Application of mannose salt in preparation of medicine for treating congenital myasthenia syndrome or congenital glycosylation disorder

Abstract

The invention belongs to the technical field of medicine application, and discloses application of mannose salt in preparation of medicine for treating congenital myasthenia syndrome or congenital glycosylation disorder. The application of the mannose salt in preparing the medicine for treating the congenital myasthenia syndrome or the congenital glycosylation disorder disease, provided by the invention, particularly has a good treatment effect on the congenital myasthenia syndrome or the congenital glycosylation disorder disease caused by Alg2 gene defects.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and particularly relates to the application of mannose salt in preparing drugs for treating congenital myasthenic syndrome or congenital glycosylation disorder. Background Art

[0002] Protein glycosylation is responsible for the modification and folding of proteins in cells. About half of the proteins need to be modified by glycosylation to maintain their stability. Glycosylation is mainly divided into two forms: O-linked and N-linked. The core structure of N-sugar is composed of five oligosaccharide groups, namely, a "biantennary" pentasaccharide molecule Man3GlcNAc2 composed of three mannose groups (Man) and two N-acetylglucosamines (GlcNAc). Glycosylation defects can directly lead to the onset of rare diseases such as congenital myasthenic syndrome and congenital glycosylation disorders.

[0003] Congenital myasthenic syndromes (CMS) are a group of heterogeneous diseases caused by mutations in genes encoding presynaptic, intrasynaptic or postsynaptic proteins of the neuromuscular junction. The inheritance pattern of CMS is usually autosomal recessive, and the characteristic phenotype is weakness and fatigue caused by exercise. The disease often occurs in neonates or infants, with symptoms such as muscle weakness, muscle atrophy, and feeding difficulties, which can lead to respiratory arrest or even failure.

[0004] Congenital disorders of glycosylation (CDG) are genetic defects in the synthesis and attachment of glycoproteins and glycolipid glycans. The typical biochemical characteristics of CDG are mutations in genes required for the biosynthesis of N-linked oligosaccharides, resulting in protein glycosylation defects, which are usually associated with severe psychomotor and intellectual disabilities. The above gene mutations mostly occur in genes encoding glycosyltransferases, remodeling glycosidases, and sugar nucleotide transporters, directly affecting the protein glycosylation process. Since its first discovery in 1980, 105 types of CDG have been found, which is a rapidly growing type of genetic metabolic disease in recent years, and the specific types of diseases continue to rise.

[0005] In the prior art, there are many factors causing congenital myasthenic syndrome and congenital glycosylation disorder, and the congenital myasthenic syndrome and congenital glycosylation disorder caused by different factors require different therapeutic drugs.

[0006] Therefore, there is an urgent need to provide drugs for treating congenital myasthenic syndrome and congenital glycosylation disorder caused by different factors. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of mannose salt in the preparation of a drug for treating congenital myasthenic syndrome or congenital glycosylation disorder. The mannose salt has a good therapeutic effect on congenital myasthenic syndrome or congenital glycosylation disorder caused by Alg2 gene defect.

[0008] Alg2 gene defects lead to weight loss, weakened muscle strength, decreased power, decreased mobility, obstruction of the construction of the intracellular N-glycosylated pentasaccharide core, dysfunction of hematopoietic stem and progenitor cells and decreased differentiation ability, and loss of N-sugar in hematopoietic stem and progenitor cells.

[0009] To this end, the present invention proposes a drug for alleviating and treating myasthenic syndrome and congenital glycosylation deficiency caused by Alg2 deficiency (including loss-of-function mutation / reduced expression). The drug has a significant therapeutic effect, especially for sporadic and familial Alg2 deficiency diseases.

[0010] The first aspect of the present invention provides the use of mannose salt in the preparation of a drug for treating congenital myasthenic syndrome or congenital glycosylation disorder.

[0011] Specifically, the use of mannose salt in the preparation of drugs for treating congenital myasthenic syndrome or congenital glycosylation disorder.

[0012] Preferably, the mannose salt includes at least one of mannose sodium salt, mannose potassium salt, mannose hydrochloride, mannose sulfate, mannose nitrate, mannose magnesium salt, mannose calcium salt, and mannose quaternary ammonium salt; mannose sodium salt, mannose potassium salt, and mannose hydrochloride are further preferred; mannose sodium salt is more preferred.

[0013] Preferably, the mannose salt is at least one selected from GDP-D-mannose disodium salt, guanosine 5'-(trihydrogen diphosphate) P'-ALPHA-D-mannosyl ester disodium salt, D-mannosamine hydrochloride, N-acetyl-D-mannosamine, and mannitol, and more preferably, the mannose salt is GDP-D-mannose disodium salt.

[0014] The CAS number of the GDP-D-mannose disodium salt is 103301-73-1, and the molecular formula is C 16 H 23 N 5 Na 2 O 16 P 2 .

[0015] The CAS number of the guanosine 5'-(trihydrogen diphosphate) P'-ALPHA-D-mannosyl ester disodium salt is 148296-46-2, and the molecular formula is C 16H 23 N 5 Na 2 O 16 P 2 .

[0016] The CAS number of the D-mannose amine hydrochloride is 5505-63-5, and the molecular formula is C 6 H 14 ClNO 5 , or C 6 H 13 NO 5 ·HCl.

[0017] The CAS number of the N-acetyl-D-mannosamine is 7772-94-3, and the molecular formula is C 8 H 15 NO 6 .

[0018] The CAS number of the D-mannitol is 69-65-8, and the molecular formula is C 6 H 14 O 6 .

[0019] Preferably, the mannose salt is used in the preparation of a drug for treating congenital myasthenic syndrome or congenital glycosylation disorder caused by Alg2 gene defect.

[0020] Preferably, the Alg2 gene defect includes at least one of a loss-of-function mutation and a decreased expression level.

[0021] Preferably, the medicine further comprises excipients.

[0022] Preferably, the auxiliary materials include at least one of a carrier, an osmotic pressure regulator, a pH regulator, a diluent, a disintegrant, an excipient, a solubilizer, a stabilizer, and a preservative.

[0023] Preferably, the carrier is selected from at least one of liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, and mesoporous silica nanoparticles.

[0024] Preferably, during the application, the concentration of the mannose salt in the human body is 0.2-80 mg / kg, more preferably 3-5 mg / kg.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention proposes the use of mannose salt in the preparation of a drug for treating congenital myasthenic syndrome or congenital glycosylation disorder. The mannose salt has a good therapeutic effect on congenital myasthenic syndrome or congenital glycosylation disorder caused by Alg2 gene defect.

[0027] The GDP-D-mannose disodium salt of the present invention can effectively improve the decreased differentiation ability of hematopoietic stem cells, decreased motor ability, decreased grip strength and muscle endurance of Alg2-deficient mice, and improve defects such as the lack of N-glycoprotein oligosaccharides in mouse serum. These results show that GDP-D-mannose disodium salt can effectively improve the hematopoietic and immune system functions, muscle motor ability, and the synthesis and modification of glycoprotein N-glycoconjugate chains in cells and blood of congenital myasthenic syndrome caused by Alg2 deficiency (including loss-of-function mutation / decreased expression).

[0028] There are many factors that cause congenital myasthenic syndrome or congenital glycosylation disorder, and the disease mechanisms caused by different factors are different, resulting in different effective drugs required. The present invention proposes for the first time to use mannose salt to treat congenital myasthenic syndrome or congenital glycosylation disorder caused by Alg2 gene defect, and has a good therapeutic effect. At present, there are about 35 mutant genes detected in congenital myasthenic syndrome. The symptoms of each disease are similar, but the causes of complications are different, resulting in different effective therapeutic drugs required for congenital myasthenic syndrome caused by different mutant genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of GDP-D-mannose disodium salt alleviating the weight loss defect of Alg2 knockout mice in Example 1;

[0030] Figure 2 The results of GDP-D-mannose disodium salt in Example 2 improving the strength, grip duration and motor ability of Alg2-deficient mice;

[0031] Figure 3 The results of Example 3 show that disodium GDP-D-mannose effectively improves the differentiation ability of hematopoietic stem and progenitor cells in Alg2 knockout mice;

[0032] Figure 4 The results of GDP-D-mannose disodium salt increasing the N-glycoprotein complex glycoforms in the serum of knockout mice in Example 4;

[0033] Figure 5 The results of Example 5 in which D-mannitol improves the defect of decreased differentiation of hematopoietic stem cells and hematopoietic progenitor cells caused by decreased Alg2;

[0034] Figure 6The results of the comparative experiment on the behavior of GDP-D-mannose disodium salt and compound M3N2-glycan in Example 7;

[0035] Figure 7 The results of the comparative experiment on the differentiation ability of GDP-D-mannose disodium salt and compound M3N2-glycan on hematopoietic stem and progenitor cells in Example 8 are shown. DETAILED DESCRIPTION

[0036] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0037] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0038] definition

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs.

[0040] The drug is dissolved in water or an organic solvent according to the designed concentration.

[0041] To verify the in vitro therapeutic effect of the drug, mouse hematopoietic stem cells were used. The experimental group used shRNA targeting the Alg2 gene (abbreviated as shAlg2) to knock down the Alg2 gene in hematopoietic stem cells, and the control group used hematopoietic stem cells treated with non-targeted shRNA (shLuciferase, abbreviated as shLuci). The treated hematopoietic stem cells were cultured at 37°C and 5% CO 2 The cells were cultured in a constant temperature and humidity cell culture incubator for 5 days; the drugs were added to the hematopoietic stem cell expansion medium at the designed concentration, and the hematopoietic stem cell culture data were statistically analyzed and plotted using Prism Graphpad software.

[0042] In order to verify the therapeutic effect of the drug in vivo, an Alg2 inducible knockout mouse model was bred. The Alg2 flox mice were bred by CRISPR-Cas9 knockout technology, and then the Alg2 inducible knockout mice were bred by crossing this mouse with Cre-ERT mice, namely Alg2 fl / fl-CAGG-CreERTM mice. This mouse strain was injected with tamoxifen (Tamoxifen, Catalog No.: T5648, Sigma-Aldrich) intraperitoneally to induce activation of Cre-loxP recombinase in mouse cells, knock out the second exon of the Alg2 gene in cells, and induce Alg2 gene knockout; the knockout group mice were injected with 40 mg / kg tamoxifen intraperitoneally to induce Alg2 knockout; the control group mice were injected with corn oil intraperitoneally; after Alg2 knockout was induced, the treatment group mice were injected with the drug at the designed concentration. The data of the control group, knockout group and treatment group were compared in the supplementary experiment, and the PrismGraphpad software was used for analysis and plotting after statistics.

[0043] Example 1: In vivo supplementation of GDP-mannose disodium salt alleviates weight loss in Alg2 knockout mice

[0044] In the in vivo treatment experiment, Alg2fl / fl-CAGG-CreERTM inducible knockout mice were used as the subjects. The Alg2 gene was knocked out by intraperitoneal injection of tamoxifen, and the control group was injected with corn oil. Three groups of mice were used in the experiment, one group was the control group; one group was the knockout group; and the other group was the mannose supplementation group (or GDP mannose rescue group), that is, GDP-D-mannose disodium salt was injected in vivo after induction of knockout. GDP-D-mannose disodium salt PBS solution was prepared before injection. The concentration of GDP-D-mannose disodium salt in the PBS solution was 1μg / μL, and the injection volume was 100μL. In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose supplementation group, and 5 mice (n=5) in the control group.

[0045] Figure 1 This is the result of GDP-D-mannose disodium salt alleviating the body weight loss defect of Alg2 knockout mice in Example 1. Figure 1 In the figure, the gray line is the control group; the red line is the knockout group; the green line is the mannose replenishment group (or GDP mannose rescue group). Figure 1 The abscissa represents time (day). In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose supplementation group, and 5 mice (n=5) in the control group.

[0046] In the experiment, the mice in the mannose supplementation group were injected with GDP-D-mannose disodium salt on the 1st, 10th and 16th day, respectively, while the mice in the control group and the knockout group were injected with PBS phosphate buffer. The weight of the mice in the knockout group and the mannose supplementation group began to decrease from the 10th day, and the mice in the mannose supplementation group received the second GDP-D-mannose disodium salt injection. By the 16th day, the weight loss of the mice in the knockout group and the mannose supplementation group was close to 15%. At this time, the mannose supplementation group was injected with 100μg GDP-D-mannose disodium salt for the third time. 24 hours after the injection, the weight loss of the mice in the knockout group and the mannose supplementation group exceeded 15%, and the experiment was terminated.

[0047] The experiment found that although the mice in the mannose supplementation group lost weight after the knockout was induced, their weight loss trend (y=-0.3771x+30.1395) was lower than that of the knockout group (y=-0.5528x+34.5998). This result shows that GDP-D-mannose disodium salt improves the weight loss defect of Alg2 knockout mice.

[0048] Example 2: In vivo supplementation of GDP-D-mannose disodium salt improves muscle strength and motor ability in Alg2 knockout mice

[0049] In the in vivo GDP-D-mannose disodium salt intervention experiment (ie, Example 1), the experimental mice were subjected to behavioral tests such as rotarod, grasping and inverted hanging.

[0050] Figure 2 These are the results of Example 2 in which GDP-D-mannose disodium salt improves the strength, grip duration and motor ability of Alg2-deficient mice. Figure 2 In the figure, the gray column is the control group (control); the red column is the knockout group (KO); and the green column is the mannose supplementation group (Mannose). In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose supplementation group, and 5 mice (n=5) in the control group. Figure 2 “Reverse hanging” means reverse hanging, “time” means time, “maximal grip” means maximal grip, “Grip strength” means grip strength, “Grip duration” means grip duration, and “Rotarod” means rotating rod.

[0051] The duration of suspension in the inversion suspension test in the mannose supplementation group was longer than that in the knockout group ( Figure 2 In the mouse grasping experiment, the maximum grip strength of the mice in the mannose supplementation group was higher than that in the knockout group and close to that in the control group ( Figure 2 B), and the grasping time was also longer than that of the knockout mice ( Figure 2In addition, in the rotarod test, the motor ability of the mannose-supplemented mice also exceeded that of the knockout mice ( Figure 2 These results indicate that GDP-D-mannose disodium salt can improve muscle atrophy and weakness and enhance the exercise capacity of Alg2-deficient mice.

[0052] Example 3: In vivo supplementation of GDP-D-mannose disodium salt improves the differentiation ability of hematopoietic stem and progenitor cells in Alg2 knockout mice

[0053] Inhibition of the Alg2 gene will affect the differentiation ability of hematopoietic stem cells. Therefore, in the mannose intervention experiment of Example 1, blood and bone marrow cells were extracted from the control group, knockout group and mannose supplementation group of mice, and flow FACS analysis showed that GDP-D-mannose disodium salt can improve the functional defects of hematopoietic stem cells and immune cells in the hematopoietic and immune systems of Alg2 knockout mice.

[0054] The flow cytometry sample preparation and analysis methods are as follows:

[0055] Blood flow cytometry sample preparation: Blood samples were extracted from mice in the control group, knockout group, and mannose complement group. The blood sample volume was 50-100 μl, and EDTA (ethylenediaminetetraacetic acid) anticoagulant (blood: EDTA = 9: 1 v / v) was added. Fluorescence-coupled CD45-PE, CD4-APC, CD8-APC, B220-APC, B220-APC-Cy7, Gr1-APC-Cy7, CD11b-APC-Cy7 antibodies were used. Each sample was added with 1 μL of the above fluorescence-coupled antibodies. The cells were incubated on ice for 30 minutes, rinsed with PBS, and red blood cell lysis buffer was added and placed on ice for 5 minutes. Centrifuged at 4°C, 1200 rpm, the upper lysate was removed, and the bottom cells were retained for flow cytometry analysis.

[0056] Preparation of bone marrow immune cell flow cytometry samples: extract bone marrow cells from mice in the control group, knockout group, and mannose complementation group, use fluorescently coupled CD45-PE, CD4-APC, CD8-APC, B220-APC, B220-APC-Cy7, Gr1-APC-Cy7, CD11b-APC-Cy7 antibodies, and add 1 μL of the above fluorescently coupled antibodies to each sample. Incubate the cells on ice for 30 minutes, rinse the cells with PBS, centrifuge at 4°C, 1200rpm to remove the upper cell staining solution, and retain the bottom cells for flow cytometry analysis.

[0057] Preparation of hematopoietic stem / progenitor cell flow cytometry samples: extract bone marrow cells from mice in the control group, knockout group, and mannose complementation group, perform hematopoietic lineage antibody staining, rinse cells with PBS, use fluorescence-coupled Streptavidin-APC-Cy7 (abbreviation: SA), cKit-APC, ScaI-PE-Cy7, Flt3-PE, CD16 / 32-FITC, CD150-BV605, CD34-AF700, IL7-PerCP-Cy5.5 antibodies, and add 1 μL of the above fluorescence-coupled antibodies to each sample. Incubate cells on ice for 120 minutes. Centrifuge at 4°C, 1200rpm to remove the upper cell staining solution, and retain the bottom cells for flow cytometry analysis.

[0058] Flow cytometric analysis of LSK hematopoietic stem / progenitor cells and LK hematopoietic stem / progenitor cells.

[0059] Among the above-stained bone marrow cells, Lin / Streptavidin, cKit, and ScaI antibodies were used to label hematopoietic stem / progenitor cells in the bone marrow cells, among which LSK hematopoietic stem / progenitor cells expressed SA - cKit + ScaI + Bone marrow cells, LK hematopoietic progenitor cells express SA - cKit + ScaI - of bone marrow cells.

[0060] Flow cytometric analysis of hematopoietic stem and multipotent progenitor cells.

[0061] In the above-stained LSK cells, CD34 and CD150 antibodies were used to label hematopoietic stem cells (HSC) and multipotent progenitor cells (MPP) in LSK cells. + CD34 - LSK cells, multipotent progenitor cells expressing CD34 + of LSK cells.

[0062] Flow cytometric analysis of myeloid differentiated progenitor cell samples.

[0063] In the above-stained LK cells, CD16 / 32 and CD34 were used to label myeloid common progenitor cells (CMP), erythroid-megakaryocyte progenitor cells (MEP) and granulocyte-macrophage progenitor cells (GMP). CMP is the expression of CD34 + CD16 / 32 - LK cells; MEP cells express CD34 - CD16 / 32 - LK cells; GMP expresses CD34 + CD16 / 32+ of LK cells.

[0064] Flow cytometric analysis of lymphoid hematopoietic progenitor cells.

[0065] Streptavidin, cKit, ScaI, Flt3, and IL7 antibodies were used to label bone marrow cells and lymphoid hematopoietic progenitor cells (CLP). CLP cells express Flt3 + IL7 + lin - cKit middle ScaI middle of bone marrow cells.

[0066] Figure 3 This is the result of Example 3 that GDP-D-mannose disodium salt effectively improves the differentiation ability of hematopoietic stem and progenitor cells in Alg2 knockout mice. Figure 3 In the figure, the gray column is the control group; the red column is the knockout group; and the green column is the mannose supplementation group. In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose supplementation group, and 5 mice (n=5) in the control group.

[0067] from Figure 3 The results showed that the number of various hematopoietic progenitor cells in the knockout group was lower than that in the control group. However, in the mannose replenishment group, the number of MPP, LSK and CLP cells was significantly higher than that in the knockout group, with MPP increasing by 40%, LSK increasing by 27% and CLP increasing by 55% ( Figure 3 In addition, some disordered lymphocytes in the knockout group were also alleviated in the mannose replenishment group. For example, the decrease of B cells in the blood and the abnormal expansion of T cells in the bone marrow were significantly alleviated ( Figure 3 D and E). This result indicates that GDP-mannose disodium salt can improve the defect of decreased differentiation of hematopoietic stem and progenitor cells caused by Alg2 deficiency, especially the decreased differentiation of lymphoid hematopoietic stem and progenitor cells, thereby improving the disorder of lymphoid immune cells and improving the immune system.

[0068] Example 4: In vivo supplementation of GDP-D-mannose disodium salt alleviates the lack of complex sugar chains in the serum of Alg2 knockout mice

[0069] The blood of the mice in the mannose intervention experiment of Example 1 was extracted, and the serum was separated and prepared. The glycoprotein spectrum analysis showed that GDP-D-mannose disodium salt increased the N-sugar glycoform in the serum of Alg2 knockout mice.

[0070] Figure 4 This is the result of increasing the N-glycoprotein complex glycoforms in the serum of knockout mice by disodium GDP-D-mannose in Example 4.

[0071] Compared with the N-glycan glycoforms in the serum of the control group, most of the N-glycan complex chains in the serum of the knockout group disappeared. However, the types of N-glycans in the serum of the mice in the mannose supplementation group increased significantly by supplementing GDP-D-mannose disodium salt, and the abundance of typical N-glycans was close to that of the control mice. These results indicate that supplementing GDP-D-mannose disodium salt significantly alleviated the abnormal lack of complex sugar chains in the serum of Alg2 knockout mice, effectively increased the reserve of oligosaccharide precursors in the serum, provided more substrates for the synthesis of various complex sugar chains in the later stage of N-glycosylation, and maintained the continuation of protein glycosylation modification.

[0072] Example 5: D-mannitol promotes differentiation of hematopoietic stem cells in vitro

[0073] In order to identify the effect of D-mannitol on hematopoietic stem cells after knocking down the Alg2 gene, mouse bone marrow cells were extracted in vitro and placed in flow cytometry staining solution (1xPBS with 2% fetal bovine serum FBS and 1% penicillin and streptomycin), and the suspension was added with fluorescent antibodies containing APC fluorescent conjugated mouse anti-cKit, incubated on ice for 30 minutes, rinsed the cells, and the cell suspension was centrifuged at 4°C, 1200rpm, and 5 minutes. After centrifugation, the supernatant was removed, the lower layer was retained, and 200μLl of cell staining solution containing anti-APC micromagnetic beads (Miltenyi, Germany) was added, and incubated on ice for 30 minutes. After the incubation, the cells were rinsed, centrifuged at 4°C, 1200rpm, and 5 minutes, and the cells were resuspended with 3mL of cell staining solution, and cKit-positive hematopoietic stem cells were isolated using LS magnetic bead separation columns and magnetic bead separation systems (Miltenyi, Germany). A mixture of blood lineage cell lineage flow cytometry antibodies, including Biotin-coupled CD3, CD8, CD11b, Gr-1, B220, and Ter119 antibodies, was added to cKit-positive hematopoietic stem and progenitor cells, mixed thoroughly, incubated on ice for 30 minutes, rinsed the cells, and centrifuged to retain the lower cell mass. The cells were resuspended in cell staining solution, and APC fluorescent-coupled anti-cKit, PE fluorescent-coupled anti-Sca1, and APC / cy7 fluorescent-coupled anti-streptavidin were added for staining. The cells were mixed thoroughly, placed on ice for 120 minutes, rinsed, and the fluorescent antibodies in the cell suspension were removed by centrifugation. LSK cells (i.e., cells expressing lineage negative-Sca1 positive-cKit positive cells) were separated by flow cytometry and FACS AriaIII flow cytometer. LSK cells were infected with lentivirus containing shAlg2 to knock down the expression of the Alg2 gene, and set as the experimental group.

[0074] The experimental control group was LSK cells infected with shLuci (i.e., shLuci group). At the same time, a group of LSK cells after Alg2 knockdown (i.e., shAlg2 group) was set up, and D-mannose was added to the cell culture medium as a rescue intervention drug, as a mannitol intervention group (i.e., shAlg2+5uM mannitol group). The GFP fluorescent indicator protein in the stable expression plasmid used in this experiment began to be activated on the third day after cell infection. Therefore, this time was set as the first analysis time point of flow analysis, i.e., day 0 (day0); thereafter, LSK cells continued to be cultured for 5 days, and this time was set as the second analysis time point (day 5, abbreviated as d5). On d0 and d5 days, the cells were subjected to flow cytometry analysis, and relevant experimental data on in vitro differentiation and regeneration of hematopoietic stem cells and early progenitor cells were collected.

[0075] Figure 5 This is the result of D-mannitol improving the defect of decreased differentiation of hematopoietic stem cells and hematopoietic progenitor cells caused by decreased Alg2 in Example 5.

[0076] The rescue experiment showed that at the beginning of the experiment (d0), the growth and differentiation of hematopoietic stem and progenitor cells in the three groups were similar. On d5 of the experiment, the number of hematopoietic stem and progenitor cells in the Alg2 knockdown group decreased compared with the experimental control group. It was also found that after the addition of D-mannitol, the hematopoietic stem and progenitor cells in the mannose intervention group showed an upward trend. In particular, it was pointed out that D-mannitol improved the decline in hematopoietic progenitor cells caused by Alg2 knockdown, and their number reached or exceeded the number of hematopoietic progenitor cells in the control group. This shows that the addition of D-mannitol during in vitro culture can improve the defect of decreased differentiation of hematopoietic stem and progenitor cells caused by Alg2 knockdown.

[0077] Example 6: Similar effects of D-mannitol and GDP-D-mannose disodium salt on hematopoietic stem cells in vitro

[0078] In order to identify the effects of D-mannitol and GDP-D-mannose disodium salt on hematopoietic stem cells after knocking down the Alg2 gene. A comparative experiment of the two compounds was carried out according to the method of Example 5. The extraction and induction knockdown process of hematopoietic stem cells in the experiment were consistent with Example 5. The control group of this comparative experiment was the shLuci group. The knockout group was the shAlg2 group, the D-mannose rescue group was the shAlg2+5uM mannitol group, and the GDP-D-mannose disodium salt rescue group added 5uM GDP-D-mannose disodium salt to the cell culture medium, i.e. (shAlg2+5uM GDP-D-mannose disodium salt group). The flow cytometry analysis of hematopoietic stem cells was carried out according to the method of Example 5.

[0079] Comparative rescue experiments showed that at the beginning of the experiment (d0), the growth and differentiation of the four groups of hematopoietic stem and progenitor cells were similar. On d5 of the experiment, the number of hematopoietic stem and progenitor cells in the Alg2 knockdown group decreased compared with the experimental control group. In addition, it was found that the hematopoietic stem cells and progenitor cells in the D-mannitol group and the GDP-D-mannose disodium salt group showed an upward trend, and the results of the two were similar. This shows that the addition of D-mannitol and GDP-D-mannose disodium salt during in vitro culture can improve the defect of decreased differentiation of hematopoietic stem and progenitor cells caused by Alg2 knockdown.

[0080] Example 7: GDP-D-mannose disodium salt is more effective than compound M3N2-glycan in rescuing the motor behavior of Alg2-deficient mice

[0081] M3N2-glycan in mammals is a pentasaccharide core complex type N-glycan, which is a product of Alg2 protein enzyme catalysis. In order to identify the rescue effect of GDP-D-mannose disodium salt and compound M3N2-glycan on the motor ability of Alg2 gene knockout mice, Alg2fl / fl-CAGG-CreERTM induced knockout mice were used to perform a complementation experiment after inducing Alg2 gene knockout (as in Example 1). The behavioral data of the experimental mice were collected and analyzed.

[0082] Four groups of mice were used in the experiment, one group was the control group; one group was the knockout group; one group was the mannose anastomosis group, and a PBS solution of GDP-D-mannose disodium salt was prepared before injection, the concentration of GDP-D-mannose disodium salt in the PBS solution was 1μg / μL, and the injection volume was 100μL; another group was the M3N2 group (or M3N2 rescue group), and a PBS solution of M3N2-glycan was prepared before injection, the concentration of M3N2-glycan in the PBS solution was 0.1μg / μL, and the injection volume was 100μL. In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose anastomosis group, 3 mice (n=3) in the control group, and 3 mice (n=3) in the M3N2 group.

[0083] Figure 6 These are the results of a behavioral comparison experiment between GDP-D-mannose disodium salt and compound M3N2-glycan in Example 7. Figure 6 In the figure, the gray column is the control group; the red column is the knockout group; the green column is the mannose supplementation group (or GDP mannose rescue group), and the blue column is the M3N2-glycan supplementation group (or M3N2 rescue group). In the experiment, 3 mice (n=3) were used in the knockout group, 3-5 mice (n=3) in the control group, 3 mice (n=3) in the mannose supplementation group, and 3 mice (n=3) in the M3N2 group. Figure 6“Reversehanging” means reverse hanging, “time” means time, and “Rotarod” means rotating rod.

[0084] from Figure 6 Comparison of experimental results shows that the duration of the rotarod exercise of the M3N2-glycan group mice is the same as that of the knockout group, but the duration of both the inversion hanging and the rotarod of the mannose supplementation group mice is longer than that of the M3N2-glycan group mice ( Figure 6 A and B). Therefore, although M3N2-glycan is an enzymatic product of Alg2 protein, reinfusion of M3N2-glycan did not significantly improve the motor behavior of Alg2-deficient mice. It is worth noting that the effect of GDP-D-mannose disodium salt is better than that of M3N2-glycan, indicating that the mannose salts of the present invention have significant advantages in alleviating motor and muscle disorders caused by Alg2 deficiency.

[0085] Example 8: GDP-D-mannose disodium salt is more effective than M3N2-polysaccharide in rescuing the differentiation ability of hematopoietic stem cells in Alg2-deficient mice

[0086] During the experiment of Example 7, bone marrow cells of four groups of mice in the experiment (i.e., control group, knockout group, mannose supplementation group, and M3N2 group) were collected and analyzed, and the effects of GDP-D-mannose disodium salt and M3N2-glycan on hematopoietic stem and progenitor cells were analyzed and compared. In the experiment, 3 mice (n=3) were used in the knockout group, 3 mice (n=3) in the mannose supplementation group, 3 mice (n=3) in the control group, and 3 mice (n=3) in the M3N2 group.

[0087] Figure 7 The results of the comparative experiment on the differentiation ability of GDP-D-mannose disodium salt and compound M3N2-glycan on hematopoietic stem and progenitor cells in Example 8 are shown. Figure 7 In the experiment, 3 mice (n=3) were used in the knockout group, 3-5 mice (n=3) in the control group, 3 mice (n=3) in the mannose supplementation group, and 3 mice (n=3) in the M3N2 group.

[0088] from Figure 7 The results show that the number of various hematopoietic stem and progenitor cells in the M3N2-glycan supplementation group is lower than that in the knockout group. However, the number of corresponding hematopoietic stem and progenitor cells in the mannose supplementation group is higher than that in the M3N2-glycan supplementation group, such as early hematopoietic stem and progenitor cells, multipotent progenitor cells, common lymphoid progenitor cells ( Figure 7A, B and C in the figure). This result shows that the effect of GDP-mannose disodium salt on improving the decline in differentiation of hematopoietic stem and progenitor cells caused by Alg2 deficiency is significantly better than that of M3N2-glycan. Similarly, from the perspective of hematopoietic stem cells, the advantages of the mannose salt compounds of the present invention in alleviating the hematopoietic system caused by Alg2 deficiency are further explained.

[0089] In summary, the present invention finds that D-mannitol has a significant improvement effect on the decline in differentiation of hematopoietic stem cells and hematopoietic progenitor cells caused by Alg2 deficiency. At the same time, through animal in vivo experiments, it was found that GDP-D-mannose disodium salt effectively alleviated the decline in differentiation ability of hematopoietic stem cells, reduced motor ability, decreased grip strength and muscle endurance caused by Alg2 deficiency, and improved the phenomenon of N-glycoprotein oligosaccharide deficiency. In addition, it was found that mannose salt compounds have advantages over polysaccharide compounds in vivo reactions. These results show that D-mannitol and GDP-D-mannose disodium salt can effectively improve the hematopoietic immune system, muscle motor ability, and the synthesis and modification of N-sugar complex sugar chains in vivo in congenital myasthenic syndrome and congenital glycosylation disorders caused by Alg2 deficiency, and at the same time, due to the small structural characteristics of salt molecules, it is beneficial for salt compounds to play a role in the rescue process. Therefore, the application of D-mannitol, GDP-D-mannose disodium salt and mannose salts can provide new ideas for the development of drugs for the treatment of congenital myasthenic syndrome and congenital glycosylation disorders caused by Alg2 deficiency.

Claims

1. The use of mannose salt in the preparation of drugs for treating congenital myasthenic syndrome or congenital glycosylation disorder.

2. The use according to claim 1, characterized in that: The mannose salt includes at least one of mannose sodium salt, mannose potassium salt, mannose hydrochloride, mannose sulfate, mannose nitrate, mannose magnesium salt, mannose calcium salt and mannose quaternary ammonium salt.

3. The use according to claim 2, characterized in that: The mannose salt is selected from at least one of mannose sodium salt, mannose potassium salt and mannose hydrochloride.

4. The use according to claim 1, characterized in that: The mannose salt is at least one selected from GDP-D-mannose disodium salt, guanosine 5'-(trihydrogen diphosphate) P'-ALPHA-D-mannosyl ester disodium salt, D-mannose amine hydrochloride, N-acetyl-D-mannosamine, and mannitol.

5. The use according to any one of claims 1 to 4, characterized in that: The mannose salt is used in the preparation of a drug for treating congenital myasthenic syndrome or congenital glycosylation disorder caused by Alg2 gene defect.

6. The use according to claim 1, characterized in that: The Alg2 gene defect includes at least one of a loss-of-function mutation and a decreased expression level.

7. The use according to any one of claims 1 to 4, characterized in that: The medicine also includes excipients.

8. The use according to claim 7, characterized in that: The auxiliary materials include at least one of a carrier, an osmotic pressure regulator, a pH regulator, a diluent, a disintegrant, an excipient, a solubilizer, a stabilizer, and a preservative.

9. The use according to claim 8, characterized in that: The carrier is selected from at least one of liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, and mesoporous silica nanoparticles.

10. The use according to claim 5, characterized in that: During the application, the concentration of the mannose salt used in the body is 0.2-80 mg / kg.

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