Application of L-threonic acid hemicalcium salt in preparation of medicine for preventing and / or treating diseases related to retinal neovascularization
By using the HCV injection of L-threonate hemicalcium salt, it inhibits the formation of retinal neovascularization and improves retinal ischemia, solving the problem of low treatment efficiency of existing anti-VEGF drugs and providing a new retinal neovascular disease Treatment options.
Patent Information
- Application Number
- CN202411923493.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-02-18
AI Technical Summary
The existing treatment methods for retinal neovascular diseases mainly rely on anti-VEGF drugs, but their effective efficiency is only 40% to 60%, and repeated injections are required, which cannot completely solve the problem of metabolic disorders of the disease.
The drug is administered by L-threonate hemicalcium salt and by vitreal injection to inhibit the formation of retinal neovascularization and improve retinal ischemia.
In a mouse model of oxygen-induced retinopathy, the HCL-threonate hemical salt significantly inhibited the formation of retinal neovascularization, reduced the area of avascular area, and showed better performance than traditional anti-VEGF drugs in relieving retinal vascular ischemia Effect.
Smart Images
Figure CN119454679A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medicine, and specifically relates to a new use of L-threonate hemicalcium salt, and more specifically relates to the use of L-threonate hemicalcium salt in the preparation of a drug for preventing and / or treating retinal neovascularization-related diseases. Background Art
[0002] Retinal choroidal neovascular disease is a comprehensive phenotype of a group of diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), wet age-related macular degeneration (nAMD), and choroidal neovascularization secondary to pathological myopia. The common pathological feature is that the existing vascular structures of the retina and choroid generate new blood vessels in a sprouting manner. Retinal neovascularization-related diseases involve a large number of people and can be seen in all age groups such as infants and middle-aged and elderly people. It is currently the most important fundus vascular disease causing moderate to severe visual impairment worldwide; the number of blind people caused by this disease each year accounts for 45%-50% of all blind eye patients. At present, the treatment of retinal choroidal neovascular disease is mainly based on anti-vascular endothelial growth factor (VEGF), but this treatment method requires repeated injections and the effective rate is only 40% to 60%. Therefore, it is necessary to find new intervention targets to develop alternative therapeutic drugs. The retina is a highly metabolically active tissue, and the occurrence and development of retinal neovascularization diseases are closely related to metabolic disorders. L-threonic acid is a degradation product of ascorbic acid after oxidation, hydrolysis, and decarboxylation. It often exists in the form of calcium and magnesium in vitro. Studies have shown that its calcium preparation exhibits a certain degree of pharmacological activity on the proliferation and mineralization of osteoblasts, so it is often used in the treatment and prevention of diseases such as osteoporosis; its magnesium preparation has a certain function of regulating neuroimmune inflammation. However, little is known about the role of L-threonic acid in eye diseases. There is only one article in the search on PUBMED using L-threonic acid and eye as keywords, and the article only mentions L-threonic acid metabolic disorders in patients with autosomal recessive estradiol disease. When searching with L-threonic acid as the keyword in the Patent Information Center and Freepatentsonline (FPO), a non-profit website for foreign patents, no relevant patents for the application of L-threonic acid in eye diseases were found. Summary of the invention
[0003] The purpose of the present invention is to provide a new medical use of L-threonic acid.
[0004] The new medical use of L-threonic acid provided by the present invention is the use of L-threonic acid or its pharmaceutically acceptable salt, ester, hydrate or prodrug in the preparation of drugs for preventing and / or treating retinal neovascularization related diseases.
[0005] The retinal neovascularization-related diseases include proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), wet age-related macular degeneration (nAMD), choroidal neovascularization secondary to pathological myopia, etc.
[0006] In the application, L-threonic acid or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is used to inhibit retinal neovascularization and improve retinal ischemia.
[0007] The pharmaceutically acceptable salt of L-threonic acid may be a calcium preparation of L-threonic acid, specifically L-threonic acid hemi-calcium salt, and its structural formula is as follows:
[0008]
[0009] In the application, L-threonate hemi-calcium salt is administered by intravitreal injection.
[0010] In order to make up for the shortcomings of existing drug treatments for retinal neovascularization diseases, the present invention integrates the metabolomics data of retinal neovascularization diseases and discovers the role of calcium preparations of the small molecule metabolite L-threonic acid in inhibiting retinal neovascularization and improving retinal ischemia.
[0011] The animal experiment results of the present invention show that L-threonic acid hemi-calcium salt can effectively inhibit the formation of retinal neovascularization in the OIR model and reduce the area of the avascular region. The present invention has discovered a new use of L-threonic acid hemi-calcium salt, providing a new option for the treatment of retinal neovascularization diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 To integrate the metabolomics results of serum and vitreous humor in patients with proliferative diabetic retinopathy, a small molecule metabolite L-threonic acid was discovered. A: Intersection analysis of integrated metabolomics. B: Receiver-effect curve of small molecule metabolites in intersection analysis.
[0013] Figure 2The effect of L-threonate hemicalcium on retinal neovascularization and retinal hypoxia in the oxygen-induced retinopathy mouse model in Example 1 of the present invention. A: Specific operation flow chart. B: The effect of L-threonate hemicalcium salt on retinal ischemia, **** indicates P < 0.001. C: The effect of L-threonate hemicalcium salt on retinal neovascularization, **** indicates P < 0.001. D: Retinal flat mount vascular staining of each group after the action of L-threonate hemicalcium salt. The part circled by the red line shows the avascular area, and the red arrow indicates the retinal neovascularization.
[0014] Figure 3 This is a comparative analysis of the therapeutic effects of L-threonate hemi-calcium and anti-VEGF drugs in the oxygen-induced retinopathy mouse model in Example 2 of the present invention. A: Retinal flat mount vascular staining of the negative control group, L-threonate hemi-calcium salt group, and anti-VEGF treatment group. The part circled by the red line shows the avascular area, and the red arrow indicates the retinal neovascularization. B: The area ratio of the retinal avascular area of each group, **** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05. C: The area ratio of retinal neovascularization of each group, **** indicates P < 0.001, and * indicates P < 0.05. DETAILED DESCRIPTION
[0015] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0016] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0017] The present invention firstly integrates the metabolomics data of serum and vitreous humor of patients with proliferative diabetic retinopathy and conducts parallel intersection analysis. Among them, serum metabolomics collected plasma from 100 patients with diabetic retinopathy and 100 patients in the control group (diabetic non-DR) for non-targeted metabolomics determination, and reduced the data dimension through statistical methods such as least absolute shrinkage and selection operator (LASSO), and screened out 8 characteristic small molecule metabolites; while vitreous humor metabolomics with 25 PDR patients (10 patients with epiretinal membrane matched by gender and age as controls) as sample sources obtained 160 differential metabolites; finally, through intersection analysis, two substances with common changes were found: L-arginine and L-threonic acid ( Figure 1A); The time-receiver operating curves of L-arginine and L-threonic acid showed that both of them could negatively predict the progression of PDR, with a diagnostic efficacy of 0.92 ( Figure 1 (B) The role of L-arginine in ocular diseases has been widely reported, so the present invention focuses on providing the role of L-threonate in retinal neovascularization diseases.
[0018] The drug used in the present invention is a calcium preparation of L-threonic acid: L-threonic acid hemi-calcium salt (Sigma, item number: 380644). It is a white, tasteless particle insoluble in alcohol, ether and chloroform, with a molecular formula of C8H 14 O 10 Ca, the chemical structure is as follows:
[0019]
[0020] The preparation method of L-threonate hemi-calcium is relatively mature and has been reported by domestic and foreign patents. Previous studies have shown that L-threonate calcium is soluble in water, so it is made into tablets and administered orally for treatment. In our invention, it is found that L-threonate hemi-calcium salt is not easily soluble in water and is easy to precipitate.
[0021] We changed the dissolution method of L-threonic acid hemi-calcium salt. The specific operation process is: first dissolve 300mg L-threonic acid hemi-calcium salt in 1ml DMSO to make a 300mg / ml storage solution, then take 100ul of the storage solution and dissolve it in 900ul of serum-free DMEM culture medium, and finally make a working solution with a final concentration of 30mg / ml. The remaining concentrations of the working solution can be reduced accordingly. The drug preparation method of the control group is to take 100ul DMSO and dissolve it in 900ul of serum-free DMEM culture medium.
[0022] The administration method of L-threonate hemi-calcium salt in the present invention is intravitreal injection, and the drug concentration is 30 mg / ml and 3 mg / ml.
[0023] In order to demonstrate the effect of L-threonate hemi-calcium salt on retinal neovascularization diseases, the present invention performs treatment in an oxygen-induced retinopathy mouse model.
[0024] Example 1. Effect of L-threonate hemi-calcium salt in oxygen-induced retinopathy mouse model
[0025] Objective: To explore whether L-threonate hemi-calcium salt can inhibit the formation of retinal neovascularization in the oxygen-induced retinopathy mouse model.
[0026] Experimental reagents and animals: L-threonate hemi-calcium salt (Sigma, catalog number: 380644), DMSO (MERCK, catalog number: D2650), DMEM culture medium (Gibco, catalog number: C11885500BT), C57BL / 6J pups, oxygen box.
[0027] Experimental methods
[0028] 1. Drug preparation: Dissolve 300 mg of L-threonic acid hemi-calcium salt in 1 ml of DMSO to make a 300 mg / ml storage solution, then take 100 ul of the storage solution and dissolve it in 900 ul of serum-free DMEM medium to finally prepare a working solution with a final concentration of 30 mg / ml; take 10 ul of the storage solution and dissolve it in 990 ul of serum-free DMEM medium to finally prepare a working solution with a final concentration of 3 mg / ml. The drug preparation method for the control group is to take 100 ul of DMSO and dissolve it in 900 ul of serum-free DMEM medium.
[0029] 2. Construction of oxygen-induced retinopathy mouse (OIR) model: For detailed construction methods, see Figure 2 In the operation flow chart of A, 7-day-old C57BL / 6J pups and their mothers were placed in an oxygen chamber with an oxygen content of 75%. After being raised in this environment for 5 days, the pups were placed in a normal environment with an oxygen content of 21% and continued to be raised. Under the 75% high oxygen state, avascular areas gradually formed, and after returning to the normal environment, retinal neovascularization gradually formed.
[0030] 3. Experimental treatment and grouping: Drug injection will be performed on the day when the mouse pups return to 21% normal oxygen content. The drug will be administered by intravitreal injection. Two drug concentrations are used, 30mg / ml and 3mg / ml. The specific groups are as follows: ① Normal control group, n=5, ② OIR+control drug group, n=5, ③ OIR+30mg / ml L-threonate hemi-calcium salt, n=5, ④ OIR+3mg / ml L-threonate hemi-calcium salt, n=5. After the fifth day of drug administration, the eyeballs of the mice in each group were fixed with 4% paraformaldehyde and the retinas were taken.
[0031] 4. Immunofluorescence staining of retinal flat mounts: The ex vivo eyeballs were fixed in 4% paraformaldehyde for 30 minutes and rinsed three times with 1×PBS for 10 minutes each time. The entire cornea and lens were removed along the corneoscleral limbus. The vitreous was peeled off with microscopic toothless forceps. The retina was separated along the white retina and the black sclera, and the retina was washed, punched, and blocked (punching and blocking solution: 0.1% TritonX-100, 5% donkey serum in 1×PBS) for 1 hour at room temperature. The retina was exposed to CD31 antibody dilution at 4°C overnight, washed, incubated with secondary antibody at room temperature for 1 hour, washed, cut into four retinal petals, spread on a slide, covered with 1×PBS, and observed under a fluorescence microscope.
[0032] 5. Statistics of the area ratio of retinal neovascularization: ImageJ software was used to calculate the total area of the retinal region, the area of retinal neovascularization and the area of retinal avascular area. The specific operation method can be referred to the previously published literature (PMID: 35343957). Finally, the area ratio was calculated by dividing the total area of the retina by the area of neovascularization or the area of avascular area.
[0033] Experimental results: From Figure 2 The experimental results show that 30mg / ml L-threonate hemi-calcium salt significantly inhibits the formation of retinal neovascularization. Figure 2 D in the middle shows that the retina of the normal control group of young mice developed normally, with no obvious avascular area and neovascular membrane formation. A large number of retinal neovascularization appeared in the OIR+control drug group (red arrows indicate retinal neovascularization). Five days after intravitreal injection of 30 mg / ml L-threonate hemi-calcium salt, the area of retinal neovascularization decreased from 12.34% (standard deviation 5.23%) to 1.80% (standard deviation 1.04%), and the effect of inhibiting retinal neovascularization was significant ( Figure 2 At the same time, we also found that L-threonate hemi-calcium salt can improve retinal hypoxia ( Figure 2 In the middle C, P < 0.001), the percentage of retinal avascular area decreased from 87.74% (standard deviation 4.72%) to 80.21% (standard deviation 5.12%).
[0034] The animal experiment results of the present invention show that L-threonic acid hemi-calcium salt can effectively inhibit the formation of retinal neovascularization in the OIR model and reduce the area of the avascular region. The present invention has discovered a new use of L-threonic acid hemi-calcium salt, providing a new option for the treatment of retinal neovascularization diseases.
[0035] Example 2: Comparative analysis of the therapeutic effects of L-threonate hemi-calcium salt and traditional anti-VEGF drugs in the OIR model
[0036] Objective: To compare the therapeutic effects of L-threonate hemi-calcium salt and traditional anti-VEGF drugs in the OIR model
[0037] Experimental reagents and animals: L-threonate hemi-calcium salt (Sigma, catalog number: 380644), DMSO (MERCK, catalog number: D2650), DMEM culture medium (Gibco, catalog number: C11885500BT), anti-VEGF drug (Conbercept), C57BL / 6J pups, oxygen box.
[0038] Experimental methods
[0039] 1. Drug preparation: The preparation of L-threonate hemi-calcium salt is the same as above, and the anti-VEGF drug Conbercept is a 10 mg / ml fusion protein injection.
[0040] 2. Construction of oxygen-induced retinopathy mouse (OIR) model: same as Example 1.
[0041] 3. Experimental treatment and grouping: Drug injection will be performed on the day when the mouse pups return to 21% normal oxygen content. The drug will be administered by intravitreal injection. L-threonate hemi-calcium salt uses a drug concentration of 30mg / ml; Conbercept uses an original concentration of 10mg / ml. The injection volume is 1ul, and the specific groups are as follows: ①OIR+negative control group, n=5, ②OIR+30mg / ml L-threonate hemi-calcium salt, n=5, ③OIR+anti-VEGF drug group, n=5. After the fifth day of drug administration, the eyeballs of the mice in each group were fixed with 4% paraformaldehyde and the retinas were taken.
[0042] 4. Immunofluorescence staining of retinal flat mounts: Same as Example 1.
[0043] 5. Statistics on the percentage of retinal neovascularization area: same as case 1.
[0044] Experimental results: From Figure 3 The experimental results show that L-threonate hemi-calcium salt has more advantages than traditional anti-VEGF drugs in alleviating retinal vascular ischemia and inhibiting retinal neovascularization. The area of retinal neovascularization in the 30mg / ml L-threonate hemi-calcium salt group was 1.67% (standard deviation 0.94%), while the area of neovascularization in the anti-VEGF drug group was 3.08% (standard deviation 1.26%). The area of neovascularization in the L-threonate hemi-calcium salt group was significantly reduced; at the same time, compared with traditional anti-VEGF drugs, the area of avascular area in the L-threonate hemi-calcium salt group was also smaller.
[0045] Conclusion: The experiments in this part show that L-threonate hemi-calcium salt is superior to traditional anti-VEGF drugs in inhibiting the formation of retinal neovascularization.
[0046] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. Use of L-threonic acid or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof in the preparation of a drug for preventing and / or treating retinal neovascularization-related diseases.
2. The use according to claim 1, characterized in that: In the application, the retinal neovascularization-related diseases include proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, and choroidal neovascularization secondary to pathological myopia.
3. The use according to claim 1, characterized in that: In the application, L-threonic acid or a pharmaceutically acceptable salt, ester, hydrate or prodrug thereof is used to inhibit retinal neovascularization and improve retinal ischemia.
4. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt of L-threonate is a calcium agent of L-threonate.
5. The use according to claim 4, characterized in that: The pharmaceutically acceptable salt of L-threonic acid is L-threonic acid hemi-calcium salt, and its structural formula is shown below:
6. The use according to claim 5, characterized in that: In the application, L-threonate hemi-calcium salt is administered by intravitreal injection.