro-inflammatory factors IL-6 and TNF-α, indicating that the pharmaceutical composition of the present invention can improve the inflammatory response of allergic conjunctivitis in children by reducing the expression of pro-inflammatory factors.

[0054] Experiment Example 2 Following the method of Fan Hua (Study on the anti-inflammatory and immune mechanism of Qiwei Shumin Fang on ovalbumin-induced allergic conjunctivitis mice [D]. China Academy of Chinese Medical Sciences), a mouse model of allergic conjunctivitis in children was prepared: Young BALB / c mice were intraperitoneally injected with a mixture containing 100 μg of high-purity ovalbumin and 5 mg of Al(OH)3 adjuvant on days 0, 7, and 14 of the experiment, with a total injection dose of 200 μL, as the basic sensitization stage; then, 300 μg of high-purity ovalbumin dissolved in 100 μL of physiological saline was applied to the right eye every day for 3 consecutive weeks as the modeling and stimulation stage. The successful establishment of the model was determined based on the ocular symptoms of the mice starting from day 7 of the modeling and stimulation stage.

[0055] The experiment included a control group, a model group, an experimental group (Example 1), and a control group (Comparative Examples 1-3). Starting from day 8 of the modeling and activation phase, the experimental and control groups received 100 μL of the corresponding drug composition instilled into the right eye daily, along with an intraperitoneal injection of 200 μL of the corresponding drug composition, for two consecutive weeks. The control group received no modeling and only received an equal volume of physiological saline in the same manner. The model group received both eye drops and an intraperitoneal injection of an equal volume of physiological saline starting from day 8 of the modeling and activation phase. After the experiment, serum samples were collected from mice, and conjunctival tissue was separated.

[0056] Table 1. Serum inflammatory markers in mice ; Note: *p<0.05 compared to the model group, #p<0.05 compared to the experimental group. The expression of the inflammatory protein p-ERK1 / 2 in isolated mouse conjunctival tissue was detected by Western blotting. The results are shown below. Figure 2 Lanes 1-6 correspond to the blank group, model group, experimental group, control group 1, control group 2, and control group 3, respectively.

[0057] Data from this experiment demonstrates the successful establishment of a mouse model of allergic conjunctivitis in children. The serum levels of pro-inflammatory factors TNF-α, IFN-γ, and IL-1β were significantly increased in successfully modeled mice, and the p-ERK1 / 2 protein content in the conjunctival tissue was significantly elevated. Treatment with hsa-miR-19b mimic and the fusion peptide NGF-FGF significantly reduced the serum levels of pro-inflammatory factors TNF-α, IFN-γ, and IL-1β, as well as the p-ERK1 / 2 protein content in mice. This indicates that hsa-miR-19b mimic and the fusion peptide NGF-FGF can alleviate the course of allergic conjunctivitis in children and reduce allergic symptoms by inhibiting the inflammatory response.

[0058] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A pharmaceutical composition for treating allergic conjunctivitis in children, characterized in that, It contains hsa-miR-19b mimic and an immune peptide; the immune peptide is a fusion peptide NGF-FGF, which is a combination of nerve growth factor (NGF) and fibroblast growth factor (FGF).

2. The pharmaceutical composition according to claim 1, characterized in that, The mature miRNA sequence of the hsa-miR-19b mimic is UUGGCAAAUCCAUGCAAAACUGA.

3. The pharmaceutical composition according to claim 1, characterized in that, The amino acid sequence of the nerve growth factor (NGF) is shown in SEQ ID NO.

3.

4. The pharmaceutical composition according to claim 1, characterized in that, The amino acid sequence of the fibroblast growth factor (FGF) is shown in SEQ ID NO.

4.

5. The pharmaceutical composition according to claim 1, characterized in that, The fusion polypeptide NGF-FGF consists of NGF and FGF linked by a linker.

6. The pharmaceutical composition according to claim 1, characterized in that, The ratio of hsa-miR-19b mimic to immune peptides is (10~200 nM): (10~200) ng / mL.

7. The method for preparing the pharmaceutical composition for treating allergic conjunctivitis in children as described in claim 1, characterized in that, The hsa-miR-19b mimic is prepared by mixing it with immune peptides in the specified ratio.

8. The preparation method according to claim 7, characterized in that, The ratio of hsa-miR-19b mimic to immune peptides is (10~200 nM): (10~200) ng / mL.

9. A pharmaceutical preparation for treating allergic conjunctivitis in children, characterized in that, The pharmaceutical composition comprising the treatment of allergic conjunctivitis in children as described in claim 1, and a pharmaceutically acceptable carrier and diluent.

10. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating conjunctivitis.

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