Application of mannose salts in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders

Abstract

This invention belongs to the field of pharmaceutical application technology and discloses the application of mannose salts in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders. The application of mannose salts proposed in this invention in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders is particularly effective for congenital myasthenia gravis or congenital glycosylation disorders caused by Alg2 gene defects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, and specifically relates to the application of mannose salts in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders. Background Technology

[0002] Protein glycosylation plays a crucial role in the modification and folding of proteins within cells; approximately half of all proteins require glycosylation for synthesis and modification to maintain their stability. Glycosylation primarily occurs in two forms: O-linked and N-linked. The core structure of N-glycans is composed of five oligosaccharide groups, specifically the "double-antennae" pentasaccharide molecule Man3GlcNAc2, consisting of three mannose groups (Man) and two N-acetylglucosamine groups (GlcNAc). Glycosylation defects directly contribute to the development of rare diseases such as congenital myasthenia gravis and congenital glycosylation disorders.

[0003] Congenital myasthenic syndromes (CMS) are a heterogeneous group of diseases caused by mutations in genes encoding presynaptic, intrasynaptic, or postsynaptic proteins at the neuromuscular junction. CMS is typically inherited in an autosomal recessive pattern, with characteristic phenotypes of weakness and fatigue upon exertion. Onset is usually in the neonatal or infancy period, with symptoms including muscle weakness, muscle atrophy, feeding difficulties, and potentially respiratory arrest or even respiratory failure.

[0004] Congenital disorders of glycosylation (CDG) are genetic defects in the synthesis and attachment of glycoproteins and glycolipoproteins. The typical biochemical feature of CDG is a defect in protein glycosylation caused by mutations in genes required for the biosynthesis of N-linked oligosaccharides, often associated with severe psychomotor and intellectual disabilities. These gene mutations are frequently found in genes encoding glycosyltransferases, remodeling glycosidases, and glyconucleotide transporters, directly affecting the protein glycosylation process. Since its initial discovery in 1980, 105 types of CDG have been identified, representing a rapidly growing category of inherited metabolic diseases in recent years, and the number of specific types continues to rise.

[0005] There are many factors that cause congenital myasthenia gravis and congenital glycosylation disorders in the existing technology, and the treatment drugs required for congenital myasthenia gravis and congenital glycosylation disorders caused by different factors are different.

[0006] Therefore, there is an urgent need to provide drugs for treating congenital myasthenia gravis and congenital glycosylation disorders caused by different factors. Summary of the Invention

[0007] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the use of mannose salts in the preparation of medicaments for treating congenital myasthenia gravis or congenital glycosylation disorders. The mannose salts are particularly effective in treating congenital myasthenia gravis or congenital glycosylation disorders caused by Alg2 gene defects.

[0008] Alg2 gene defects lead to weight loss, weakened muscle strength, decreased power, reduced athletic ability, impaired construction of intracellular N-glycosylated pentasaccharide core, dysfunction of hematopoietic stem and progenitor cells and decreased differentiation capacity, and N-glucose deficiency in hematopoietic stem and progenitor cells.

[0009] Therefore, this invention proposes a drug for alleviating and treating congenital myasthenia gravis and congenital glycosylation defects caused by Alg2 deficiency (including loss-of-function mutations / decreased expression). The drug, in particular, has significant therapeutic effects on sporadic and familial Alg2 deficiency diseases.

[0010] The first aspect of the present invention proposes the use of mannose salts in the preparation of medicaments for treating congenital myasthenia gravis syndrome or congenital glycosylation disorders.

[0011] Specifically, mannose salts are used in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders.

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

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

[0014] The CAS number of the GDP-D-mannose disodium salt is 103301-73-1, and its molecular formula is C. 16 H 23 N5Na2O 16 P2.

[0015] The CAS number of the disodium guanosine 5'-(trihydrodiphosphate)P'-ALPHA-D-mannosyl ester is 148296-46-2, and the molecular formula is C 16 H 23 N5Na2O 16 P2.

[0016] The CAS number of the D-mannose amino hydrochloride is 5505-63-5, and its molecular formula is C6H. 14 ClNO5, or C6H 13 NO5·HCl.

[0017] The N-acetyl-D-mannosamine has the CAS number 7772-94-3 and the molecular formula C8H. 15 NO.6.

[0018] The CAS number of the D-mannitol is 69-65-8, and its molecular formula is C6H. 14 O6.

[0019] Preferably, the mannose salt is used in the preparation of a medicament for treating congenital myasthenia gravis syndrome or congenital glycosylation disorder caused by Alg2 gene deficiency.

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

[0021] Preferably, the drug further includes excipients.

[0022] Preferably, the excipients include at least one of the following: carrier, osmotic pressure regulator, pH regulator, diluent, disintegrant, excipient, solubilizer, stabilizer, and preservative.

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

[0024] Preferably, during the application process, the concentration of mannose salt used 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] This invention proposes the use of mannose salts in the preparation of drugs for treating congenital myasthenia gravis or congenital glycosylation disorders. The mannose salts are particularly effective in treating congenital myasthenia gravis or congenital glycosylation disorders caused by Alg2 gene defects.

[0027] The GDP-D-mannose disodium salt of this invention can effectively improve the decreased differentiation capacity of hematopoietic stem and progenitor cells, reduced motor ability, decreased grip strength and muscle endurance in Alg2-deficient mice, and improve defects such as the absence of N-glycoprotein oligosaccharides in mouse serum. These results indicate that GDP-D-mannose disodium salt can effectively improve hematopoietic and immune system function, muscle motor ability, and the synthesis and modification of glycoprotein N-glycoprotein complexes in cells and blood in congenital myasthenia gravis caused by Alg2 deficiency (including loss-of-function mutations / decreased expression)-induced myasthenia gravis.

[0028] There are many factors that cause congenital myasthenia gravis or congenital glycosylation disorders, and the disease mechanisms caused by different factors are different, resulting in different effective drugs required. This invention is the first to propose the use of mannose salts to treat congenital myasthenia gravis or congenital glycosylation disorders caused by Alg2 gene defects, and it has shown good therapeutic effects. Currently, about 35 mutated genes have been detected in congenital myasthenia gravis. The symptoms of each disease are similar, but the underlying causes are different, leading to different effective therapeutic drugs required for congenital myasthenia gravis caused by different mutated genes. Attached Figure Description

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

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

[0031] Figure 3 This is the result of the effective enhancement of hematopoietic stem and progenitor cell differentiation ability of GDP-D-mannose disodium salt in Example 3;

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

[0033] Figure 5 This is the result of D-mannitol improving the deficiency of hematopoietic stem cell and hematopoietic progenitor cell differentiation caused by Alg2 deficiency in Example 5;

[0034] Figure 6 The results of the behavioral comparison experiment between GDP-D-mannose disodium salt and compound M3N2-polysaccharide in Example 7;

[0035] Figure 7 This is the result of a comparative experiment on the differentiation ability of hematopoietic stem and progenitor cells between GDP-D-mannose disodium salt and compound M3N2-polysaccharide in Example 8. Detailed Implementation

[0036] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

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

[0038] definition

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

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

[0041] To verify the in vitro therapeutic effect of the drug, mouse hematopoietic stem and progenitor cells were used. The experimental group received Alg2 gene knockdown in hematopoietic stem and progenitor cells using shRNA (shAlg2) targeting the Alg2 gene, while the control group received hematopoietic stem and progenitor cells treated with untargeted shRNA (shLuciferase, shLuci). The treated hematopoietic stem and progenitor cells were cultured for 5 days in a 37°C, 5% CO2 constant temperature and humidity cell culture incubator. The drug was added to the hematopoietic stem cell expansion medium at the designed concentration. The hematopoietic stem and progenitor cell culture data were statistically analyzed and plotted using Prism Graphpad software.

[0042] To verify the therapeutic effect of the drug in vivo, an Alg2 knockout mouse model was developed. This model mouse was an Alg2 flox mouse bred using CRISPR-Cas9 knockout technology. This mouse strain was then crossed with Cre-ERT mice to create Alg2-induced knockout mice, namely the Alg2 fl / fl-CAGG-CreERTM mouse. This mouse strain was induced to knock out the Alg2 gene by intraperitoneal injection of tamoxifen (product code: T5648, Sigma-Aldrich), which activated the Cre-loxP recombinase in mouse cells, knocking out exon 2 of the Alg2 gene. Mice in the knockout group were intraperitoneally injected with 40 mg / kg tamoxifen to induce Alg2 knockout. Mice in the control group were intraperitoneally injected with corn oil. In the treatment group, after Alg2 knockout, the drug was reinfused into the mice at the designed concentration. Comparative data from the control, knockout, and treatment groups were statistically analyzed and plotted using PrismGraphpad software.

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

[0044] In vivo treatment experiments used Alg2fl / fl-CAGG-CreERTM-induced knockout mice as subjects. Alg2 gene knockout was induced by intraperitoneal injection of tamoxifen, while the control group received corn oil. Three groups of mice were used: a control group, a knockout group, and a mannose recovery group (or GDP-D-mannose rescue group), where GDP-D-mannose disodium salt was injected in vivo after knockout. A PBS solution of GDP-D-mannose disodium salt with a concentration of 1 μg / μL was prepared before injection, and the injection volume was 100 μL. In the experiment, the knockout group used 3 mice (n=3), the mannose recovery group used 3 mice (n=3), and the control group used 5 mice (n=5).

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

[0046] In the experiment, mice in the mannose replenishment group were injected with GDP-D-mannose disodium salt on days 1, 10, and 16, while mice in the control and knockout groups were injected with PBS phosphate buffer. From day 10, the mice in the knockout and mannose replenishment groups began to lose weight, and the mice in the mannose replenishment group received a second infusion of GDP-D-mannose disodium salt. By day 16, the weight loss of mice in both the knockout and mannose replenishment groups was close to 15%. At this point, the mannose replenishment group received a third injection of 100 μg of GDP-D-mannose disodium salt. Twenty-four hours after the injection, the weight loss of mice in both the knockout and mannose replenishment groups exceeded 15%, and the experiment was terminated.

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

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

[0049] In the in vivo GDP-D-mannose disodium salt intervention experiment (i.e. Example 1), the mice underwent behavioral tests such as rotarod, grasping, and inverted hanging.

[0050] Figure 2 This is the result of GDP-D-mannose disodium salt improving the strength, grip duration, and motor ability of Alg2-deficient mice in Example 2. Figure 2 In the diagram, gray bars represent the control group; red bars represent the knockout group (KO); and green bars represent the mannose replenishment group (Mannose). In the experiment, the knockout group used 3 mice (n=3), the mannose replenishment group used 3 mice (n=3), and the control group used 5 mice (n=5). Figure 2 In this context, "reverse hanging" means reverse hanging, "time" means time, "maximal grip" means maximum grip strength, "Grip strength" means grip strength, "Grip duration" means grip duration, and "Rotarod" means rotator.

[0051] The mannose-replenished mice had a longer suspension duration in the inverted suspension test than the knockout mice. Figure 2 (A). In the mouse grasping experiment, the maximum grip strength of mice in the mannose-replenished group was higher than that in the knockout group and close to that in the control group. Figure 2 The B group mice (of type B) also had a longer grasping time than the knockout group mice (of type B). Figure 2 (C). Additionally, in the rotarod experiment, the mannose-replenished group mice also exhibited superior motor function compared to the knockout group mice (C). Figure 2 These results indicate that GDP-D-mannose disodium salt can improve muscle atrophy and weakness in Alg2-deficient mice and enhance their motor function.

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

[0053] Inhibition of the Alg2 gene affects the differentiation capacity of hematopoietic stem and progenitor cells. Therefore, in the mannose intervention experiment of Example 1, blood and bone marrow cells were extracted from mice in the control group, knockout group, and mannose replenishment group. Flow cytometry FACS analysis revealed that GDP-D-mannose disodium salt can improve the functional defects of hematopoietic stem and progenitor cells and immune cells in the hematopoietic and immune systems of Alg2 knockout mice.

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

[0055] Blood Flow Cytometry Sample Preparation: Blood samples were extracted from the control group, knockout group, and mannose replenishment group, with a sample volume of 50-100 μl. EDTA (ethylenediaminetetraacetic acid) anticoagulant was added (blood:EDTA = 9:1 v / v). Fluorescently conjugated antibodies against CD45-PE, CD4-APC, CD8-APC, B220-APC, B220-APC-Cy7, Gr1-APC-Cy7, and CD11b-APC-Cy7 were added to each sample at a concentration of 1 μL. Cells were incubated on ice for 30 minutes, washed with PBS, and incubated on ice for 5 minutes with erythrocyte lysis buffer. The cells were then centrifuged at 4°C and 1200 rpm, the supernatant lysate was removed, and the basal layer of cells was retained for flow cytometry analysis.

[0056] Bone marrow immune cell flow cytometry sample preparation: Bone marrow cells were extracted from mice in the control group, knockout group, and mannose replenishment group. Fluorescently conjugated antibodies against CD45-PE, CD4-APC, CD8-APC, B220-APC, B220-APC-Cy7, Gr1-APC-Cy7, and CD11b-APC-Cy7 were added to each sample at 1 μL. Cells were incubated on ice for 30 minutes, washed with PBS, and centrifuged at 4°C and 1200 rpm to remove the supernatant staining solution, retaining the basal cells for flow cytometry analysis.

[0057] Preparation of hematopoietic stem / progenitor cell flow cytometry samples: Bone marrow cells were extracted from mice in the control, knockout, and mannose-replenished groups. Hematopoietic lineage antibody staining was performed, and cells were washed with PBS. Fluorescently coupled Streptavidin-APC-Cy7 (SA), cKit-APC, ScaI-PE-Cy7, Flt3-PE, CD16 / 32-FITC, CD150-BV605, CD34-AF700, and IL7-PerCP-Cy5.5 antibodies were added to each sample at 1 μL. Cells were incubated on ice for 120 minutes. After incubation at 4°C and 1200 rpm, the supernatant staining solution was removed by centrifugation, retaining the bottom layer of cells for flow cytometry analysis.

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

[0059] In the stained bone marrow cells described above, hematopoietic stem / progenitor cells were labeled with Lin / Streptavidin, cKit, and ScaI antibodies. LSK hematopoietic stem / progenitor cells were those expressing SA. - cKit + ScaI + Bone marrow cells, LK hematopoietic progenitor cells express SA - cKit+ ScaI - Bone marrow cells.

[0060] Flow cytometry analysis of hematopoietic stem cells and pluripotent progenitor cells.

[0061] In the stained LSK cells described above, hematopoietic stem cells (HSCs) and pluripotent progenitor cells (MPPs) within the LSK cells were further labeled using CD34 and CD150 antibodies. Hematopoietic stem cells were labeled with cells expressing CD150. + CD34 - LSK cells, pluripotent progenitor cells expressing CD34 + LSK cells.

[0062] Flow cytometry analysis of progenitor cells in myeloid differentiation.

[0063] In the LK cells stained above, CD16 / 32 and CD34 were further used to label myeloid common progenitor cells (CMP), erythroid-megakaryocytic progenitor cells (MEP), and granulocyte-macrophage progenitor cells (GMP). CMP cells expressed CD34... + CD16 / 32 - LK cells; MEP cells express CD34. - CD16 / 32 - LK cells; GMP is a CD34-expressing cell. + CD16 / 32 + LK cells.

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

[0065] Bone marrow cells were labeled with antibodies against Streptavidin, cKit, ScaI, Flt3, and IL7. CLP cells expressed Flt3 were used to label lymphoid hematopoietic progenitor cells (CLPs). + IL7 + lin - cKit middle ScaI middle Bone marrow cells.

[0066] Figure 3 This is the result of the effective enhancement of hematopoietic stem cell differentiation ability of GDP-D-mannose disodium salt in Alg2 knockout mice in Example 3. Figure 3 In the diagram, gray bars represent the control group; red bars represent the knockout group; and green bars represent the mannose replenishment group. In the experiment, the knockout group used 3 mice (n=3), the mannose replenishment group used 3 mice (n=3), and the control group used 5 mice (n=5).

[0067] from Figure 3The 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 by 27%, and CLP by 55%. Figure 3 (A, B, and C). Furthermore, some disordered lymphocytes in the knockout group were alleviated in the mannose replenishment group; for example, the decrease in blood B cells and the abnormal expansion of bone marrow T cells were significantly alleviated. Figure 3 (D and E). This result indicates that GDP-mannose disodium salt can improve the Alg2 deficiency-induced decline in hematopoietic stem and progenitor cell differentiation, especially the decline in lymphoid hematopoietic stem and progenitor cell differentiation, thereby improving the disorder of lymphoid immune cells and improving the immune system.

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

[0069] Blood was extracted from mice in the mannose intervention experiment of Example 1, serum was prepared, and glycoprotein proteomic analysis revealed that GDP-D-mannose disodium salt increased the N-glucose glycoform in the serum of Alg2 knockout mice.

[0070] Figure 4 The results of GDP-D-mannose disodium salt increasing the N-glycoprotein complex glycoform in the serum of knockout mice in Example 4.

[0071] Compared to the control group, the knockout group showed a significant loss of N-glycan complexes in serum. However, in the mannose replenishment group, supplementation with GDP-D-mannose disodium salt significantly increased the variety of N-glycans in mouse serum, with the abundance of typical N-glycans approaching that of the control group. These results indicate that GDP-D-mannose disodium salt supplementation significantly alleviated the abnormal loss of complex glycans in Alg2 knockout mice serum, effectively increased the reserve of oligosaccharide precursors in serum, provided more substrates for the synthesis of various complex glycans in the later stages of N-glycosylation, and maintained the continued progress of protein glycosylation modification.

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

[0073] To identify the effect of D-mannitol on hematopoietic stem and progenitor cells after Alg2 gene knockdown, mouse bone marrow cells were extracted in vitro and placed in flow cytometry staining solution (1xPBS with 2% fetal bovine serum FBS, 1% penicillin, and streptomycin). A fluorescent antibody containing APC conjugated to mouse anti-cKit was added to the suspension, and the cells were incubated on ice for 30 minutes. After washing, the cell suspension was centrifuged at 4°C, 1200 rpm, and 5 minutes. The supernatant was removed, and the lower cell mass was retained. 200 μL of cell staining solution containing anti-APC microbeads (Mitteni, Germany) was added, and the cells were incubated on ice for 30 minutes. After incubation, the cells were washed, centrifuged at 4°C, 1200 rpm, and 5 minutes. The cells were resuspended in 3 mL of cell staining solution, and cKit-positive hematopoietic stem and progenitor cells were isolated using an LS magnetic bead separation column and magnetic bead separation system (Mitteni, Germany). cKit-positive hematopoietic stem and progenitor cells were mixed with a blood lineage cell flow cytometry antibody containing biotin-conjugated CD3, CD8, CD11b, Gr-1, B220, and Ter119 antibodies. The mixture was thoroughly mixed, incubated on ice for 30 minutes, rinsed, centrifuged, and the lower cell cluster was retained. Cells were resuspended in cell staining solution and stained with APC fluorescently conjugated anti-cKit, PE fluorescently conjugated anti-Sca1, and APC / cy7 fluorescently conjugated anti-streptavidin. The mixture was thoroughly mixed, incubated on ice for 120 minutes, rinsed, and the fluorescent antibodies in the cell suspension were removed by centrifugation. LSK cells (i.e., lineage-negative, Sca1-positive, and cKit-positive cells) were isolated using flow cytometry and a FACS AriaIII flow cytometer. LSK cells were infected with a lentivirus containing shAlg2 to knock down Alg2 gene expression and were designated as the experimental group.

[0074] The control group consisted of LSK cells infected with shLuci (shLuci group). Simultaneously, an Alg2 knockdown LSK cell group (shAlg2 group) was established, with D-mannose added to the cell culture medium as the mannitol intervention group (shAlg2 + 5 μM mannitol group). The GFP fluorescent indicator protein in the stable expression plasmid used in this experiment was activated on day 3 after cell infection; therefore, this was set as the first analysis time point for flow cytometry, day 0 (day0). LSK cells were then cultured for another 5 days, which was set as the second analysis time point (day 5, abbreviated as d5). Flow cytometry analysis was performed on days 0 and 5 to collect relevant experimental data on the in vitro differentiation and regeneration of hematopoietic stem cells and early progenitor cells.

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

[0076] The rescue experiment showed that at the start of the experiment (d0), the growth and differentiation of hematopoietic stem and progenitor cells were similar in all three groups. On day 5, the number of hematopoietic stem cells and progenitor cells in the Alg2 knockdown group was lower than that in the control group. It was also found that the addition of D-mannitol increased the number of hematopoietic stem cells and progenitor cells in the mannose intervention group. Specifically, D-mannitol improved the decline in hematopoietic progenitor cells caused by Alg2 knockdown, with the number reaching or exceeding that in the control group. This indicates 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: The same effects of D-mannitol and GDP-D-mannose disodium salt on hematopoietic stem cells in vitro

[0078] To identify the effects of D-mannitol and GDP-D-mannose disodium salt on hematopoietic stem and progenitor cells after Alg2 gene knockdown, a comparative experiment was conducted using the two compounds according to the method in Example 5. The extraction and knockdown induction procedures for hematopoietic stem and progenitor cells were consistent with those in Example 5. The control group in this comparative experiment was the shLuci group. The knockout group was the shAlg2 group, the D-mannose rescue group was the shAlg2 + 5 μM mannitol group, and the GDP-D-mannose disodium salt rescue group was prepared by adding 5 μM GDP-D-mannose disodium salt to the cell culture medium, i.e., (shAlg2 + 5 μM GDP-D-mannose disodium salt group). Flow cytometry analysis of hematopoietic stem and progenitor cells was performed according to the method in Example 5.

[0079] The comparative rescue experiment showed that at the start of the experiment (d0), the growth and differentiation of hematopoietic stem and progenitor cells were similar in all four groups. On day 5, the number of hematopoietic stem cells and progenitor cells in the Alg2 knockdown group was lower than that in the control group. Furthermore, it was found that the number of hematopoietic stem cells and progenitor cells in both the D-mannitol group and the GDP-D-mannose disodium salt group showed an increasing trend, with similar effects from both groups. This indicates that the addition of D-mannitol and GDP-D-mannose disodium salt during in vitro culture can improve the defect of decreased hematopoietic stem and progenitor cell differentiation caused by Alg2 knockdown.

[0080] Example 7: GDP-D-mannose disodium salt showed better rescue effect on motor behavior in Alg2-deficient mice than compound M3N2-polysaccharide.

[0081] In mammals, M3N2-glycan is a pentasaccharide-core complex of N-glycans and a product of Alg2 protease. To determine the rescue effects of GDP-D-mannose disodium salt and compound M3N2-glycan on the motor function of Alg2 gene knockout mice, Alg2fl / fl-CAGG-CreERTM-induced knockout mice were used. After Alg2 gene knockout (as in Example 1), a recovery experiment was conducted using both GDP-D-mannose disodium salt and compound M3N2-glycan. Behavioral data from the experimental mice were collected and analyzed.

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

[0083] Figure 6 The results of the behavioral comparison experiment between GDP-D-mannose disodium salt and compound M3N2-glycan in Example 7 are shown. Figure 6 In the diagram, gray bars represent the control group; red bars represent the knockout group; green bars represent the mannose replenishment group (or GDP-mannose rescue group); and blue bars represent the M3N2-glycan replenishment group (or M3N2 rescue group). In the experiment, the knockout group used 3 mice (n=3), the control group used 3-5 mice (n=3), the mannose replenishment group used 3 mice (n=3), and the M3N2 group used 3 mice (n=3). Figure 6 In the text, "reversehanging" means reverse hanging, "time" means time, and "Rotarod" means rotating rod.

[0084] from Figure 6 The comparative experimental results show that the rotarod exercise time of the M3N2-glycan group mice was the same as that of the knockout group. However, the mannose-replenished group mice had longer durations in both inverted suspension and rotarod exercises than the M3N2-glycan group mice. Figure 6(A and B in the original text). Therefore, it is believed that although M3N2-glycan is an enzyme-catalyzed product of Alg2 protein, the reinfusion of M3N2-glycan did not significantly improve the motor behavior of Alg2-deficient mice. It is worth noting that GDP-D-mannose disodium salt is more effective than 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 showed better rescue effect on the differentiation capacity of hematopoietic stem cells in Alg2-deficient mice than M3N2-glycan.

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

[0087] Figure 7 This is the result of a comparative experiment on the differentiation ability of hematopoietic stem and progenitor cells between GDP-D-mannose disodium salt and compound M3N2-polysaccharide in Example 8. Figure 7 In the diagram, gray bars represent the control group; red bars represent the knockout group; green bars represent the mannose replenishment group; and blue bars represent the M3N2-glycan replenishment group. In the experiment, the knockout group used 3 mice (n=3), the control group used 3-5 mice (n=3), the mannose replenishment group used 3 mice (n=3), and the M3N2 group used 3 mice (n=3).

[0088] from Figure 7 The results showed that the number of various hematopoietic stem and progenitor cells in the M3N2-glycan replenishment group was lower than that in the knockout group. However, the number of corresponding hematopoietic stem and progenitor cells in the mannose replenishment group was higher than that in the M3N2-glycan replenishment group, such as early hematopoietic stem and progenitor cells, pluripotent progenitor cells, and common lymphoprogenitor cells. Figure 7 (A, B, and C). This result indicates that GDP-mannose disodium salt is significantly more effective than M3N2-glycan in improving the decline in hematopoietic stem and progenitor cell differentiation caused by Alg2 deficiency. Similarly, from the perspective of hematopoietic stem cells, this further clarifies the advantages of the mannose salt compounds of this invention in alleviating the hematopoietic system caused by Alg2 deficiency.

[0089] In summary, this invention demonstrates that D-mannitol significantly improves the decreased differentiation of hematopoietic stem cells and progenitor cells induced by Alg2 deficiency. Furthermore, in vivo animal experiments revealed that GDP-D-mannose disodium salt effectively alleviates the decreased differentiation capacity of hematopoietic stem and progenitor cells, reduced motor ability, decreased grip strength, and decreased muscle endurance caused by Alg2 deficiency, and improves the deficiency of N-glycoprotein oligosaccharides. Additionally, the advantages of mannose salts over polysaccharide compounds in in vivo response were observed. These results indicate that D-mannitol and GDP-D-mannose disodium salt can effectively enhance the hematopoietic immune system, muscle motor ability, and the synthesis and modification of N-glycosylation complexes in Alg2-deficient congenital myasthenia gravis and congenital glycosylation disorders. Moreover, the small molecular size of salts facilitates their 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 to treat congenital myasthenia gravis and congenital glycosylation disorders caused by Alg2 deficiency.

Claims

1. Use of a mannose salt in the preparation of a medicament for treating congenital myasthenic syndrome or congenital disorder of glycosylation caused by Alg2 gene defect. The mannose salt is selected from GDP-D-mannose disodium salt.

2. Use according to claim 1, characterized in that, The Alg2 gene defect comprises at least one of loss-of-function mutation, decreased expression.

3. Use according to claim 1 or 2, characterized in that, The medicament further comprises an excipient.

4. Use according to claim 3, characterized in that, The excipient comprises at least one of a carrier, an osmotic pressure regulator, a pH regulator, a diluent, a disintegrant, a solubilizer, a stabilizer, a preservative.

5. Use according to claim 4, characterized in that, The carrier is selected from at least one of a liposome, an ethosome, a polymeric micelle, a nanostructured lipid carrier, a solid lipid nanoparticle, a mesoporous silica nanoparticle.

6. Use according to claim 1, characterized in that, In the process of the use, the concentration of the mannose salt used in vivo is 0.2-80 mg / kg.

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