CN110051656A - Purposes of half hydrochloride of O- (carboxymethyl) azanol in preparation treatment uveitis drug - Google Patents

Abstract

The invention belongs to the field of medicine, and particularly relates to the use of O- (carboxymethyl)hydroxylamine hemihydrochloride in preparing a medicine for treating uveitis. The medicament of the invention is used for treating uveitis, and has the advantages of less side effect and high curative effect.

Description

Use of O-(carboxymethyl)hydroxylamine hemihydrochloride in the preparation of medicine for treating uveitis

technical field

The invention belongs to the field of medicine, in particular to the use of O- (carboxymethyl)hydroxylamine hemihydrochloride in preparing a medicine for treating uveitis.

Background technique

Uveitis, also known as uveitis, is a general term for inflammation of the iris, ciliary body and choroid tissue. It is a group of inflammatory diseases characterized by intraocular inflammation, which can cause some serious complications and sequelae. one of the blinding eye diseases. The disease is characterized by: photophobia, floaters, vision loss, visual field blind spots and systemic symptoms. The etiology and mechanism of uveitis are complex. According to the anatomical site of inflammation, it can be divided into: 1. Anterior uveitis; 2. Intermediate uveitis; 3. Posterior uveitis. Uveitis is the main cause of blindness worldwide. It is a common ophthalmological disease and occurs mostly in young adults. In recent years, some studies have shown that its incidence is increasing.

Experimental autoimmune uveitis (EAU, Experimental autoimmune uveitis) is an animal model of human endogenous uveitis. EAU can be induced by immunizing susceptible animals with retinal antigens. EAU and human uveitis have similar immunological features, as both result from T cell-mediated damage to the retina and its associated tissues.

The traditional treatment of uveitis is usually immunosuppressive therapy such as glucocorticoids, plus immunosuppressive agents such as cyclosporine and rapamycin. Due to the particularity of the disease, topical or periocular corticosteroids cannot achieve therapeutic effect and therefore need to be taken orally. However, systemic corticosteroids have serious side effects and cannot be used in children and cannot prevent the recurrence of uveitis, especially are HLA-B27-related patients. Traditional treatment methods not only bring serious side effects, but also some patients are not sensitive to treatment.

O- (carboxymethyl)hydroxylamine hemihydrochloride has the effect of inhibiting the development of Alzheimer's disease, and recent experiments have shown that it can also treat the development of experimental allergic encephalomyelitis (EAE, Experimenta autoimmuneencephalomyelitis) and its effect of the degree of inflammation. Summarizing previous literature and patent reports, O- (carboxymethyl)hydroxylamine hemihydrochloride is mainly used for anti-inflammatory treatment and treatment of Alzheimer's disease. So far no reports of O- (carboxymethyl)hydroxylamine hemihydrochloride in the treatment of uveitis have been reported.

SUMMARY OF THE INVENTION

The object of the present invention is to provide the use of O- (carboxymethyl) hydroxyhemihydrochloride for treating uveitis. The medicine is used for treating uveitis, and has the advantages of less side effect and high curative effect.

The technical scheme of the present invention is:

Application of O- (carboxymethyl)hydroxylamine hemihydrochloride in the treatment of uveitis In the present invention, the used AOA is used for the treatment of uveitis, and can be administered in the form of injection without oral administration.

The dosage of the drug was 31.25ng/kg per day.

The O- (carboxymethyl) hydroxylamine hemihydrochloride of the present invention has a molecular formula of NH ₂ OCH ₂ COOH · 0.5HCl, a molecular weight of 109.30, an English name (Aminooxy-acetic acid), and an English abbreviation of AOA.

The chemical structure of O- (carboxymethyl)hydroxylamine hemihydrochloride is:

The above-mentioned AOA can also be obtained by chemical synthesis.

The applicant found for the first time that AOA has a strong inhibitory effect on inflammation in the body. It has been verified through experiments that the drug has a reducing effect on inflammatory cells, while anti-inflammatory cells have an increasing effect; it also has a reducing effect on inflammatory factors in the body; The connection function of the damaged retinal cell barrier in inflammatory mice is restored, and its physicochemical properties are stable and the toxic and side effects are small, so it can be prepared as a drug for the treatment of uveitis.

Description of drawings

Figure 1 shows that AOA reduces anterior chamber inflammation;

Figure 2 shows that AOA reduces the clinical score of uveitis;

Figure 3 shows that AOA reduces the aggregation of retinal inflammatory cells;

Figure 4 is the pathological score of AOA to alleviate uveitis;

Figure 5 shows that AOA restores tight junctions between retinal cell barriers;

Figure 6 shows that the proportion of inflammatory cells decreased and the proportion of anti-inflammatory cells increased after AOA treatment;

Figure 7 shows the decrease in the secretion of inflammatory factors and the increase in the secretion of anti-inflammatory factors in vivo after AOA treatment;

Figure 8 shows that the expression of inflammatory factors decreased and the expression of anti-inflammatory factors increased after AOA treatment.

Detailed ways

The following implementation materials illustrate the invention in detail, but do not limit the invention in any way.

Example 1: Induction of Experimental Autoimmune Uveitis (EAU)

Select 6-8-week-old C57BL/6J female mice (the mice were purchased from the Animal Experiment Center of Chongqing Medical University and raised in an environment where the mice are independently air-supplied and isolated) and treated with sodium pentobarbital (Merck, Germany) anesthesia. After the mice were anesthetized, 100ul of 3.5ng/ul IRBP651-670 (LAQGAYRTAVDLESLASQLT, Sangon, Shanghai) solution was mixed with 100ul of complete Freund's adjuvant (5 mg/ml Mycobacterium tuberculosis H37RA (Difco, USA)) ( Sigma, USA) was connected through a sterile three-way tube and mixed thoroughly into a white emulsion with a total volume of 200ul, and then the white emulsion was used on the back of the neck, the outer sides of both thighs, the base of the tail and the underarms of each mouse. Subcutaneous injection. Then, 1ug pertussis toxin (Sigma, USA) dissolved in 200ul sterile phosphate buffered saline PBS (Dingguochangsheng, Beijing) was intraperitoneally injected to obtain the EAU model.

Example 2: EAU model grouping and AOA injection, clinicopathological scoring

2.1 Experimental method:

On the 9th day of the above EAU model, a slit lamp (Shangbang, Chongqing) was used to observe whether there was congestion in the anterior chamber of the eyeball. If congestion was present, the modeling was successful. Mice with successful modeling were selected and randomly divided into PBS control group and AOA (Sigma, USA) (Aminooxy-acetic acid) (500ug/mice) treatment group, with 10-20 model mice in each group. The AOA treatment group was intraperitoneally injected with 200ul of O- (carboxymethyl)hydroxylamine hemihydrochloride (ie AOA), and the control group was injected with an equal volume of phosphate buffered saline (PBS) for 5 consecutive days. Mice were clinically scored on day 14 post-immunization according to the level of intraocular inflammation. The clinical score of intraocular inflammation is based on five signs of conjunctival hyperemia, ciliary hyperemia, corneal opacity, anterior chamber inflammation and retroiris adhesion, and is divided into 0-5 points. Then, 6 mice were taken out of each group, and the pentobarbital sodium was overdose to death. The eyeballs were taken out and the surrounding tissues were carefully removed. They were fixed with eyeball fixative (Sevier Bio, Wuhan), dehydrated, transparent, immersed in wax, cooled, and packaged. After the paraffin section was embedded, the cornea-optic nerve axis was cut into 4-6um thick sections with a microtome (Leica, Germany). After deparaffinization and alcohol hydration, hematoxylin-eosin staining was performed, and the pathological changes of the retinal tissue were observed under a light microscope. The severity of EAU was evaluated according to Caspi's scoring standard, which was divided into 0-4 points, and the inflammation degree of mice in each group was pathologically scored by double-blind method.

2.2 Experimental results: AOA significantly reduced the severity of inflammation in the EAU model. A schematic diagram of the results is shown in Figure 1-4. Figure 1 shows that there is no pupillary adhesion in the AOA treatment group and the vitreous congestion is relieved, while the PBS group has adhesion and severe congestion; Figure 2 shows that after treatment, compared with the PBS group, the clinical score of the AOA group decreased and was statistically significant; 3. The results of pathological sections showed that the number of inflammatory cells, folds and vasculitis in the AOA treatment group was lower than that in the PBS group; Figure 4 shows that the pathological score of the AOA treatment group was significantly lower than that of the PBS group.

Example 3: AOA restores tight junctions between retinal cell barriers

3.1 Experimental method: Take 3 mice that were successfully modeled and treated in Example 2, wipe the tail of each mouse with an alcohol cotton ball to fully dilate the tail vein, immobilize the mouse and pull its tail to make it in the In a tense state, the needle was inserted at 1/3 of the tip of the tail. If the blood vessel swayed with the needle, the blood was drawn back and then injected with 100ul of 2% Evans blue (Sigma, USA). Two hours later, the neck was severed and sacrificed. Mice were removed and their eyeballs were placed in eye fixative solution (Sevier Bio, Wuhan) for 2 hours. The fixed eyeball was placed in a petri dish, cut along the corneoscleral limbus under a dissecting microscope, and the retina was bluntly separated from the eyeball and rinsed with PBS. The isolated retina was radially cut under a dissecting microscope, and it was evenly divided into four quadrants and spread on a clean glass slide, paying attention to the vitreous surface facing upward, and then dropwise added 90% glycerol ( Bioshop, Canada) coverslips. The distribution of retinal blood vessels and the leakage of Evans blue in each group of mice were observed under a fluorescence microscope (Leica, Germany) with a filter with a wavelength of 546 nm.

3.2 Experimental results: As shown in Figure 5, the retinal cell barrier in the control group was severely damaged and the blood vessels were exuded more; the retinal blood vessels in the AOA treatment group were slightly exuded.

Example 4: After AOA treatment, the proportion of inflammatory cells Th17 and Th1 cells decreased, and the proportion of anti-inflammatory cells Treg increased

4.1 Experimental method: Take six mice that were successfully modeled in Example 2, take the spleen and lymph nodes and put them into the filter screen in the petri dish, add 1 ml of lymphocyte dilution solution (Haoyang, Tianjin), and gently grind with a syringe Afterwards, 1 ml of diluent was added and the cell suspension was aspirated several times with a pipette (Eppendorf, Germany), and the spleen residue remaining on the filter was ground again until it was invisible to the naked eye. Transfer the cell suspension into a glass tube with 3ml of lymphocyte separation solution, then add 1ml of diluent to the dish to wash the adherent cells in the dish and transfer it into a glass tube again, seal the glass tube and put it in a centrifuge for centrifugation. , 400g, 25min, up 4 down 0. After centrifugation, suck the milky white lymphocyte layer in the glass tube into the centrifuge tube, add 10 ml of PBS, and then put it into a centrifuge (Eppendorf, Germany) at 250 g, 4 and 0, and centrifuge for 10 minutes to wash the cells. Phorbol ester (Sigma, USA), inoxomycin (Sigma, USA), and Golgi blocker (BD Bioscience, USA) were added to the isolated cells to stimulate the cells for 5 hours, and then the cells were collected and divided into equal parts. Blank tube, CD4 single-stained tube, IL-17 single-stained tube, IFN-r single-stained tube, IL-10 single-stained tube, CD4/IL-17 double-labeled tube, CD4/IFN-r double-labeled tube, CD4/Foxp3 double-labeled tube Standard tube. After the staining was completed, the ratio of inflammatory cells and anti-inflammatory cells was detected by a flow machine (BD Bioscience, USA), and the ratios of inflammatory cells and anti-inflammatory cells were detected by Flowjo (Ashland, USA).

Analyze the results.

4.2 Experimental results: As shown in Figure 6, the proportion of inflammatory cells Th1 and Th17 in the mice after AOA treatment decreased, and the proportion of anti-inflammatory cells Treg increased.

Example 5: The secretion of inflammatory factors in vivo decreased after AOA treatment, and the secretion of anti-inflammatory factors increased

5.1 Experimental method: The lymphocytes isolated in Example 4 were seeded in a 24-well plate (Bai Le, Chengdu), with 5× ^{10 6} cells per well, and 10ug/ml of IRBP651-670 (Sangong, Shanghai) was added to the plate. Perform secondary stimulation. After 48 hours, the supernatant was collected and the levels of IL-17, IL-10 and IFN-r in the supernatant were detected with corresponding ELISA kits (Xinbosheng, Guangdong).

5.2 Experimental results: As shown in Figure 7, after AOA treatment, the secretion of inflammatory factors IL-17 and IFN-r decreased, and the secretion of anti-inflammatory factor IL-10 increased.

Example 6: After AOA treatment, the mRNA expression levels of inflammatory factors were decreased, and the mRNA expression levels of anti-inflammatory factors were increased.

6.1 Experimental method: 6 mice that were successfully modeled in Example 2 were taken from each of the mice after excessive anesthesia with sodium pentobarbital (Merck, Germany). The retinas were bluntly dissected and transferred into centrifuge tubes (Aozer, Chongqing), and Trizol (Invitrogen, Carlsbad, USA) was added to each sample. After the RNA was extracted, its concentration was measured with a quantitative spectrophotometer, and it was reverse transcribed into cDNA with a reverse transcription kit (Takara, Dalian), which was mixed with the corresponding primers for quantitative detection.

6.2 Experimental results: After AOA treatment, the mRNA expression levels of inflammatory factors IL-17 and IFN-r decreased, and the mRNA expression levels of anti-inflammatory factors IL-10 increased. Figure 8 shows that the levels of IL-17 mRNA and IL-10 mRNA in inflammatory cells in the AOA group were lower than those in the PBS group, while the levels of IL-10 mRNA in anti-inflammatory cells were higher than those in the PBS group.

Conclusion: O- (carboxymethyl)hydroxylamine hemihydrochloride can inhibit the occurrence and development of uveitis model and the severity of inflammation, and has a protective effect on uveitis mice.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention; if the present invention is modified or equivalently replaced without departing from the spirit and scope of the present invention, it shall be covered by the rights of the present invention. within the scope of protection required.

Claims (3)

1. the application of a kind of O- (carboxymethyl) hydroxylamine hemihydrochloride in the preparation of medicine for the treatment of uveitis. 2. The application according to claim 1, wherein the medicine is administered by injection. 3. application according to claim 1 is characterized in that: the dosage of described medicine is 31.25ng/kg every day.