Glycosylation inhibitors as therapeutic agents for stroke

Abstract

A pharmaceutical composition for treating an ischemic condition in a subject may include a protein glycosylation inhibitor. Methods can include administering to the subject an effective amount of a protein glycosylation inhibitor. The administration is effective to promote endothelial cell proliferation and angiogenesis in the subject. The subject may require inducible angiogenesis due to ischemic conditions caused by stroke. A pharmaceutical composition for inhibiting protein glycosylation in a cell may include ManN. A method for inhibiting protein glycosylation in a cell can include administering an effective amount of ManN to the cell.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 332,932, filed on April 20, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporated by Reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] Angiogenesis is a complex process involving the growth of new blood vessels from existing vasculature, and occurs in both physiological and pathological environments. In tumors, angiogenesis promotes rapid growth and metastasis by delivering nutrients and oxygen and removing metabolic waste. The development of the vasculature requires the coordinated activation of multiple signal transduction pathways, including VEGF / VEGFR, angiopoietin (Ang) / Tie2, Notch, Ephrin / Eph and PDGF / PDGFR. Stimulating angiogenesis is likely to promote the treatment of many conditions characterized by reduced perfusion, including diabetic ulcers, myocardial and limb ischemia. On the contrary, blocking angiogenesis is a clinically validated strategy for the treatment of malignant tumors and intraocular neovascularization disorders.

[0006] Endothelial cell (EC) metabolism is hypothesized to play a key role in the regulation of angiogenesis in normal and pathological situations. Metabolic switches in EC, such as fatty acid, glucose, and glutamine metabolism, have been reported to trigger angiogenesis. EC in tumor vasculature are known to rely on glycolysis for ATP production, for example through enhanced expression of the glucose transporter GLUT1. Reducing glycolysis in tumor EC blocks their proliferation. Additionally, aberrant glycosylation patterns have been documented during oncogenic transformation and progression of cancer, and it has been proposed that inhibition of glycosylation may inhibit key angiogenic pathways, including VEGF / VEGFR2 and Notch. Evidence has emerged in recent years suggesting that glycans act as novel regulators of angiogenesis due to changes in protein glycosylation. For example, it has been reported that the glycan-binding protein galectin-1 interacts with VEGFR2, leading to ligand-independent receptor activation, which may contribute to tumor resistance to anti-VEGF therapy. Therefore, EC metabolism has been identified as a novel target for anti-angiogenic therapy, particularly through inhibition of energy metabolism and glycosylation. Summary of the invention

[0007] There remains a need for effective treatments and therapies for stroke. Stroke remains a debilitating condition that can result in a lengthy recovery period, reduced ability to perform activities of daily living, social and economic difficulties, and can be fatal. Preventing a first stroke, preventing recurrent stroke, mitigating the effects of ongoing stroke, or improving treatment and recovery from stroke would provide enormous benefits to society and improve the quality of life for many individuals.

[0008] The present disclosure provides pharmaceutical compositions and methods for treating ischemic conditions of subjects caused by stroke. In some embodiments, a pharmaceutical composition for treating stroke, the pharmaceutical composition comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castaneospermine (Cas) or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the subject has experienced ischemic stroke. In some embodiments, the ischemic stroke comprises thrombotic stroke or embolic stroke. In some embodiments, the subject has experienced hemorrhagic stroke. In some embodiments, the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage. In some embodiments, the subject has experienced brainstem stroke. In some embodiments, the subject has experienced transient ischemic attack. In some embodiments, the subject has experienced cryptogenic stroke.

[0009] Described herein is a pharmaceutical composition that provides an effective amount of an N-glycosylation inhibitor to treat stroke. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a single-dose treatment regimen. In some embodiments, the single-dose treatment regimen comprises pre-treatment, concurrent treatment, or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a multiple-dose treatment regimen. In some embodiments, the multiple-dose treatment regimen comprises pre-treatment, concurrent treatment, and / or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 100 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 40 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 20 g of the N-glycosylation inhibitor.

[0010] Described herein is a pharmaceutical composition that provides an effective amount of an N-glycosylation inhibitor that can be combined with other factors that regulate angiogenesis to treat stroke. In some embodiments, the pharmaceutical composition described herein further comprises an effective amount of an angiogenic factor. In some embodiments, the angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof. In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF) or a combination thereof. In some embodiments, the VEGF is recombinant VEGF. In some embodiments, the VEGF is administered locally near the brain site of a subject affected by an ischemic event.

[0011] Described herein is a method for treating stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castaneospermine (Cas) or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the subject has experienced ischemic stroke. In some embodiments, the ischemic stroke is a thrombotic stroke or an embolic stroke. In some embodiments, the subject has experienced hemorrhagic stroke. In some embodiments, the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage. In some embodiments, the subject has experienced brainstem stroke. In some embodiments, the subject has experienced transient ischemic attack. In some embodiments, the subject has experienced cryptogenic stroke.

[0012] Described herein is a method for treating stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a single-dose treatment regimen. In some embodiments, the single-dose treatment regimen comprises pre-treatment, concurrent treatment, or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a multiple-dose treatment regimen. In some embodiments, the multiple-dose treatment regimen comprises pre-treatment, concurrent treatment, and / or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 100 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 40 g of the N-glycosylation inhibitor. In some embodiments, the single-dose treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 20 mg to about 2000 mg / kg of subject body weight. In some embodiments, the single-dose treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 100 mg to about 300 mg / kg of subject body weight. In some embodiments, the multiple-dose treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 10 mg to about 2000 mg / kg of subject body weight. In some embodiments, the multiple-dose treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 100 mg to about 300 mg / kg of subject body weight.

[0013] Described herein is a method for treating stroke, the method comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor that can be combined with other factors that regulate angiogenesis. In some embodiments, the administration to the subject further comprises administering an effective amount of an angiogenic factor. In some embodiments, the angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof. In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF) or a combination thereof. In some embodiments, the VEGF is recombinant VEGF. In some embodiments, the VEGF is administered locally near the brain site of a subject affected by an ischemic event.

[0014] Described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein N-linked protein glycosylation is inhibited in endothelial cells of the subject. Also described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein the administration is effective to stimulate endothelial cell proliferation and angiogenesis of blood vessels near the brain. Also described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein the administration is effective to stimulate brain endothelial cell proliferation and angiogenesis. Also described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein the administration is effective to activate JNK signaling and upregulate an unfolded protein response caused by endoplasmic reticulum stress. Also described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein the subject exhibits an improvement in one or more symptoms of stroke after the administration. In some embodiments, the one or more symptoms are selected from the group consisting of muscle weakness, facial numbness, facial paralysis, limb paralysis, paralysis on one side of the body, slurred speech, disorganized speech, difficulty understanding others, sudden blindness in one or both eyes, double vision, vertigo, confusion, lack of mental alertness, loss of balance, loss of coordination, stroke-induced death, anoxic damage to one or more areas of the brain, stroke-induced memory impairment, stroke-induced dysautonomia, stroke-induced language impairment, stroke-induced cognitive decline, stroke-induced decreased mobility. In some embodiments of the methods of treating stroke described herein, the subject exhibits a significant increase in vascular density in or near the stroke-induced brain injury area after the administration.

[0015] In some embodiments, a pharmaceutical composition for treating an ischemic condition caused by stroke in a subject includes an N-glycosylation inhibitor. In some embodiments, a pharmaceutical composition for treating an ischemic condition caused by stroke in a subject includes hexosamine D-mannosamine (ManN). In some embodiments, a method for treating an ischemic condition caused by stroke in a subject includes administering an effective amount of hexosamine D-mannosamine (ManN) to the subject in need. The present disclosure also provides pharmaceutical compositions and methods for preventing stroke in a subject. The present disclosure also provides pharmaceutical compositions and methods for alleviating the effects of persistent ischemic events affecting the brain.

[0016] In some embodiments, the administration is effective in promoting endothelial cell proliferation and angiogenesis in the subject. In some embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the method further comprises administering an effective amount of VEGF to a subject in need thereof.

[0017] Described herein are methods for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, N-linked protein glycosylation is inhibited in endothelial cells of the subject. In some embodiments, the administration is effective to stimulate endothelial cell proliferation and angiogenesis of blood vessels near the brain. In some embodiments, the administration is effective to stimulate brain endothelial cell proliferation and angiogenesis. In some embodiments, the administration is effective to activate JNK signaling and upregulate the unfolded protein response caused by endoplasmic reticulum stress.

[0018] Described herein is a method for preventing stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castaneospermine (Cas) or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a single-dose treatment regimen. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises a multiple-dose treatment regimen. In some embodiments, the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 100 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 40 g of the N-glycosylation inhibitor.

[0019] Described herein is a method for preventing stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the risk of recurrent stroke in the subject is reduced. In some embodiments, the subject has previously experienced ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack or cryptogenic stroke. In some embodiments, the risk of recurrent stroke includes the risk of ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack or cryptogenic stroke. In some embodiments, the measurement of CRP in the blood of the subject is reduced compared to the previous measurement of the blood C-reactive protein (CRP) level of the subject. In some embodiments, the measurement of the CRP in the blood of the subject is reduced to about 10 mg / L or less.

[0020] Described herein is a method for reducing persistent ischemic events affecting the brain, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castaneospermine (Cas) or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the subject is experiencing ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack or cryptogenic stroke. In some embodiments, the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor. In some embodiments, the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 40 g of the N-glycosylation inhibitor. In some embodiments, the neurological assessment of acute stroke in the subject is improved compared to a previous assessment. In some embodiments, the blood flow to the brain of the subject is improved. In some embodiments, said blood flow to the brain is assessed by transcranial Doppler ultrasound.

[0021] In some embodiments, the ischemic condition affecting the brain is caused by disease or trauma. In some embodiments, the subject needs to induce angiogenesis because the ischemic condition is caused by disease or trauma. In some embodiments, the administration effectively reduces the local ischemia of the subject. In some embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need. In some embodiments, the method further comprises administering an effective amount of VEGF to a subject in need. In some embodiments, the administration is performed orally, intravenously, intrathecally or intraperitoneally.

[0022] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, the method comprising administering an effective amount of hexosamine D-mannosamine (ManN) to the subject in need. In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, the method comprising administering an effective amount of kif to the subject in need. In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, the method comprising administering an effective amount of castanospermine (Cas) to the subject in need.

[0023] In some embodiments, the administration is performed in vivo. In some embodiments, the subject to which the N-glycosylation inhibitor is administered is a mammal. In some embodiments, the subject is a rodent. In some embodiments, the subject belongs to the genus Rattus. In some embodiments, the subject belongs to the genus Mus. In some embodiments, the subject belongs to the genus Canis. In some embodiments, the subject belongs to the genus Felis. In some embodiments, the subject belongs to the genus Equus. In some embodiments, the subject is a human. In some embodiments, the administration of the N-glycosylation inhibitor can effectively stimulate EC proliferation and angiogenesis. In some embodiments, the administration can effectively activate JNK signaling and unfolded protein responses caused by ER stress. In some embodiments, the administration can effectively induce changes in N-glycan and O-glycan profiles in endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] This patent application contains at least one drawing drawn in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The novel features of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments.

[0026] Figures 1A to 1F An example showing the effect of mannosamine (ManN) on the proliferation of bovine choroidal microvascular endothelial cells (BCEC) is presented. Figure 1A: is an image showing crystal violet stained BCEC samples treated with ManN in the presence or absence of VEGF. BCEC were treated with different concentrations of ManN in the range of 0.5 μM to 1 mM for 5-6 days with or without 5 ng / ml VEGF. At the end of the experiment, the cells were fixed and stained with crystal violet. The cell coverage area of ​​the different treatment groups was quantified by ImageJ software. Figure 1B is a graph showing the effect of ManN on cell number in the presence or absence of VEGF. Cell number was quantified by adding AlamarBlue and measuring fluorescence at 530nm / 590nm. n=3 independent samples were used. Figure 1C is a graph showing the effect of ManN on the proliferation of bovine retinal microvascular endothelial cells (BRECs). n=3 biologically independent samples were used. Figure 1D are images showing the effects of hexosamines other than ManN on BCEC proliferation in samples. Each treatment group was tested in duplicate. Figure 1E are graphs and images showing the effect of ManN on wounded BCEC samples. BCEC confluent monolayers were scraped with a 1 ml pipette tip, washed, and then incubated for 40 hours in low glucose DMEM containing 1% FBS. n = 3 independent samples were used. Scale bar = 400 μm. Images were taken using AxioVision LE Rel.4.4 software and the gap between the anterior wounds was quantified. Representative images of crystal violet staining are shown. Figure 1F is a chart showing the effect of ManN in BCEC transwell migration assay. n=4 independent samples were used. Asterisks indicate significant differences compared to controls. When different controls were used for statistical analysis, a line was used between specific groups. Representative experiments are shown based on 2 independent studies. Data are mean + / - SD, and statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0027] Figures 2A to 2C is a Western blot image showing that the activation of ERK, AKT, mTOR, AMPKα, CREB, ACC and eNOS is not unique to ManN. After ManN was treated with VEGF for different time periods, the activation of ERK (Thr 202 / Tyr 204), AKT (Ser 473) and CREB (Ser 133) in BCEC was enhanced ( Figure 2A ) or after 8-hour pretreatment with ManN followed by 15-minute VEGF stimulation ( Figure 2B ).for Figure 2CThe samples shown in , BCEC were treated with 40 μM ManN, ManNAc or mannose for different times. Total mTOR, ACC, eNOS, AMPKα, ERK, AKT, CREB and mTOR (Ser 2448), ACC (Ser 79), eNOS (Ser 1177), AMPKα (Thr 172), ERK (Thr 202 / Tyr 204), AKT (Ser 473) and CREB (Ser 133) were detected by Western blot analysis. f3-actin served as a loading control. Molecular weight (KDa) is marked on the right. Representative experiments are shown according to 2 independent studies.

[0028] Figures 3A to 3E An example is shown in which ManN specifically activates the JNK pathway in BCECs. Figure 3A It is a western blot image. BCEC grown in growth medium (GM: low glucose DMEM containing 10% calf serum (BCS), 10ng / ml VEGF and 5ng / ml bFGF) was switched to growth factor-free medium and then treated with 4μM-4mM ManN or mannose. Four hours later, cell lysates were collected and western blot analysis was performed for phosphorylated JNK (Thr183 / Tyr 185), p38 (Thr 180 / Tyr 182) and ERK (Thr 202 / Tyr 204) as well as total JNK, p38 and ERK. Figure 3B is a Western blot image showing that ManN, but not mannose, can activate JNK and its downstream c-Jun. f3-actin served as a loading control. For each study, a representative experiment is shown from 2-3 independent studies. Figure 3C is a graph showing the effect of ManN on pretreated samples. BCECs adherently plated in 96-well plates were pretreated with specific JNK inhibitor SP600125 (5 μM) for 2 hours with or without 5 ng / ml VEGF, followed by 40 μM or 2 mM ManN. Six days later, cell proliferation was quantified using AlamarBlue. n=3 independent samples were used. Figure 3D Is a Western blot image showing siRNA screening for JNK1 and JNK2. 24 hours after siRNA transfection, BCECs were lysed and Western blot analysis was performed on proteins. f3-actin served as a loading control. Quantification of target knockout is shown. Figure 3Eis a graph showing example results, showing that about 80% knockout of JNK1 and / or JNK2 by two independent siRNAs is associated with a significant reduction in the stimulatory effect of ManN on BCEC proliferation. n=3 independent samples were used. Data are mean + / - SD; asterisks indicate significant differences compared to controls. When different controls were used for statistical analysis, a line was used between specific groups. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0029] Figures 4A to 4G Shown are examples showing that ManN affects protein glycosylation. Figure 4A is a Western blot image showing a decrease in the molecular weight of VEGFR2 after ManN treatment. BCECs were treated with 40 μM of various hexosamines, their derivatives and monosaccharides or 5 ng / ml of VEGF for 24 hours. VEGFR2 Western blot analysis was performed. Figure 4B is a Western blot image showing the dose-dependent effect of ManN on the molecular weight of VEGFR2 in BCECs. Figure 4C are Western blot images showing that mannose can dose-dependently reverse the effect of 2 mM ManN on the molecular weight change of VEGFR2, while mannose alone has no effect even at 10 mM. Figure 4D is a graph showing that 5 mM mannose can completely reverse the bell-shaped effect of ManN on BCEC proliferation with or without 5 ng / ml VEGF. BCECs were plated in 96 wells and then ManN was added. Two hours later, cells were treated with different concentrations of mannose with or without VEGF. Six days later, cell proliferation was quantified using AlamarBlue. n=3 independent samples were used. Figure 4E is a Western blot image showing that the effect of ManN is reversible. After treatment with 40 μM ManN for 24 hours, BCECs were washed three times with low glucose DMEM. Cells were maintained in low glucose DMEM for another 8 or 24 hours. VEGFR2 Western blot analysis was performed. Figure 4F are Western blot images showing the decrease in molecular weight of VEGFR2, Neuropilin-1, CD31, and c-met in HUVECs after treatment with different concentrations of ManN. Figure 4Gis a Western blot image showing that the molecular weight of VEGFR2, f31 integrin and bFGFR1 in hDMVECs was reduced by different concentrations of ManN. f3-actin served as a loading control. Data are mean + / - SD; asterisks indicate significant differences compared to controls. For each study, representative experiments are shown based on 2-5 independent studies. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0030] Figures 5A to 5D Examples showing that ManN specifically induces expression of unfolded protein response (UPR)-responsive proteins are shown. Figure 5A is a Western blot image. BCECs were grown in growth medium (GM) until about 80% confluence. The medium was replaced with growth factor-free medium containing 10% BCS in the presence or absence of 40 or 400 μM ManN or mannose for different times. At the end of each incubation, cell lysates were collected and proteins were separated on 4-12% Bis-Tris gels for Western blot analysis. Figure 5B are Western blot images of cells treated with different concentrations of ManN, mannose, 5 ng / ml VEGF or a combination of ManN and VEGF for 24 hours. Cell lysates were separated on NuPAGE 3-8% Tris-acetate gels for Western blot analysis. Figure 5C 4-PBA but not TUDCA can effectively block the induction of CHOP in BCEC, accompanied by the restoration of the expression of transcription factor ATF-6 after 400 μM ManN treatment. BCEC were pretreated with 2mM 4-PBA or 500 μM TUDCA (two chemical partners). Sixteen hours later, cells were switched to growth factor-free medium for 4 hours in the presence of ManN. GM: growth medium. Figure 5D 4-PBA significantly blocked the bell-shaped effect of ManN on BCEC proliferation. Pretreatment of cells with 1 mM 4-PBA for 8 hours eliminated the additive effects of 40 μM ManN and 5 ng / ml VEGF and protected cells from toxic effects induced by 2 mM ManN. n=3 independent samples were used. For each study, a representative experiment is shown based on 2-3 independent studies. Data are mean + / - SD; asterisks indicate significant differences compared to the control. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0031] Figures 6A to 6Jis a graph showing the effect of ManN on non-endothelial cells of bovine, mouse or human origin. ManN did not promote Calu6 ( Fig. 6A )、A673( Figure 6B )、U87MG( Figure 6C ) and 4T1( Fig.6D ) tumor cell growth. 10% FBS was used as a positive control for Calu6 and A673, while 10 ng / ml bFGF and 1 μg / ml human apo-transferrin were used as positive controls for U87MG and 4T1, respectively. Similarly, ManN alone or in combination with growth factors did not induce AML12 ( Fig. 6E ), bovine pituitary cells ( Fig. 6F ), NIH3T3 cells ( Figure 6G )、Human RPE( Figure 6H ), human skin fibroblasts ( Fig.6I ) and 10 human keratinocytes ( Figure 6J ) increased proliferation. Proliferation was quantified using AlamarBlue or MTS (for 4T1 cells). n = 3 independent samples were used. Figures 6A to 6J The graphs in are representative Western blot analyses showing the dose-dependent effects of 400 μM (2, 4) and 2 mM (3, 5) of ManN and mannose on the molecular weight of bFGFR1 or f31 integrin (for 4T1, AML12, NIH3T3 cells, human skeletal muscle cells, human skin fibroblasts, and human keratinocytes) compared to untreated controls (1). f3-actin served as a loading control. GM: growth medium. Proteins were separated on NuPAGE 3-8% Tris-acetate gels for Western blot analysis. For each study, a representative experiment from 2 independent studies is shown. Asterisks indicate significant differences compared to controls. When different controls were used for statistical analysis, brackets were used between specific groups. When n=2, data are means or mean + / - SD of the means. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0032] Figures 7A to 7F Shown are examples showing the effects of protein glycosylation inhibitors on BCEC proliferation. Fig. 7AIncluded are images of samples showing dose-dependent stimulation of BCEC proliferation by various glycosylation inhibitors. Inhibitors were added at concentrations ranging from 0.01 to 100 μM for 3 days with or without 5 ng / ml VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. A representative experiment is shown. Kif, an inhibitor of ERα-1,2-mannosidase I and Golgi α-mannosidase I; Castanospermine (Cas), an α-glucosidase inhibitor. The cell coverage area of ​​different treatment groups was quantified by ImageJ software. Figure 7B is a graph showing the dose-dependent effects of Kif and Cas in promoting BCEC proliferation with or without 5 ng / ml VEGF. n=3 independent samples were used. Figure 7C Western blot images showing that both inhibitors reduced VEGFR2 molecular weight in a dose-dependent manner and induced Bip expression (as assessed by Western blot analysis) are included. Proteins were isolated from total cell lysates using 3-8% Tris-acetate gels. BCECs were treated with various inhibitors for 24 hours. Western blots were quantified by densitometry. β-actin was a loading control. Fig.7D Graph showing accelerated closure of monolayer gaps by Kif and Cas in BCEC scratch assay, with Kif (H2O) and Cas (DMSO) as controls. Gaps were quantified using AxioVision LE Rel. 4.4 software. n=3 independent samples used. Scale bar=400 μm. Fig. 7E Is a Western blot image showing activation of AKT and JNK in BCEC by 40 μM of glycosylation inhibitor and 10 ng / ml VEGF. However, Cas did not activate ERK. Phosphorylated AKT, JNK and ERK were quantified by density analysis relative to total protein. Figure 7F is a graph showing that pretreatment of BCEC with 5 μM SP600125 for 2 hours significantly blocked the effects of two glycosylation inhibitors on BCEC proliferation. n=3 independent samples were used. Representative experiments are shown based on 2-4 independent studies. Data are shown as mean + / - SD. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0033] Figures 8A to 8D Examples are shown showing that local application of ManN and VEGF stimulates angiogenesis and accelerates wound healing in mice. Fig. 8Ais a chart showing the effect of ManN on wounds. Wounds were made on the dorsal skin of mice by a 6 mm punch. In the first 4 days, VEGF and ManN were applied at a dose of 20 μg per wound in 25 μL PBS every day, with PBS as a control. A 10-day wound healing study was conducted in 5 mice per group. In two independent studies, wound closure rate (%) was quantified by ImageJ software. Asterisks indicate significant differences compared to controls at each time point. Figure 8B Included are images generated from a 4-day wound healing study as well as images of the wound healing process on days 1, 2, and 4. n = 5 animals / treatment group. Figure 8C Representative images of CD31 immunohistochemical staining in the PBS control group and the VEGF and ManN combination group are included (scale bar = 200 μm). Fig.8D : is a quantitative chart showing the density of CD31 positive blood vessels (red dotted circles) around the wound area counted by eye under microscopy (20 times magnification). Data are mean + / - SD. The statistical significance between the treatment groups of interest was further confirmed using the Wilcoxon rank sum test. Asterisks indicate significant differences compared to the PBS control. For each study, a representative experiment is shown. n = 3 animals / treatment group were used. When different controls were used for statistical analysis, a line was used between specific groups. Statistical analysis was completed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0034] Figures 9A to 9D An example is shown in which ManN accelerates blood perfusion recovery in a mouse ischemic hindlimb model. Fig. 9A Included are images obtained from continuous laser Doppler analysis of blood perfusion in the hindlimbs of ManN-treated, Kif-treated, and control mice. Different colors are used to indicate blood perfusion in the ischemic limb (ligated; left) to the non-ischemic limb (sham; right). Representative images from week 0 and week 1 are shown. Fig. 9B is a graph showing the quantification of the blood perfusion ratio between region 2 (ischemic; left limb) and region 1 (non-ischemic; right limb), n=8 animals / treatment group. Fig. 9C Included are images of sample tissues. Three weeks after surgery, skeletal muscle tissue was harvested and fixed. CD31 immunostaining was performed on these tissue sections to mark the vasculature. H&E staining was also performed. Representative CD31 staining and H&E histological images of ischemic hindlimbs 21 days after surgery are shown. Scale bar = 50 μm. Fig.9Dis a graph showing quantification of vascular density by CD31 immunostaining using ImageJ software, n=8 animals / treatment group, 3 independent experiments; data are mean + / - SEM. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0035] Fig. 10A and 10B Shown is an example showing that ManN promotes retinal neovascularization in mice. Fig. 10A Images of tissues are included. Intravitreal injection of ManN increases vascular density. Adult mice were injected once intravitreally with 500ng ManN, Kif, or 200ng bFGF. PBS was used as a vehicle control. Seven days after injection, PFA-fixed retinas were subjected to CD31 immunofluorescence. Representative images of CD31-positive vessels are shown. n=10 animals / treatment group, 3 independent experiments, scale bar=50μm. Fig. 10B is a graph showing vascular density measured using ImageJ software, n=10 animals / treatment group, 3 independent experiments. Data represent mean + / - SEM. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01, *** p<0.001.

[0036] Figures 11A to 11D Examples showing that ManN (but not structurally related molecules) stimulate endothelial cell proliferation are shown. Fig.11A is a graph showing the additive effect of ManN and bFGF on BCEC proliferation. Bell-shaped effect of ManN on BCEC proliferation. BCECs were treated with ManN in the range of 0.4-400 μM for 5-6 days with or without 20 ng / ml bFGF. At the end of the experiment, proliferation was quantified using AlamarBlue. Fig. 11B BE is a chart showing that the additive effect of VEGF and ManN on BCEC proliferation depends on the glycolytic pathway. Proliferation assays were performed in low glucose DMEM medium without growth factors or in DMEM medium without glucose and pyruvic acid. Asterisks indicate significant differences compared to the untreated control. Statistical analysis was also performed to compare VEGF alone and VEGF plus ManN treatment groups for cells grown in two different assay media. Fig. 11C and 11D is shown in the absence of ( Fig. 11C ) or exists( Fig.11D) Graph showing the effect of various agents from 0.04 μM to 5 mM on BCECs in the presence of 5 ng / ml VEGF. n=3 independent samples were used. For each study, a representative experiment from 2 independent studies is shown. Data are mean + / - SD. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. * p<0.05, ** p<0.01.

[0037] Figures 12A to 12D Figure 4 shows the experimental setup and analysis of the effects of ManN treatment on cerebral infarction. Fig. 12A is a summary of the timing of the ManN treatment regimen and the timing of the induced neural ischemia and post-infarction assessment. Fig. 12B The first experimental results of an acute middle cerebral artery occlusion (MCAO) stroke model in mice testing the effect of ManN treatment are presented. Fig. 12C Results from a secondary experiment in an acute middle cerebral artery occlusion (MCAO) stroke model in mice testing the effects of ManN treatment are presented. Fig.12D express Fig. 12B and Fig. 12C Graphs and analysis of the combined results of the experiments in .

[0038] Figures 13A to 13C Represents the analysis of the effect of ManN treatment on cerebral infarction. Fig.13A Representative MRI coronal sections of ManN-treated and vehicle-treated mouse brains are shown, illustrating the infarct area at days 2 and 4 of the MCAO stroke model. Fig. 13B is a graph and analysis of combined results of infarct volume at day 4 of MCAO stroke model in ManN-treated and vehicle-treated mouse brains. Fig. 13C Shown are TTC staining in coronal sections of ManN-treated and vehicle-treated mouse brains on day 4 of the MCAO stroke model. Figures 14A to 14B Represents CD31 staining and vascular density analysis in MCAO stroke model. Fig.14A Shown is CD31 immunostaining labeling endothelial cells in the area of ​​the cerebral cortex affected by infarction in ManN-treated and vehicle-treated mouse brains at day 6. Fig. 14B Graphs and analysis of CD31 staining density of representative sections of infarct-affected cerebral cortex of vehicle-treated and ManN-treated mice at day 6 are shown, demonstrating an increase in vascular density in the ischemic area of ​​the brain after ManN administration.

[0039] Fig.15 Shown are histopathological analyses of the cerebral cortex and striatum of vehicle-treated and ManN-treated mice at day 4 in the MCAO stroke model. DETAILED DESCRIPTION

[0040] Overview

[0041] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0042] Pharmaceutical composition

[0043] Composition for preventing, alleviating and / or treating stroke

[0044] Described herein is a composition for preventing, alleviating and / or treating stroke, the composition comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanespermine (Cas), or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN and Kif. In some embodiments, the N-glycosylation inhibitor comprises ManN and Cas. In some embodiments, the N-glycosylation inhibitor comprises Kif and Cas. In some embodiments, the N-glycosylation inhibitor comprises ManN, Kif, and Cas. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the N-glycosylation inhibitor comprises Kif. In some embodiments, the N-glycosylation inhibitor comprises Cas. In some embodiments, the composition for preventing stroke is formulated as a pharmaceutical composition. In some embodiments, the composition for alleviating stroke is formulated as a pharmaceutical composition. In some embodiments, the composition for treating stroke is formulated as a pharmaceutical composition.

[0045] In some embodiments, the subject in need of administration of an N-glycosylation inhibitor is a mammal. In some embodiments, the subject is a rodent. In some embodiments, the subject is of the genus Rat. In some embodiments, the subject is of the genus Musculus. In some embodiments, the subject is of the genus Canis. In some embodiments, the subject is of the genus Feline. In some embodiments, the subject is of the genus Equus. In some embodiments, the subject is a human.

[0046] In some embodiments, the pharmaceutical composition for treating stroke is used for use after a subject has a stroke. In some embodiments, the subject has experienced ischemic stroke. In some embodiments, the ischemic stroke comprises thrombotic stroke or embolic stroke. In some embodiments, the subject has experienced hemorrhagic stroke. In some embodiments, the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage. In some embodiments, the subject has experienced brainstem stroke. In some embodiments, the subject has experienced transient ischemic attack (TIA). In some embodiments, the subject has experienced one or more transient ischemic attacks. In some embodiments, the one or more transient ischemic attacks have occurred before the stroke event treated by the subject. TIA may last for a short period of time, including no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 minutes. TIA may temporarily interrupt the blood supply to a certain area of ​​the subject's brain. In some embodiments, the subject suddenly experiences or has experienced the onset of TIA symptoms. In some embodiments, the symptoms of the TIA of the subject subside after a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more than 30 minutes. In some embodiments, the TIA does not cause permanent damage to the brain of the subject. In some embodiments, one or more TIAs in the subject indicate a warning that the subject has an increased risk of stroke. In some embodiments, based on the previous occurrence of one or more TIAs, a pharmaceutical composition comprising an N-glycosylation inhibitor is administered to the subject. In some embodiments, the subject has experienced a cryptogenic stroke. In some embodiments, the subject has been diagnosed with a stroke. In some embodiments, it is assumed that the subject has suffered a stroke. In some embodiments, the subject is suspected of having suffered a stroke. In some embodiments, it is not known whether the subject has experienced a stroke. In some embodiments, it is not known whether the subject has experienced a stroke, but has experienced one or more TIAs. In some embodiments, a pharmaceutical composition comprising one or more N-glycosylation inhibitors is administered to a subject based on an increased risk of stroke after one or more TIAs. In some embodiments, a pharmaceutical composition comprising one or more N-glycosylation inhibitors is administered to a subject based on an increased risk of recurrent stroke after one or more TIAs. In some embodiments, a pharmaceutical composition comprising one or more N-glycosylation inhibitors is administered to a subject based on an increased risk of recurrent stroke after one or more prior strokes.

[0047] In some embodiments, the pharmaceutical composition for treating stroke is administered orally. In some embodiments, the pharmaceutical composition for treating stroke is administered intravenously. In some embodiments, the pharmaceutical composition for treating stroke is administered intrathecally. In some embodiments, the pharmaceutical composition for treating stroke is administered intraperitoneally. In some embodiments, the pharmaceutical composition for treating stroke is administered to the cerebrospinal fluid. In some embodiments, the pharmaceutical composition for treating stroke is administered to the choroid plexus. In some embodiments, the pharmaceutical composition for treating stroke is administered locally near the ischemic site. In some embodiments, the pharmaceutical composition for treating stroke is administered to a blood vessel that directly supplies blood to the brain. In some embodiments, the pharmaceutical composition for treating stroke is administered to the carotid artery.

[0048] Therapeutically effective amount of pharmaceutical composition

[0049] In some embodiments, the pharmaceutical composition for preventing, alleviating or treating stroke comprises an effective amount of an N-glycosylation inhibitor. In some embodiments, the pharmaceutical composition for treating stroke comprises an effective amount of one or more N-glycosylation inhibitors. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif (Kif) or castaneospermine (Cas). In some embodiments, the N-glycosylation inhibitor is hexosamine D-mannosamine (ManN), kif (Kif) or castaneospermine (Cas). In some embodiments, the one or more N-glycosylation inhibitors are hexosamine D-mannosamine (ManN), kif (Kif) or castaneospermine (Cas) or any combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the N-glycosylation inhibitor comprises Kif. In some embodiments, the N-glycosylation inhibitor comprises Cas. In some embodiments, the N-glycosylation inhibitor is ManN. In some embodiments, the N-glycosylation inhibitor is Kif. In some embodiments, the N-glycosylation inhibitor is Cas. In some embodiments, the N-glycosylation inhibitor consists essentially of ManN. In some embodiments, the N-glycosylation inhibitor consists essentially of Kif. In some embodiments, the N-glycosylation inhibitor consists essentially of Cas.In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 111 mg, 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 20mg. The effective amount of the N-glycosylation inhibitor is at least about 40mg. The effective amount of the N-glycosylation inhibitor is at least about 1 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 2 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 3 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 4 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 5 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 6 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 7 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 8 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 9 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 10 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 12 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 14 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 16 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 35 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 111 mg, 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 50g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 45g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 40 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 35 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 30 g.In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 16 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 14 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 12 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 10 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 8 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 6 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 4 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 2 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 1 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 100 mg. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 50 mg. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 40 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1 mg and about 10 mg, about 3 mg and about 10 mg, about 5 mg and about 15 mg, about 10 mg and about 20 mg, about 15 mg and about 45 mg, about 20 mg and about 55 mg, about 25 mg and about 70 mg, about 35 mg and about 75 mg, about 40 mg and about 105 mg, about 45 mg and about 85 mg, about 50 mg and about 80 mg, about 55 about 125mg, about 60mg and about 150mg, about 65mg and about 185mg, about 70mg and about 125mg, about 75mg and about 200mg, about 80mg and about 225mg, about 85mg and about 300mg, about 90mg and about 180mg, about 100mg and about 150mg, about 100mg and about 225mg, about 100mg and about 350mg, about 100mg and about 500mg, about 110mg and about 200mg, about 110mg and about 275mg, about 150mg and about 375mg , about 175mg and about 500mg, about 190mg and about 315mg, about 225mg and about 600mg, about 250mg and about 700mg, about 250mg and about 500mg, about 260mg and about 800mg, about 275mg and about 600mg, about 300mg and about 900mg, about 350mg and about 750mg, about 400mg and about 2g, about 425mg and about 1.1g, about 450mg and about 1.5g, about 500mg and about 850mg, about 525mg and about 1.3g, about 600 mg and about 2g, about 700mg and about 3g, about 800mg and about 4g, about 900mg and about 5g, about 1g and about 3g, about 1.1g and about 6g, about 1.2g and about 8g, about 1.4g and about 10g, about 2.5g and about 7.5g, about 3g and about 12g, about 4g and about 15g, about 4.5g and about 16g, about 5g and about 20g, about 6g and about 18g, about 7g and about 21g, about 8g and about 23g, about 9g and about 25g, about 10g and about 30g, about 12g and about 35g, about 14g and about 28g, Between about 15 g and about 45 g, about 18 g and about 36 g, about 20 g and about 50 g, about 25 g and about 65 g, about 30 g and about 70 g, about 35 g and about 80 g, about 40 g and about 100 g, about 45 g and about 115 g, about 50 g and about 135 g, about 20 g and about 300 g, about 20 g and about 500 g, about 25 g and about 800 g, about 70 g and about 385 g, about 75 g and about 450 g, about 85 g and about 600 g, about 100 g and about 800 g, about 200 g and about 1 kg, or about 475 g and about 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 20 mg and about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 60g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 30g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 30g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 50g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 30g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 20g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5.8g and about 7.3g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 7.8g and about 10g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 11.8 g and about 14.8 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 23.7 g and about 29.7 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 31.7 g and about 39.6 g.

[0050] Angiogenic factors

[0051] In some embodiments described herein are pharmaceutical compositions comprising pro-angiogenic factors that synergistically increase angiogenic growth when combined with a therapeutically effective amount of an N-glycosylation inhibitor. In some embodiments, pro-angiogenic factors synergistically increase endothelial cell proliferation when combined with a therapeutically effective amount of an N-glycosylation inhibitor. In some embodiments, when pro-angiogenic factors are combined with a therapeutically effective amount of an N-glycosylation inhibitor, angiogenic growth is synergistically increased without significant amounts of vascular leakage or vascular edema. In some embodiments, the pro-angiogenic factor comprises VEGF. In some embodiments, VEGF comprises VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF) or a combination thereof. In some embodiments, VEGF comprises recombinant VEGF. In some embodiments, the VEGF is locally administered near a brain site of a subject affected by an ischemic event. In some embodiments, VEGF is administered systemically. In some embodiments, VEGF is administered intravenously. In some embodiments, VEGF is administered intrathecally. In some embodiments, VEGF is administered to the cerebrospinal fluid. In some embodiments, VEGF is administered to the choroid plexus. In some embodiments, the angiogenic factor and the N-glycosylation inhibitor are separately formulated in a pharmaceutical composition. In some embodiments, the angiogenic factor and the N-glycosylation inhibitor are formulated together in a pharmaceutical composition.

[0052] Drug formulations

[0053] Described herein is a pharmaceutical composition for preventing, alleviating and / or treating stroke, comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof. In some embodiments, the pharmaceutical composition for preventing, alleviating and / or treating stroke comprises an effective amount of one or more N-glycosylation inhibitors administered to a subject in need thereof. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN, Kif or Cas or any combination thereof. In some embodiments, the one or more N-glycosylation inhibitors comprise a compound having a chemical structure similar to ManN. In some embodiments, Table 1 lists the one or more N-glycosylation inhibitors having a chemical structure similar to ManN. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises ManN. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises ManN and one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to a subject consists essentially of ManN. In some embodiments, the N-glycosylation inhibitor administered to a subject consists of ManN. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more N-glycosylation inhibitors dissolved in water. In some embodiments, the pharmaceutical composition comprises. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more N-glycosylation inhibitors formulated as a pharmaceutical solution. In some embodiments, the pharmaceutical solution is a liquid preparation in which the one or more N-glycosylation inhibitors and one or more pharmaceutically acceptable excipients are dissolved in a selected solvent system. In some embodiments, the pharmaceutically acceptable excipient is purified water. In some embodiments, the vehicle solution is purified water. In some embodiments, the pharmaceutical solution comprises a cosolvent. In some embodiments, the cosolvent is propylene glycol, glycerol, propylene glycol, glycerol, poly(ethylene glycol) or ethanol. In some embodiments, the pharmaceutical solution comprises one or more agents specifically for increasing the solubility of the N-glycosylation inhibitor in the vehicle. In some embodiments, the one or more agents specifically for increasing the solubility of the N-glycosylation inhibitor in the vehicle are surfactants. In some embodiments, the pharmaceutical solution comprises one or more preservatives. In some embodiments, the one or more preservatives are parabens (methyl and propyl parabens), boric acid and borate salts, sorbic acid and sorbate salts, phenols, or any combination thereof. In some embodiments, benzoic acid and salts (0.1-0.3%) are used as preservatives. In some embodiments, sorbic acid and salts (0.05-0.2%) are used as preservatives. In some embodiments, alkyl esters of parabens (0.001-0.2%) are used as preservatives.In some embodiments, methylparaben and propylparaben (ratio of 9:1) are used as preservatives. In some embodiments, the pharmaceutical solution comprises one or more viscosity modifiers. In some embodiments, the one or more viscosity modifiers are hydrophilic polymers, such as cellulose derivatives (e.g., methylcellulose, hydroxyethylcellulose or hydroxypropylcellulose), alginic acid, sodium alginate and / or polyvinylpyrrolidone. In some embodiments, the pharmaceutical solution comprises one or more antioxidants. In some embodiments, the one or more antioxidants are sodium formaldehyde sulfoxylate, butylated hydroxyanisole, butylated hydroxytoluene or any combination thereof. In some embodiments, the pharmaceutical solution comprises one or more colorants. In some embodiments, the pharmaceutical solution comprises one or more flavoring agents. In some embodiments, the pharmaceutical solution comprises one or more sweeteners. In some embodiments, the one or more sweeteners are aspartame, sucralose, acesulfame K, saccharin, xylitol, sucrose, liquid glucose, glycerol, sorbitol or any combination thereof. In some embodiments, the one or more sweeteners are added to increase the palatability of the one or more N-glycosylation inhibitors. In some embodiments, the pharmaceutical solution comprises one or more buffer systems to regulate the pH of the formulation. In some embodiments, the pH of the pharmaceutical solution is between about pH 5.0-8.0. In some embodiments, the pH of the pharmaceutical solution is between about pH 6.0-7.5. In some embodiments, the pH of the pharmaceutical solution is selected to optimize the solubility of the one or more N-glycosylation inhibitors. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more N-glycosylation inhibitors, and the N-glycosylation inhibitor is formulated as a pharmaceutical oral suspension comprising a suspending agent and one or more of the following: a vehicle, a solvent, a co-solvent, a preservative, a sweetener, a defoaming agent, a wetting agent, a buffer and a flavoring agent. In some embodiments, the formulation of the pharmaceutical composition is selected depending on the route of administration of the one or more N-glycosylation inhibitors to the subject.

[0054] In some embodiments, a pharmaceutical composition for preventing, alleviating and / or treating stroke comprises an effective amount of one or more N-glycosylation inhibitors administered to a subject in need thereof, the pharmaceutical composition being formulated with the one or more N-glycosylation inhibitors dissolved in water for oral administration. In some embodiments, the one or more N-glycosylation inhibitors are formulated to be dissolved in water at a concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.075%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45% or 50%. In some embodiments, the pharmaceutical composition for preventing, reducing and / or treating stroke comprises ManN dissolved in purified water. In some embodiments, ManN is dissolved in purified water at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45% or 50%. In some embodiments, ManN is dissolved in purified water at a concentration of about 20%. In some embodiments, a pharmaceutical composition for preventing, alleviating and / or treating stroke comprising an effective amount of one or more N-glycosylation inhibitors administered to a subject in need thereof comprises a pharmaceutical solution of 20% ManN dissolved in water. In some embodiments, a pharmaceutical composition for preventing, alleviating and / or treating stroke is used in a method comprising administering a therapeutically effective amount of a pharmaceutical solution to a subject in need thereof, the pharmaceutical solution comprising about 20% ManN dissolved in water. In some embodiments, a pharmaceutical composition for preventing, alleviating and / or treating stroke is used in a method comprising administering a therapeutically effective amount of a pharmaceutical solution to a subject in need thereof, wherein the pharmaceutical solution consists essentially of about 20% ManN dissolved in water. In some embodiments, a pharmaceutical composition for preventing, alleviating and / or treating stroke is used in a method comprising administering a therapeutically effective amount of a pharmaceutical solution to a subject in need thereof, wherein the pharmaceutical solution consists of about 20% ManN dissolved in water. In some embodiments, the pharmaceutical solution for the method for preventing, alleviating and / or treating stroke comprises a therapeutically effective amount of ManN dissolved in a beverage for oral administration. In some embodiments, a therapeutically effective amount of ManN is dissolved in water for oral administration for use in a method for preventing, alleviating and / or treating stroke.In some embodiments, a therapeutically effective amount of ManN is dissolved in milk for oral administration for use in methods of preventing, alleviating and / or treating stroke. In some embodiments, a therapeutically effective amount of ManN is dissolved in a protein nutritional shake (e.g., ) for oral administration, for use in methods of preventing, alleviating and / or treating stroke. In some embodiments, a therapeutically effective amount of ManN is dissolved in a balanced electrolyte beverage (e.g., or Pedialyte Plus) for oral administration for use in a method for preventing, alleviating and / or treating stroke.

[0055] method

[0056] Treatments for stroke

[0057] Described herein is a method for treating stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castaneospermide (Cas), or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN and Kif. In some embodiments, the N-glycosylation inhibitor comprises ManN and Cas. In some embodiments, the N-glycosylation inhibitor comprises Kif and Cas. In some embodiments, the N-glycosylation inhibitor comprises ManN, Kif, and Cas. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the N-glycosylation inhibitor comprises Kif. In some embodiments, the N-glycosylation inhibitor comprises Cas. In some embodiments, the method for treating stroke comprises administering an effective amount of one or more N-glycosylation inhibitors to a subject in need thereof. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, or castaneospermide (Cas). In some embodiments, the N-glycosylation inhibitor is hexosamine D-mannosamine (ManN), kif or castaneospermine (Cas). In some embodiments, the one or more N-glycosylation inhibitors are hexosamine D-mannosamine (ManN), kif or castaneospermine (Cas) or any combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the N-glycosylation inhibitor comprises Kif. In some embodiments, the N-glycosylation inhibitor comprises Cas. In some embodiments, the N-glycosylation inhibitor is ManN. In some embodiments, the N-glycosylation inhibitor is Kif. In some embodiments, the N-glycosylation inhibitor is Cas. In some embodiments, the N-glycosylation inhibitor consists essentially of ManN. In some embodiments, the N-glycosylation inhibitor consists essentially of Kif. In some embodiments, the N-glycosylation inhibitor consists essentially of Cas. In some embodiments, the one or more N-glycosylation inhibitors comprise a compound having a chemical structure similar to ManN. In some embodiments, Table 1 lists the one or more N-glycosylation inhibitors having a chemical structure similar to ManN. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises ManN. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises ManN and one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to a subject comprises one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor is formulated in a pharmaceutical composition.

[0058] In some embodiments, the subject in need of administration of an N-glycosylation inhibitor is a mammal. In some embodiments, the subject is a rodent. In some embodiments, the subject is of the genus Rat. In some embodiments, the subject is of the genus Musculus. In some embodiments, the subject is of the genus Canis. In some embodiments, the subject is of the genus Feline. In some embodiments, the subject is of the genus Equus. In some embodiments, the subject is a human.

[0059] In some embodiments, the method for treating stroke is used after a subject has had a stroke. In some embodiments, the subject has experienced an ischemic stroke. In some embodiments, the ischemic stroke comprises a thrombotic stroke or an embolic stroke. In some embodiments, the subject has experienced a hemorrhagic stroke. In some embodiments, the hemorrhagic stroke comprises a subarachnoid hemorrhage or an intracerebral hemorrhage. In some embodiments, the subject has experienced a brainstem stroke. In some embodiments, the subject has experienced a transient ischemic attack (TIA). In some embodiments, the subject has experienced a cryptogenic stroke. In some embodiments, the subject has been diagnosed with a stroke. In some embodiments, it is assumed that the subject has suffered from a stroke. In some embodiments, the subject is suspected of having suffered from a stroke. In some embodiments, the subject has experienced one or more transient ischemic attacks. In some embodiments, the one or more transient ischemic attacks have occurred before the stroke event treated by the subject. TIA may last for a short period of time, including no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 minutes. TIA may temporarily interrupt the blood supply to a certain area of ​​the subject's brain. In some embodiments, the subject suddenly experiences or has experienced the onset of TIA symptoms. In some embodiments, the symptoms of the subject's TIA disappear after a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more than 30 minutes. In some embodiments, TIA does not cause permanent damage to the subject's brain. In some embodiments, one or more TIAs in the subject indicate a warning that the subject's risk of stroke is elevated. In some embodiments, based on the previous occurrence of one or more TIAs, a pharmaceutical composition comprising an N-glycosylation inhibitor is administered to the subject.

[0060] In some embodiments described herein are methods for treating stroke, wherein a pharmaceutical composition comprising an effective amount of an N-glycosylation inhibitor is administered by various routes of administration. In some embodiments, the pharmaceutical composition for treating stroke is administered intravenously. In some embodiments, the pharmaceutical composition for treating stroke is administered intrathecally. In some embodiments, the pharmaceutical composition for treating stroke is administered intraperitoneally. In some embodiments, the pharmaceutical composition for treating stroke is administered to the cerebrospinal fluid. In some embodiments, the pharmaceutical composition for treating stroke is administered to the choroid plexus. In some embodiments, the pharmaceutical composition for treating stroke is administered locally near the ischemic site. In some embodiments, the pharmaceutical composition for treating stroke is administered to a blood vessel that directly supplies blood to the brain. In some embodiments, the pharmaceutical composition for treating stroke is administered to the carotid artery.

[0061] In some embodiments described herein are methods for treating stroke, wherein a pharmaceutical composition comprising an effective amount of an N-glycosylation inhibitor is administered according to a treatment regimen. In some embodiments, the treatment regimen comprises a single-dose treatment regimen. In some embodiments, a single-dose treatment regimen comprises a pre-treatment regimen. In some embodiments, a single-dose treatment regimen comprises a concurrent treatment regimen. In some embodiments, a single-dose treatment regimen comprises a post-treatment regimen. In some embodiments, the treatment regimen comprises a multiple-dose treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a pre-treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a concurrent treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a post-treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a pre-treatment and a post-treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a pre-treatment and a concurrent regimen. In some embodiments, a multiple-dose treatment regimen comprises a concurrent and post-treatment regimen. In some embodiments, a multiple-dose treatment regimen comprises a pre-treatment, concurrent, and post-treatment regimen. In some embodiments, the multiple treatment regimen comprises 2 or more treatments, 3 or more treatments, 4 or more treatments, 5 or more treatments, 6 or more treatments, 7 or more treatments, 8 or more treatments, 9 or more treatments, 10 or more treatments, 12 or more treatments, 15 or more treatments, 20 or more treatments, or 25 or more treatments.In some embodiments, the multiple treatment regimen will comprise 1-2 treatments, 1-3 treatments, 1-4 treatments, 1-5 treatments, 1-6 treatments, 1-7 treatments, 1-8 treatments, 1-9 treatments, 1-10 treatments, 1-12 treatments, 1-15 treatments, 1-20 treatments, 2-3 treatments, 2-4 treatments, 2-5 treatments, 2-6 treatments, 2-7 treatments, 2-8 treatments, 2-9 treatments, 2-10 treatments, 2-12 treatments, 2-15 treatments, 2-25 treatments, 3-4 treatments, 3-5 treatments, 3-6 treatments, 3-7 treatments, 3-8 treatments, 3-9 treatments, 3-10 treatments, 3-14 treatments, 3-18 treatments, 3- 30 treatments, 4-5 treatments, 4-6 treatments, 4-7 treatments, 4-8 treatments, 4-9 treatments, 4-10 treatments, 4-15 treatments, 5-6 treatments, 5-7 treatments, 5-8 treatments, 5-10 treatments, 5-14 treatments, 5-23 treatments, 5-34 treatments, 6-7 treatments, 6-8 treatments, 6-9 treatments, 6-10 treatments, 6-12 treatments, 6-17 treatments, 6-22 treatments, 6-30 treatments, 7-8 treatments, 7-9 treatments, 7-12 treatments, 7-18 treatments, 9-18 treatments, 10-20 treatments, 12-25 treatments, 14-35 treatments, 15-50 treatments, or 20-100 treatments. In some embodiments, the pretreatment regimen comprises one or more treatments, comprising administering a pharmaceutical composition described herein to a subject, wherein pretreatment is started after determining that the subject has an increased risk of stroke. In some embodiments, the pretreatment regimen comprises one or more treatments, comprising administering a pharmaceutical composition described herein to a subject, wherein pretreatment is started after determining that the subject has an increased risk of recurrent stroke. In some embodiments, the pretreatment regimen comprises one or more treatments, comprising administering a pharmaceutical composition described herein to a subject, wherein pretreatment is started after one or more TIAs of the subject. In some embodiments, the pretreatment regimen comprises one or more pretreatments, comprising administering a pharmaceutical composition described herein to a subject, wherein the one or more pretreatments are started at least about 48 hours, 44 hours, 40 hours, 36 hours, 32 hours, 28 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour before a stroke event.In some embodiments, the concurrent regimen comprises one or more concurrent treatments comprising administering to a subject a pharmaceutical composition as described herein, wherein the one or more concurrent treatments are initiated within about 0.1 hour, 0.2 hour, 0.3 hour, 0.4 hour, 0.5 hour, 0.75 hour, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, or 48 hours after the estimated start time of the subject's stroke event. In some embodiments, the post-treatment regimen comprises one or more post-treatments comprising administering to the subject a pharmaceutical composition as described herein, wherein the one or more post-treatments are about 0.1 hour, 0.2 hour, 0.3 hour, 0.4 hour, 0.5 hour, 0.75 hour, 1 hour, 1.25 hour, 1.5 hour, 1.75 hour, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours after restoration of substantially normal blood flow to the subject's brain area affected by the recent stroke event. , 5.5 hours, 6 hours, 6.5 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 46 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 84 hours, 96 hours, 108 hours or 120 hours. In some embodiments, the method comprises the subject receiving a pre-treatment regimen and a post-treatment regimen. In some embodiments, the method comprises the subject receiving a pre-treatment regimen and a concurrent treatment regimen. In some embodiments, the method comprises the subject receiving a pre-treatment regimen, a concurrent treatment regimen, and a post-treatment regimen. In some embodiments, the method comprises the subject receiving a concurrent treatment regimen and a post-treatment regimen. In some embodiments, the method comprises the subject receiving a post-treatment regimen.In some embodiments, the pre-treatment regimen, concurrent treatment regimen, or post-treatment regimen comprises administering a pharmaceutical composition described herein to a subject at a determined dose at a frequency of about 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours. In some embodiments, the post-treatment regimen comprises administering to the subject a pharmaceutical composition described herein at a determined dose at a frequency of about once every other day. In some embodiments, the post-treatment regimen comprises administering to the subject a pharmaceutical composition described herein at a determined dose at a frequency of about once every other day until one or more stroke symptoms of the subject show improvement or complete resolution. In some embodiments, the method comprises administering to a subject a pharmaceutical composition described herein within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 84 hours, 96 hours, or 120 hours after the ischemic event. In some embodiments, the method comprises administering to a subject a pharmaceutical composition described herein within about 1 hour to about 12 hours after the ischemic event. In some embodiments, the method comprises administering to a subject a pharmaceutical composition described herein within about 2 hours to about 6 hours after the ischemic event. In some embodiments, the method comprises starting to administer to the subject a pharmaceutical composition described herein within about 2 hours to about 48 hours after the ischemic event. In some embodiments, the method comprises starting to administer to the subject a pharmaceutical composition described herein within about 2 hours to about 24 hours after the ischemic event.

[0062] In some embodiments described herein, the method of a single-dose treatment regimen comprises administering a therapeutically effective amount of one or more N-glycosylation inhibitors described herein. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN, Kif, Cas, or any combination thereof. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN. In some embodiments, the one or more N-glycosylation inhibitors comprise Kif. In some embodiments, the one or more N-glycosylation inhibitors comprise Cas. In some embodiments, the one or more N-glycosylation inhibitors consist essentially of ManN. In some embodiments, the one or more N-glycosylation inhibitors are listed in Table 1.In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 111 mg, 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40mg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 1g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 2 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 3 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 4 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 5 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 6 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 7 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 8 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 9 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 10 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 11 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 12 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 13 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 14 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 18 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 22 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 27 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 35 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 111 mg, 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 35g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 20 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 18g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 12g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 10g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 8g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 6g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 4g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 2g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 1g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 40mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1 mg and about 10 mg, about 3 mg and about 10 mg, about 5 mg and about 15 mg, about 10 mg and about 20 mg, about 15 mg and about 45 mg, about 20 mg and about 55 mg, about 25 mg and about 70 mg, about 35 mg and about 75 mg, about 40 mg and about 105 mg, about 45 mg and about 85 mg, about 50 mg and about 80 mg, about 55 about 125mg, about 60mg and about 150mg, about 65mg and about 185mg, about 70mg and about 125mg, about 75mg and about 200mg, about 80mg and about 225mg, about 85mg and about 300mg, about 90mg and about 180mg, about 100mg and about 150mg, about 100mg and about 225mg, about 100mg and about 350mg, about 100mg and about 500mg, about 110mg and about 200mg, about 110mg and about 275mg, about 150mg and about 375mg , about 175mg and about 500mg, about 190mg and about 315mg, about 225mg and about 600mg, about 250mg and about 700mg, about 250mg and about 500mg, about 260mg and about 800mg, about 275mg and about 600mg, about 300mg and about 900mg, about 350mg and about 750mg, about 400mg and about 2g, about 425mg and about 1.1g, about 450mg and about 1.5g, about 500mg and about 850mg, about 525mg and about 1.3g, about 600 mg and about 2g, about 700mg and about 3g, about 800mg and about 4g, about 900mg and about 5g, about 1g and about 3g, about 1.1g and about 6g, about 1.2g and about 8g, about 1.4g and about 10g, about 2.5g and about 7.5g, about 3g and about 12g, about 4g and about 15g, about 4.5g and about 16g, about 5g and about 20g, about 6g and about 18g, about 7g and about 21g, about 8g and about 23g, about 9g and about 25g, about 10g and about 30g, about 12g and about 35g, about 14g and about 28g, Between about 15 g and about 45 g, about 18 g and about 36 g, about 20 g and about 50 g, about 25 g and about 65 g, about 30 g and about 70 g, about 35 g and about 80 g, about 40 g and about 100 g, about 45 g and about 115 g, about 50 g and about 135 g, about 20 g and about 300 g, about 20 g and about 500 g, about 25 g and about 800 g, about 70 g and about 385 g, about 75 g and about 450 g, about 85 g and about 600 g, about 100 g and about 800 g, about 200 g and about 1 kg, or about 475 g and about 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 20 mg and about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 30g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 20g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 9g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 15g. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for treating a stroke in a single treatment regimen. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for reducing one or more symptoms of a stroke in a single treatment regimen. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for preventing a stroke in a single treatment regimen.

[0063] In some embodiments described herein, the method of the multiple dose treatment regimen comprises administering a therapeutically effective amount of the N-glycosylation inhibitor of at least about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40mg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 1g. In some embodiments, the effective amount of an N-glycosylation inhibitor is at least about 2 g. In some embodiments, the effective amount of an N-glycosylation inhibitor is at least about 3 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 4 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 5 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 6 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 7 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 8 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 9 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 10 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 11 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 12 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 13 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 14 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 18 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 22 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 27 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 35 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 111 mg, 15mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5 g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 32g , 34g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120g, 125g, 130g, 135g, 140g, 145g, 150g, 155g, 160g, 165g, 170g, 175g, 180g, 185g, 190g, 195g, 200g, 215g, 230g, 245g, 260g, 275g, 300g, 325g, 350g, 375g, 400g, 430g, 460g, 500g, 530g, 560g, 600g, 630g, 660g, 700g, 750g, 800g, 850g, 900g, 950g or 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 35g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor does not exceed about 20 g.In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 18g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 12g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 10g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 8g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 6g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 4g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 2g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 1g. In some embodiments, the effective amount of the N-glycosylation inhibitor is no more than about 40mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1 mg and about 10 mg, about 3 mg and about 10 mg, about 5 mg and about 15 mg, about 10 mg and about 20 mg, about 15 mg and about 45 mg, about 20 mg and about 55 mg, about 25 mg and about 70 mg, about 35 mg and about 75 mg, about 40 mg and about 105 mg, about 45 mg and about 85 mg, about 50 mg and about 80 mg, about 55 about 125mg, about 60mg and about 150mg, about 65mg and about 185mg, about 70mg and about 125mg, about 75mg and about 200mg, about 80mg and about 225mg, about 85mg and about 300mg, about 90mg and about 180mg, about 100mg and about 150mg, about 100mg and about 225mg, about 100mg and about 350mg, about 100mg and about 500mg, about 110mg and about 200mg, about 110mg and about 275mg, about 150mg and about 375mg , about 175mg and about 500mg, about 190mg and about 315mg, about 225mg and about 600mg, about 250mg and about 700mg, about 250mg and about 500mg, about 260mg and about 800mg, about 275mg and about 600mg, about 300mg and about 900mg, about 350mg and about 750mg, about 400mg and about 2g, about 425mg and about 1.1g, about 450mg and about 1.5g, about 500mg and about 850mg, about 525mg and about 1.3g, about 600 mg and about 2g, about 700mg and about 3g, about 800mg and about 4g, about 900mg and about 5g, about 1g and about 3g, about 1.1g and about 6g, about 1.2g and about 8g, about 1.4g and about 10g, about 2.5g and about 7.5g, about 3g and about 12g, about 4g and about 15g, about 4.5g and about 16g, about 5g and about 20g, about 6g and about 18g, about 7g and about 21g, about 8g and about 23g, about 9g and about 25g, about 10g and about 30g, about 12g and about 35g, about 14g and about 28g, Between about 15 g and about 45 g, about 18 g and about 36 g, about 20 g and about 50 g, about 25 g and about 65 g, about 30 g and about 70 g, about 35 g and about 80 g, about 40 g and about 100 g, about 45 g and about 115 g, about 50 g and about 135 g, about 20 g and about 300 g, about 20 g and about 500 g, about 25 g and about 800 g, about 70 g and about 385 g, about 75 g and about 450 g, about 85 g and about 600 g, about 100 g and about 800 g, about 200 g and about 1 kg, or about 475 g and about 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 20 mg and about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 1g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 30g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 3g and about 20g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 15g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 5g and about 9g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 40g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 25g. In some embodiments, the effective amount of the N-glycosylation inhibitor is between about 10g and about 15g. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for treating a stroke in multiple treatment regimens. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for alleviating one or more symptoms of a stroke in multiple treatment regimens. In some embodiments, this paragraph lists an effective amount of an N-glycosylation inhibitor administered to a subject for preventing a stroke in multiple treatment regimens.

[0064] In some embodiments described herein are methods comprising administering a formulation of an N-glycosylation inhibitor at a specific dose per subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 10 mg to about 100 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 20 mg to about 200 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 25 mg to about 250 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 35 mg to about 300 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 20 mg to about 400 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 40 mg to about 500 mg / kg subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor at a dose of about 50 mg to about 200 mg / kg subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 55 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 85 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 125 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 215 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 325 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 425 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 250 mg to about 700 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 315 mg to about 750 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 355 mg to about 800 mg / kg of subject body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 435 mg to about 620 mg / kg of subject body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 1.5 mg to about 2.0 mg / gram of subject body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 50 mg to about 600 mg / kg of subject body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 75 mg to about 300 mg / kg of subject body weight.In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 100 mg to about 200 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 130 mg to about 163 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 130 mg to about 300 mg / kg of the subject's body weight. In some embodiments, the dosage of the N-glycosylation inhibitor included in the formulation is about 130 mg to about 660 mg / kg of the subject's body weight. Described herein is a method for preventing, alleviating or treating a stroke in a subject, the method comprising administering a pharmaceutical composition described herein with a specific dose of one or more N-glycosylation inhibitors in a single treatment regimen or multiple treatment regimens. In some embodiments, the dose administered to the subject is determined by dose selection. In some embodiments, the therapeutically effective dose is determined by experimental evaluation using animals. In some embodiments, the animal is a mouse, rat, rabbit, guinea pig, cat, dog or pig. In some embodiments, the dose selection is determined by calculating the human equivalent dose (HED). In some embodiments, the dose administered to the subject is a human equivalent dose (HED). In some embodiments, HED is calculated according to the factor-by-factor dosing method. In some embodiments, HED is calculated based on a body surface area comparison between species. In some embodiments, HED is calculated based on a body surface area comparison between mice and humans. In some embodiments, HED is calculated based on a body surface area comparison between mice and humans, where the animal equivalent dose (AED) in mg / kg body weight is divided by the HED in mg / kg body weight, i.e., AED divided by a factor of 12.3. In some embodiments, the factor-by-factor dosing method uses the drug no observed adverse effect level (NOAEL) from preclinical toxicology studies to estimate HED. In some embodiments, dose selection is based on the minimum risk of human toxicity. In some embodiments, dose selection is based on the minimum pharmacological activity in humans. In some embodiments, dose selection is based on existing pharmacokinetic data of another drug of the same pharmacological class. In some embodiments, another drug of the same pharmacological class is hexosamine. In some embodiments, hexosamine is mannosamine. In some embodiments, hexosamine is fructosamine. In some embodiments, hexosamine is galactosamine. In some embodiments, hexosamine is glucosamine. In some embodiments, the mannosamine is N-acetyl-D-mannosamine (ManNAc), diazirine-derivatized. sweet Glucosamine(ManNDAz), tetraacetylated N-azidoacetyl-d-mannosamine (ManNAcAz), N-glycolyl-D-mannosamine, 2-N-acetyl-6-O-acetyl-D-mannosamine, N-levulinyl mannosamine (Man2NLev), N-azido-acetyl mannosamine (Man2NAz) or L-mannosamine. In some embodiments, hexosamine is listed in Table 1. In some embodiments, another drug of the same pharmacological class is listed in Table 1. In some embodiments, the dose administered to the subject is determined based on drug activity rather than dose scaling between species. In some embodiments, the dose administered to the subject is determined by following one or more guidelines described in Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. Journal of Basic Clinical Pharmacy (JBasic Clin Pharm). 2016 March; 7 (2): 27-31, the contents of which are incorporated herein by reference.

[0065] Stroke Prevention

[0066] Described herein is a method for treating stroke, the method comprising administering an effective amount of one or more N-glycosylation inhibitors to a subject in need thereof. In some embodiments, the one or more N-glycosylation inhibitors comprise hexosamine D-mannosamine (ManN), kif, castaneospermine (Cas), or any combination thereof. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN and Kif. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN and Cas. In some embodiments, the one or more N-glycosylation inhibitors comprise Kif and Cas. In some embodiments, the one or more N-glycosylation inhibitors comprise ManN, Kif, and Cas. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the N-glycosylation inhibitor comprises Kif. In some embodiments, the N-glycosylation inhibitor comprises Cas. In some embodiments, the N-glycosylation inhibitor consists essentially of ManN. In some embodiments, the one or more N-glycosylation inhibitors are listed in Table 1.

[0067] In some embodiments, the subject in need of administration of an N-glycosylation inhibitor is a mammal. In some embodiments, the subject is a rodent. In some embodiments, the subject is of the genus Rat. In some embodiments, the subject is of the genus Musculus. In some embodiments, the subject is of the genus Canis. In some embodiments, the subject is of the genus Feline. In some embodiments, the subject is of the genus Equus. In some embodiments, the subject is a human.

[0068] In some embodiments described herein, the method of preventing stroke comprises a single-dose treatment regimen. In some embodiments described herein, the method of preventing stroke comprises a multiple-dose treatment regimen.

[0069] In some embodiments described herein, the method of preventing stroke comprises reducing the risk of recurrent stroke in a subject. In some embodiments, the subject has previously experienced ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack, or cryptogenic stroke. In some embodiments, the risk of recurrent stroke comprises the risk of ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack, or cryptogenic stroke.

[0070] Stroke risk markers

[0071] In some embodiments described herein, the method of preventing stroke comprises measuring C-reactive protein (CRP) in the subject's blood. In some embodiments, the measurement of CRP in the subject's blood is reduced compared to a previous measurement of the subject's blood C-reactive protein (CRP) level. In some embodiments, the measurement of CRP in the subject's blood is reduced to below about 10 mg / L.

[0072] Symptoms of a stroke

[0073] In some embodiments, it is a method for preventing, alleviating or treating a stroke in a subject in need, wherein a subject is selected to administer a pharmaceutical composition described herein. In some embodiments, a subject is selected due to having previously suffered from one or more TIAs. In some embodiments, after having diagnosed one or more TIAs, a subject is selected to start treatment. In some embodiments, a subject experiencing one or more symptoms common during a TIA is used to diagnose the subject as suffering from one or more TIAs. In some embodiments, the one or more symptoms common during a TIA include muscle weakness, facial numbness, facial paralysis, limb paralysis, paralysis on one side of the body, slurred speech, confusion of speech, difficulty understanding others, sudden blindness in one or both eyes, diplopia, vertigo, confusion of consciousness, lack of mental alertness, loss of balance or loss of coordination or any combination thereof. In some embodiments, a subject is selected after observing signs of carotid artery disease in a subject, which is a common cause of ischemic stroke. In some embodiments, a subject is selected after diagnosing a stroke. In some embodiments, a computed tomography (CT) scan is used to identify cerebral hemorrhage, thereby indicating the diagnosis of a stroke. In some embodiments, a CT scan is used to identify damage in the brain, thereby indicating the diagnosis of a stroke. In some embodiments, magnetic resonance imaging (MRI) scanning is used to identify damage in the brain, thereby indicating the diagnosis of stroke. In some embodiments, imaging tests that reveal narrowing of neck blood vessels, aneurysms or cerebral vascular entanglements are used for the diagnosis of stroke. In some embodiments, a subject experiencing one or more symptoms common during a stroke selects a subject for the treatment methods described herein. In some embodiments, the one or more symptoms common during a stroke include muscle weakness, facial numbness, facial paralysis, limb paralysis, paralysis on one side of the body, slurred speech, confusion of speech, difficulty understanding others, sudden blindness of one or both eyes, diplopia, dizziness, confusion, lack of mental alertness, loss of balance or loss of coordination or any combination thereof. In some embodiments, the method is included in reducing the mortality rate of the subject after administration. In some embodiments, compared with the use of anticoagulants as a monotherapy in control subjects, tissue plasminogen activator (TPA) as a monotherapy, antiplatelet drugs as a monotherapy, or antihypertensive drugs as a monotherapy, the method includes reducing the mortality rate of the subject. In some embodiments, the method comprises reducing the mortality rate of the subject compared to control subjects administered an anticoagulant, a tissue plasminogen activator, an antiplatelet drug, or an antihypertensive drug, or any combination thereof. In some embodiments, the method comprises reducing the mortality rate of the subject compared to control subjects not receiving treatment or receiving vehicle control therapy. In some embodiments, the method comprises improving one or more symptoms of stroke in the subject after administration. In some embodiments, the method comprises reducing the area or size of damaged neural tissue in the brain of the subject after administration.In some embodiments, the method comprises rescuing hypoxic damage to one or more regions of the brain of the subject after administration. In some embodiments, the method comprises reversing stroke-induced brain damage in the subject after administration. In some embodiments, the method comprises reversing stroke-induced memory impairment in the subject after administration. In some embodiments, the method comprises reversing stroke-induced voluntary movement disorders in the subject after administration. In some embodiments, the method comprises reversing stroke-induced language disorders in the subject after administration. In some embodiments, the method comprises improving cognitive abilities in the subject after administration. In some embodiments, the method comprises improving learning in the subject after administration. In some embodiments, the method comprises improving memory in the subject after administration. In some embodiments, the method comprises improving verbal or non-verbal communication in the subject after administration. In some embodiments, the method comprises improving mobility in the subject after administration. In some embodiments, the method comprises reversing stroke-induced brain damage in the subject after administration. In some embodiments, the method comprises reversing stroke-induced cognitive and / or memory impairment in the subject after administration. In some embodiments, the method comprises reversing stroke-induced voluntary muscle control disorders in the subject after administration. In some embodiments, the method comprises reversing infarction-induced brain damage in the subject after administration. In some embodiments, the method comprises reducing hemorrhagic transformation of ischemic events in the subject after administration. In some embodiments, the reduction in hemorrhagic transformation is determined by measuring the subject's hemoglobin level compared to administration of the anticoagulant alone as a monotherapy.

[0074] Reduce ongoing ischemic events

[0075] Methods of reducing persistent ischemic events affecting the brain are described herein. In some embodiments, the method comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof while experiencing an ischemic event. In some embodiments, the ischemic event comprises that the subject has experienced an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke. In some embodiments described herein, an effective amount of an N-glycosylation inhibitor is administered orally, intravenously, intrathecally, and / or intraperitoneally.

[0076] Neurologic assessment and restoration of blood flow

[0077] Described herein is a method for alleviating a persistent ischemic event that affects the brain. In some embodiments, a neurological assessment is performed on a subject during an ischemic event that affects the brain. In some embodiments, a neurological assessment is performed on a subject after an ischemic event affects the brain. In some embodiments, multiple neurological assessments are performed on a subject during an ischemic event that affects the brain. In some embodiments, multiple neurological assessments are performed on a subject after an ischemic event has affected the brain. In some embodiments, neurological assessments performed at different times are compared. In some embodiments described herein, compared to a previous assessment, a neurological assessment of acute stroke in a subject is improved. In some embodiments described herein, blood flow to the brain of a subject is improved. In some embodiments described herein, blood flow to the brain is assessed by transcranial Doppler ultrasound.

[0078] Compounds with chemical structures similar to ManN

[0079] In some embodiments of the methods for preventing, alleviating or treating stroke described herein, one or more compounds comprising a chemical structure similar to ManN are administered to a subject in need. In some embodiments, the one or more compounds comprising a chemical structure similar to ManN are administered in combination with ManN. In some embodiments, the one or more compounds comprising a chemical structure similar to ManN are administered in combination with Kif. In some embodiments, the one or more compounds comprising a chemical structure similar to ManN are administered in combination with Cas. In some embodiments, the one or more compounds comprising a chemical structure similar to ManN act as an N-glycosylation inhibitor and are administered to a subject. In some embodiments, Table 1 lists the one or more compounds comprising a chemical structure similar to ManN. In some embodiments, the compounds listed in Table 1 are administered to a subject in the methods for preventing, alleviating or treating stroke described herein.

[0080] Table 1: Compounds with chemical structures similar to ManN

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Combination with known stroke treatments

[0106] In some embodiments, the method of preventing, alleviating or treating stroke in a subject in need thereof comprises administering a pharmaceutical composition as described herein, further comprising administering to the subject one or more known stroke treatments. In some embodiments, the one or more known stroke treatments are anticoagulant drugs, tissue plasminogen activator drugs, antiplatelet drugs, or any combination thereof. In some embodiments, the anticoagulant drug is rivaroxaban, ), dabigatran ), apixaban ) or edoxaban ( In some embodiments, the tissue plasminogen activator drug is alteplase ( ), reteplase ) or tenecteplase In some embodiments, the antiplatelet drug is acetylsalicylic acid (ASA), clopidogrel, ASA, and extended release dipyridamole or ticagrelor administered at 75-325 mg / day.

[0107] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0108] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the present disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary methods, devices, and materials are described herein.

[0109] The present disclosure provides a pharmaceutical composition and method for treating an ischemic condition in a subject, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need thereof. Since ManN is converted into ManN-6-phosphate (ManN-6p) in vivo, the present disclosure provides the use of such metabolic precursors and derivatives.

[0110] ManN is a hexosamine that has the ability to inhibit protein post-translational modifications, activate stress pathways, and has additive effects with VEGF in promoting endothelial cell (EC) proliferation and angiogenesis. The effects of ManN on EC and angiogenesis have not been previously reported. Without being bound by theory, the use of well-known glycosylation inhibitors together with ManN may create a link between changes in the glycosylation pattern of mammalian EC and angiogenesis. The effects of ManN on endothelial cells may be independent of the activation of VEGFR2.

[0111] ManN is a bacterial wall component discovered in the 1960s and accounts for 5-10% of capsular polysaccharides. The related N-acetylmannosamine is considered to be an intermediate in the biosynthesis of sialic acid. Over the years, various effects of ManN on enzymes, growth factor-mediated signaling, protein stability and cell viability have been recorded. Most of these effects are not unique to ManN and may be caused by other hexosamines. In addition, it requires high concentrations. ManN is reported to have anti-tumor properties to stimulate osteogenic differentiation and protect articular cartilage. Recently, ManN has been used as an intermediate for modifying various molecules / nanoparticles and synthesizing non-natural ManNAc analogs for expressing thiols on cell surface sialic acid to facilitate high-throughput screening. However, to date, the effects of ManN on EC have not been described.

[0112] It has been previously reported that ManN may affect the formation of lipid-linked oligosaccharides (LLO) in MDCK cells by inhibiting α-1,2-mannosyltransferase. After ManN treatment, the main oligosaccharides associated with dolichol are Man5G1cNAc2 and Man6GlcNAc2, rather than Glc3Man9G1cNAc2, which is usually found in MDCK cells. In addition, ManN has been reported to alter the biosynthesis of protein GPI and the production of hybrid glycans in the ER. However, none of the angiogenesis-related proteins previously examined have GPI anchors. Without being bound by theory, the reduction of Man-9 can be a direct result of inhibiting the synthesis of LLO donors, i.e., Glc3Man9G1cNAc20PP-Dol formation, which is then transferred from the dolichol donor to the polypeptide. In some embodiments, Man-5 can be significantly increased within 24 hours by treating cells with 40 μM ManN. ManN may not affect α-mannosidase in the ER.

[0113] Activation of PI3K-AKT, PLCy-ERK and p38 is associated with EC survival, proliferation and migration mediated by VEGFR2. Other cell metabolic stress sensors, such as AMPK (AMP-activated protein kinase), can also confer stress adaptation ability and promote EC survival through eNOS. Without being bound by theory, ERK, AKT, mTOR, AMPKa, eNOS and ACC activation are common phenomena for hexosamine and mannose. However, activation of JNK / c-Jun and UPR pathways in BCEC is unique to ManN and glycosylation inhibitors. Glycosylation is required for correct protein folding in ER. It is reported that there is a connection between LLO inhibition and UPR activation. In fact, despite the complexity of ManN action, LLO inhibition followed by UPR activation seems to be a reasonable explanation for the reported ManN effect.

[0114] ECs are able to respond to acute / mild ER stress caused by glycosylation inhibition by activating the UPR pathway. The UPR detects misfolded proteins that accumulate in the ER and initiates a response that maintains cellular homeostasis by inducing the major ER chaperone Bip. BiP binds to hydrophobic patches exposed on nascent or incompletely folded, usually non-glycosylated proteins. ManN exhibits a strong induction of Bip expression relative to hexosamine. The glycosylation inhibitors Kif and Cas may have similar effects on stress pathway activation.

[0115] Glycosylation inhibition is considered a new pharmacological strategy to target metabolic pathways essential for excessive angiogenesis in various pathological conditions, and glycosylation inhibitors are expected to have anti-angiogenic and anti-metastatic properties. Glycosylation has been shown to be involved in cellular stress responses and compensatory angiogenesis to blockade of VEGF-VEGFR2 signaling. It has been previously reported that stress-induced O-G1cN acetylation promotes the survival of multiple cell types in response to DNA damage, ER stress, glucose deprivation, and hypoxia. Without being bound by theory, glycosylation inhibition may be associated with angiogenesis promotion, and inhibition of glycosylation in the tumor microenvironment may stimulate rather than inhibit tumor angiogenesis.

[0116] In some embodiments, ManN can be used to promote angiogenesis in a mouse skin injury model, accompanied by accelerated wound closure. In some embodiments, ManN can be used to stimulate angiogenesis and blood flow restoration in ischemic hind limbs of mice. The combination of ManN or other glycosylation inhibitors with VEGF-A may be more advantageous than monotherapy in the treatment of ischemic conditions. The lack of a direct permeability-enhancing effect of ManN may result in a reduction in edematous tissue. In this context, pulmonary endothelial damage is a central pathogenic event in respiratory failure associated with various infections, including SARS-CoV-2. Endothelial cell mitogens like ManN, which have no permeability effects, may help protect and stabilize blood vessels and thus limit tissue damage.

[0117] In some embodiments, intravitreal administration of ManN can be used to enhance retinal neovascularization, such as in therapeutic applications for ocular diseases. 10-15% of patients with moderate AMD progress to the neovascular form, while the remaining patients may develop geographic atrophy (GA). Previous studies have shown that the loss of choroidal capillaries is often detected in GA, which increases the possibility that regeneration / protection of choroidal capillaries may be a treatment strategy for GA. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN can be used in a method for treating dry age-related macular degeneration (AMD) and / or geographic atrophy (GA), or a combination thereof. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN can be used in a method for treating dry age-related macular degeneration (AMD), diabetic macular edema, macular edema caused by retinal vein occlusion, diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, diabetic retinal neovascularization, diabetic optic nerve neovascularization, familial exudative vitreoretinopathy, sickle cell disease, or a combination thereof. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN can be used in a method for treating wet age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV), degenerative (pathological) myopia, or a combination thereof. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN can be used in a method for treating wet age-related macular degeneration (AMD), choroidal neovascularization (CNV), polypoidal choroidal vasculopathy, degenerative (pathological) myopia, giant cell arteritis, or a combination thereof. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN can be used in a method for treating degenerative (pathological) myopia. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN slows the progression of an ocular disease. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN stops the progression of an ocular disease. The cessation or slowing of disease progression can be mediated by increasing retinal perfusion with no or minimal leakage and / or retinal vascular reconstruction with no or minimal leakage. The cessation or slowing of disease progression can be mediated by increasing choroidal perfusion with no or minimal leakage and / or choroidal vascular reconstruction with no or minimal leakage.

[0118] In some embodiments, the administration is effective to promote endothelial cell proliferation and angiogenesis in the subject. In some embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the method further comprises administering an effective amount of VEGF to a subject in need thereof.

[0119] In some embodiments, the ischemic condition is caused by disease or trauma. The present disclosure provides methods of treating a variety of conditions characterized by reduced perfusion, including but not limited to diabetic ulcers, macular degeneration, peripheral arterial disease (PAD), limb ischemia, brain or cerebral ischemia, and coronary ischemia.

[0120] In some embodiments, administration is performed orally, intravenously, intrathecally, or intraperitoneally.

[0121] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, comprising administering an effective amount of the hexosamine D-mannosamine (ManN) to a subject in need thereof.

[0122] In some embodiments, administration is effective to reduce ischemia in a subject. In some embodiments, ischemia may include cerebral ischemia. Administration can effectively prevent, reduce or treat conditions associated with cerebral ischemia, such as edema, ischemic stroke or infarction. In some embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the method further comprises administering an effective amount of VEGF to a subject in need thereof.

[0123] In some embodiments, the subject is in need of induction of angiogenesis because the ischemic condition is caused by disease or trauma.

[0124] In some embodiments, administration is performed orally, intravenously, intrathecally, or intraperitoneally.

[0125] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in cells, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to the cells.

[0126] In some embodiments, administration is performed in vivo. In some embodiments, administration is performed ex vivo. In some embodiments, administration is effective to stimulate EC proliferation and angiogenesis. In some embodiments, administration is effective to activate JNK and the unfolded protein response caused by ER stress.

[0127] In some embodiments, administration is effective to induce changes in N-glycan and O-glycan profiles. In embodiments, administration is effective to induce a decrease in the total oligomannosyl N-glycan content of Man6GlcNAc2 (Man-6), Man-8, and Man-9, induce the accumulation of Man-5 and Man-7, and reduce O-glycosylation after treatment with ManN, compared to untreated controls.

[0128] In contrast, in some embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting angiogenesis, including but not limited to methods for treating malignancies and intraocular neovascular disorders in a subject, comprising administering to a subject in need thereof an effective amount of a hexosamine D-mannosamine (ManN) inhibitor or reducing the amount of ManN available to the subject.

[0129] Unless otherwise indicated, the practice of the present disclosure will adopt conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology within the technical scope of the art. Such techniques are fully explained in the following documents, such as "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., ed., 1987 and regularly updated); PCR: polymerase chain reaction (Mullis et al., ed., 1994); "Remington: The Science and Practice of Pharmaceutical Sciences" (Remington, The Science and Practice of Pharmacy, 20th ed. (Lippincott, Williams & Wilkins, 2003); and Remington: The Science and Practice of Pharmacy, 22nd ed. (Pharmaceutical Press and Philadelphia College of Pharmacy at The University of Sciences, 2012).

[0130] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to cover the non-exclusive inclusion of the recited components, subject to any limitations expressly stated otherwise. For example, a pharmaceutical composition and / or method that "comprising" a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the pharmaceutical composition and / or method.

[0131] It should be understood that aspects and embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments. As used herein, the transitional phrases "consists of" and "consisting of" exclude any elements, steps, or components that are not specified. For example, "consists of" or "consisting of" used in a claim limits the claim to the components, materials, or steps specifically listed in the claim, except for impurities normally associated therewith (i.e., impurities within a given component). When the phrase "consists of" or "consisting of" appears in a clause of the body of a claim, rather than immediately following a preamble, the phrase "consists of" or "consisting of" is limited only to the elements (or components or steps) set forth in the clause; other elements (or components) are not excluded from the claim as a whole.

[0132] As used herein, the transitional phrases "consists essentially of" and "consisting essentially of" are used to define pharmaceutical compositions and / or methods that include materials, steps, features, components, or elements in addition to those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristics of the claimed subject matter. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of."

[0133] When introducing elements of the present disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0134] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean one or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0135] It should be understood that the description in range format is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the present disclosure. Therefore, the description of the range should be regarded as having clearly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, the description of a range such as 1 to 6 should be regarded as having specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, this applies. Values ​​or ranges can be expressed as "about" in this article, from "about" a specific value and / or to "about" another specific value. When expressing such values ​​or ranges, other disclosed embodiments include the listed specific values, from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment. It should be further understood that multiple values ​​are disclosed herein and that at each value is also disclosed herein as "about" that particular value in addition to the value itself. In some embodiments, "about" can be used to mean, for example, within 10% of the value, within 5% of the value, or within 2% of the value.

[0136] As used herein, "patient" or "subject" means a human or other mammalian subject to be treated.

[0137] As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition, wherein the composition comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be a combination of a pharmaceutically active agent and a carrier.

[0138] The term "combination" refers to a fixed combination in the form of a dosage unit or a kit for combined administration, wherein one or more active compounds and a combination partner (e.g., another drug explained below, also referred to as "therapeutic agent" or "adjuvant") can be administered independently at the same time or separately. In some cases, the combination partner shows collaboration, such as a synergistic effect. As used herein, the term "co-administration" or "combination administration" etc. means to cover the administration of a selected combination partner to a single subject (e.g., patient) in need, and is intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or simultaneously.

[0139] As used herein, the term "pharmaceutical composition" means a product that is mixed or combined with more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, such as a compound and a combination partner, are both administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, such as a compound and a combination partner, are administered to the patient simultaneously, concurrently or sequentially as separate entities without specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the patient. The latter also applies to mixture therapy, such as the administration of three or more active ingredients.

[0140] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, other generally recognized pharmacopeia, except for other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0141] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant and / or vehicle administered with the demethylating compound. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerol, propylene glycol or other synthetic solvents. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tonicity, such as sodium chloride or dextrose can also be carriers. Methods for producing pharmaceutical compositions in combination with carriers are known to those skilled in the art. In some embodiments, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th ed., (Lippincott Williams & Wilkins, 2003.) Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions is contemplated.

[0142] As used herein, "therapeutically effective amount" refers to the amount of a pharmaceutically active compound sufficient to treat or improve, or in some way alleviate the symptoms associated with a disease and medical condition. When used with reference to a method, the method is sufficient to effectively treat or improve, or in some way alleviate the symptoms associated with a disease or condition. For example, in terms of a disease, an effective amount is an amount sufficient to prevent or prevent the onset of disease; or if the disease pathology has already begun, it is sufficient to alleviate, improve, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. In any case, the effective amount can be administered in a single dose or divided doses. The phrase "therapeutically effective amount" also refers to the amount of an N-glycosylation inhibitor described herein that causes a measurable therapeutic response. The therapeutic response can be any response that a user of the method (e.g., a clinician) will identify as having an effective response to therapy, including improvement of one or more symptoms (e.g., one or more symptoms of stroke or other ischemic conditions) and alternative clinical markers (e.g., stroke biomarkers). The therapeutic response generally improves or inhibits one or more symptoms of a disease or condition (e.g., ischemic stroke, hemorrhagic stroke, or ischemic condition). A measurable therapeutic response also includes finding that the disease or one or more symptoms of a disease or condition is prevented or delayed in onset, or otherwise alleviated by a therapeutic agent described herein (e.g., an N-glycosylation inhibitor), thus, as used herein, a "therapeutically effective amount" refers to an amount sufficient to reduce one or more symptoms or conditions associated with ischemic stroke, including but not limited to hemorrhagic transformation, blood-brain barrier disruption, increased hemoglobin levels, and mortality.

[0143] As used herein, the terms "treat," "treatment," or "treating" encompass at least ameliorating symptoms associated with a patient's disease, wherein improvement is used in a broad sense to refer to at least reducing a parameter, such as the magnitude of a symptom associated with the disease or condition being treated. Thus, "treating" also includes situations where a disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) such that the patient no longer suffers from the condition or at least the symptoms characterizing the condition.

[0144] As used herein, unless otherwise indicated, the terms "prevent", "preventing" and "prevention" refer to preventing the onset, recurrence or spread of a disease or condition or one or more symptoms thereof. In some embodiments, the term refers to treating a subject who is specifically at risk of a disease or condition provided herein or administering the compound or dosage form to the subject before the onset of symptoms using a compound or dosage form provided herein with or without one or more other additional activating agents. These terms encompass the symptoms of suppressing or reducing a particular disease. In some embodiments, subjects with a family history of disease are potential candidates for a preventive regimen. In some embodiments, subjects with a history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" can be used interchangeably with the term "preventive treatment".

[0145] As used herein, and unless otherwise indicated, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or condition or prevent its recurrence. A prophylactically effective amount of a compound refers to an amount of a single therapeutic agent or a combination of the therapeutic agent and one or more other agents that provides a preventive benefit in the prevention of a disease. The term "prophylactically effective amount" can encompass an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0146] As used herein, and unless otherwise indicated, the compounds described herein are intended to encompass all possible stereoisomers unless a specific stereochemistry is specified. When the structural isomers of a compound are interconverted by a low energy barrier, the compound may exist as a single tautomer or a mixture of tautomers. This may take the form of proton tautomerism; or so-called valence tautomerism in a compound, such as a compound containing an aromatic moiety. The term "derivative" refers to a chemical substance that is structurally related to another substance, or a chemical substance that can be made from another substance (i.e., a substance from which it is derived) (e.g., by chemical or enzymatic modification).

[0147] As used herein, the term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt of a compound (such as a multi-drug conjugate) in the present disclosure. A pharmaceutically acceptable salt is any salt that retains the activity of the parent drug or compound and does not produce any harmful or undesirable effects on the subject to which it is administered and the environment in which it is administered. Pharmaceutically acceptable salts can be derived from amino acids, including but not limited to cysteine. Methods for producing compounds as salts are known to those skilled in the art (see, e.g., Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J Pharm. Sci. 66: 1, 1977). In some embodiments, "pharmaceutically acceptable salts" are intended to mean salts of free acids or bases of the agents or compounds represented herein that are non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See generally Berge et al., Journal of Pharmaceutical Sciences, 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are pharmacologically effective and suitable for contact with the tissues of a subject without excessive toxicity, irritation, or allergic reactions. The agents or compounds described herein may have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one group of each type, and therefore react with a variety of inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts.

[0148] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, hydrogensulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1, 4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [γ]-hydroxybutyrate, glycolate, tartrate, and mandelate.

[0149] The use of absolute or sequential terms, such as, "shall," "shall not," "should," "should not," "must," "may not," "first," "initially," "next," "subsequently," "before," "after," "last," and "ultimately," are not intended to limit the scope of the embodiments disclosed herein but are exemplary.

[0150] As used herein, the singular forms "a / an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, where the terms "including / includes", "having / has / with" or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0151] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both connective and disjunctive in operation. For example, one of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means only A, only B, only C, A and B together, A and C together, B and C together, or A, B, and C together.

[0152] As used herein, "or" may mean "and", "or" or "and / or" and may be used both exclusively and inclusively. For example, the term "A or B" may mean "A or B", "A but not B", "B but not A", and "A and B". In some cases, the context may dictate the particular meaning.

[0153] Any systems, methods, software, and platforms described herein are modular. Therefore, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of action.

[0154] When referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximation within the experimental variability (or within the statistical experimental error), and the number or numerical range can vary, for example, between 1% and 15% of the number or numerical range. In an example, the term "about" refers to ±10% of a given number or value.

[0155] The term "increase" is used herein to generally mean an increase in statistically significant amount. In some respects, the term "increase" means an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at most to and including 100% increase, or any increase between 10%-100%. Other examples of "increase" include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times or more compared to a reference level.

[0156] The term "reduce (decreased / decreasing / decrease)" is generally used to represent the reduction of statistically significant amount in this article.In some respects, "reduce" means reduced by at least 10% compared with a reference level, for example, reduced by at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at most and including 100% reduction (for example, non-existent level or undetectable level compared with a reference sample) or any reduction between 10-100%.In the context of markers or symptoms, these terms mean the statistically significant reduction of such levels.Reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to the level accepted in the normal range as an individual not suffering from a given disease.

[0157] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be understood in a limiting sense. Without departing from the present invention, those skilled in the art will now be aware of many variations, changes and substitutes. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions depending on various conditions and variables set forth herein. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted when practicing the present invention. It is therefore envisioned that the present invention should also cover any such substitutions, modifications, changes or equivalents. The following claims are intended to define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

[0158] Examples

[0159] The following illustrative examples represent embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.

[0160] As disclosed in the following examples, hexosamine-mannosamine (hereinafter referred to as 2-amino-2-deoxy-D-mannose or ManN) inhibits protein glycosylation and still stimulates EC proliferation in vitro. The biological effects of ManN in other in vitro and in vivo models and its possible mechanism of action were studied. ManN is an EC mitogen and survival factor for bovine and human microvascular EC, which is additive with VEGF. ManN inhibits glycosylation in EC and induces significant changes in N-glycan and O-glycan profiles. ManN and two N-glycosylation inhibitors stimulate EC proliferation through both JNK activation and unfolded protein response caused by ER stress. ManN leads to enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with faster limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways after inhibition of protein glycosylation can promote EC proliferation and angiogenesis, and may represent a therapeutic strategy for treating ischemic conditions.

[0161] Example 1

[0162] The effect of ManN on EC proliferation was evaluated. A library of 619 highly purified metabolites covering a broad spectrum of chemical entities was screened to determine the ability of VEGF to affect the growth of bovine choroidal microvascular EC (BCEC) in the presence or absence of VEGF. This and similar assays have been previously used to identify and characterize angiogenesis stimulators and inhibitors. Under the conditions tested, little or no proliferation was detected in the absence of VEGF.

[0163] A preliminary screen was performed, testing each compound at concentrations of approximately 1 and 10 μM (assuming a molecular weight of 100 Da for each compound) in the presence or absence of 5 ng / ml VEGF, which can induce an approximately 4- to 5-fold increase in cell proliferation. Six compounds of different chemical nature showed some inhibitory or stimulatory activity. The analysis focused on one of them, ManN, a hexosamine originally identified as a component of bacterial cell walls, because it showed the strongest and most consistent effects. ManN had a significant stimulatory effect in the 5-500 μM dose range and also had an additive effect with VEGF to promote BCEC proliferation. The dose-dependent effect of ManN on BCEC proliferation in the presence or absence of VEGF is shown in Figure 1A and 1BCompared with VEGF alone, ManN alone at 50 μM increased the EC-covered surface by up to 6.5-fold ( Figure 1A ), or when cells were treated with 50 μM ManN and 5 ng / mL VEGF, the fluorescence units increased by about 2.5 and 3 times after the addition of AlamarBlue ( Figure 1B AlamarBlue detects mitochondrial activity as an indicator of cell viability, which correlates with cell number within a certain range. The effect of ManN has a bell-shaped dose-response curve, with an inhibitory effect at higher concentrations ( Figure 1A and 1B The additive effect of ManN on promoting BCEC proliferation is also similar to that of bFGF ( Fig.11A ) and bovine retinal EC (BREC) Fig. 11C and 11D ) were observed together.

[0164] Various hexosamines (galactosamine, glucosamine, and their N-acetyl derivatives) were tested together with ManN in the BCEC proliferation assay. However, none of these hexosamines had a significant stimulatory effect ( Figure 1D Several structurally related molecules were also tested, such as D-isoglucosamine (fructosamine), meglumine, muramic acid, N-acetyl-neuraminic acid (sialic acid present in all mammalian cells), glucose, and mannose. None of these molecules stimulated BCEC proliferation in the presence or absence of VEGF ( Fig. 11C and D).

[0165] ManN enters and accumulates inside cells in a concentration-dependent manner. 0.66 nmol ManN was detected in 1 mg of cell lysate when BCECs were treated for 2 h with:

[0166] 400 μM ManN. Upon entry into cells, ManN, but not mannose, is rapidly converted to ManN 6-phosphate (ManN-6p). No incorporation of ManN was detected in N-glycans. Efficient uptake of ManNAc and mannose has been reported.

[0167] The effect of ManN on BCEC proliferation depends on cell glycolysis. When glucose-free medium was used, the additivity between ManN and VEGF was eliminated. On the other hand, the activity of VEGF was not dependent on the glycolytic pathway ( Fig. 11B ). However, in glucose-free medium, even in the presence of VEGF, only 4 μM ManN was significantly cytotoxic.

[0168] To further characterize the effects of ManN on EC survival, proliferation, and migration, confluent BCEC monolayers were mechanically injured. Figure 1E It was shown that after 48 hours, 40 μM ManN or 50 ng / ml VEGF significantly accelerated the migration and / or proliferation of BCECs compared to the control group, as reflected by a more complete closure of the "scratch" area. Similar to the proliferation assay, when cells were treated with both ManN and VEGF, an additive effect ( Figure 1E In addition, 40 μM ManN showed significant additive effects with VEGF in promoting BCEC migration ( Figure 1F ).

[0169] The observations were extended to human retinal microvascular EC (hRMEC), HUVEC, and skin microvascular endothelial cells (hDMVEC). ManN itself stimulated the growth of HUVEC and hDMVEC. In addition, VEGF had a dose-dependent additive effect in all EC types tested, with minimal toxicity even at a concentration of 5 mM. Similarly, stimulation of migration and wound closure was observed in HUVEC treated with 40 μM ManN alone (migration assay) and / or in combination with 50 ng / ml VEGF (scratch assay).

[0170] Example 2

[0171] Activation of ERK, AKT, mTOR, CREB, AMPK, ACC, and eNOS is not unique to ManN. Crosstalk between signaling and metabolic pathways in the vasculature, such as insulin signaling and glucose metabolism in ECs, has been reported to involve activation of AKT and STAT3. Together, they affect glycolysis, EC sprouting, proliferation, and migration. The effects of ManN and / or VEGF on the activation of major signaling pathways known to promote BCEC proliferation, such as ERK, AKT, mTOR, and CREB (cAMP response element binding protein), were evaluated. ManN activated ERK, AKT, mTOR, and CREB at 40 μM. Stimulation of ERK, AKT, and CREB was rapid and occurred within 10-30 minutes after the addition of ManN ( Figures 2A to 2C ). Further, when both ManN and VEGF were present, enhanced activation of ERK, AKT, and CREB was observed compared to ManN or VEGF alone ( Figure 2A and 2B). The effect of ManN on activation of the ACC (acetyl-CoA carboxylase) / eNOS (endothelial nitric oxide synthase 3) pathway was evaluated. Activation of the energy sensor AMPK (AMP-activated protein kinase) leads to eNOS activation and NO (nitric oxide) production; the latter has a bell-shaped effect on EC proliferation. Both eNOS and ACC were significantly activated by 40 μM ManN within 10-30 minutes (Figure 2D). However, activation of ERK, AKT, mTOR, CREB, ACC and eNOS was not unique to ManN. In fact, other hexosamines, such as ManNAc and mannose induced similar activation of these signaling pathways ( Figure 2C ). While activation of these common proliferation pathways may have a facilitatory effect, without being bound by theory, several unique mechanisms may be involved in the EC mitogenic effect of ManN.

[0172] Example 3

[0173] A series of specific pharmacological inhibitors were used to identify JNK / c-jun as the signal transduction pathway uniquely activated by ManN (in hexosamine). Western blot analysis revealed that among the three MAPK family members (ERK, p38, JNK), JNK was specifically activated by ManN. When growing BCECs were switched to a proliferation assay medium without growth factors, JNK and its downstream c-Jun were significantly activated by ManN in a dose-dependent manner, but not by mannose ( Figure 3A and 3B ). ManN, but not other hexosamines tested, activated the JNK pathway. Treatment of BCECs with the JNK-specific inhibitor SP600125 (5 μM) abolished the effect of ManN on BCEC proliferation ( Figure 3C ).

[0174] The effect of ManN on BCECs was assessed after transfection with siRNAs targeting JNK (i.e., JNK1 and JNK2, as JNK3 is not expressed in BCECs). Knockdown of JNK1 and / or JNK2 by 80% by two independent siRNAs targeting JNK1 or JNK2 abolished the mitogenic effect of ManN on BCECs at μM concentrations ( Figure 3D and 3E ), indicating that both JNK1 and JNK2 are important in transducing stress signals.

[0175] Example 4

[0176] ManN affects protein glycosylation in endothelial cells. Additivity of ManN to VEGF may occur at the transcriptional and / or translational level, or through a signal transduction pathway mediated by VEGF-VEGFR2. However, when cells were treated with different concentrations of ManN for 4 hours (gene expression level) or 24 hours (protein expression level), the transcription of VEGF, VEGFR2, and GLUT1&4 in BCECs and the total protein expression of VEGFR2 did not change significantly ( Figures 4A to 4C ; Figure 5A and 5B ; Figure 7C and 7E ). This was also true for BRECs and hRMVECs. Biotinylation studies showed that the amount of VEGFR2 on the cell surface did not change. However, phosphorylation of VEGFR2 in response to VEGF was reduced in cells pretreated with ManN, suggesting that VEGFR2 activation is hampered, rather than enhanced, in BCECs. No ligand-independent VEGFR2 activation occurred after the addition of ManN to BCECs. This was also true for HUVECs (SFIG.10a) and hDMVECs.

[0177] Starting from 40 μM, in BCEC ( Figures 4A to 4C The apparent molecular weight of VEGFR2 was significantly shifted after ManN treatment in both BCECs (Figure 4E and 4E) and BRECs. Compared with the control (major band of -230 KDa and minor band of -210 kDa), a new low molecular weight band (-170-200 KDa) appeared in BCECs treated with ManN in a dose-dependent manner (Figure 4E and 4E). Figure 4B , C and E; Figure 5A and 5B ; Figure 7C This transformation is unique to ManN in hexosamine and its derivatives ( Figure 4A VEGF alone had no effect on the molecular mass. Addition of VEGF to ManN did not induce additional shifts ( Figure 4A The lower molecular weight VEGFR2 band is unlikely to be a degradation product, because removal of ManN completely reversed the effect of ManN on molecular weight after 24 hours ( Figure 4E ). However, based on PNGase F treatment, it appears that not all glycosylation on VEGFR2 is eliminated by ManN, at least at μM concentrations. Experiments with the small molecule tyrosine kinase inhibitor axitinib, a potent VEGFR2 inhibitor, showed that the reduction in VEGFR2 molecular mass and stimulation of BCEC proliferation by ManN were independent of VEGFR2 signaling.

[0178] In the presence of 40 μM ManN, hRMVEC, HUVEC ( Figure 4F ) and hDMVEC( Figure 4G ), while the additive effects of ManN and VEGF on the proliferation of these cells occurred at the mM level.

[0179] To better understand how ManN might affect VEGFR2 post-translational modifications, cells were treated with ManN at a maximum molar ratio of 1:10 in the presence of one of four monosaccharides (mannose, glucose, galactose, or fucose). These monosaccharides are known to be important in protein N-glycosylation. The results showed that mannose could dose-dependently block the effects of ManN on VEGFR2 molecular mass as well as BCEC proliferation ( Figure 4C and 4D ). The effects of mannose may not be limited to preventing ManN from entering the cell via the same transporter, as these effects were observed when BCEC were first treated with ManN for 2 h to ensure its successful cellular uptake. Glucose, but not galactose or fucose, had similar effects to mannose.

[0180] The reduction in protein mass after ManN administration in BCECs was not limited to VEGFR2. Other N-glycosylated growth factor receptors / co-receptors or adhesion molecules, including av integrin, neuropilin-1, VE-cadherin, and bFGFR1 were also affected.

[0181] Example 5

[0182] The effect of ManN on the general protein glycosylation profile was evaluated. N-glycosylation is a complex process that depends on multiple enzymes that act sequentially during the transport of glycoproteins from the ER to the Golgi apparatus through the secretory pathway to generate hybrid and high-mannose glycan structures. It plays an important role in determining the fate of newly synthesized glycoproteins in the ER, their correct folding, cellular destination, and normal function.

[0183] Several key enzymes involved in protein N-glycosylation in both the ER and the Golgi were evaluated. α-Mannosidase from Jack bean is a broadly specific exoglycosidase that catalyzes the hydrolysis of non-reducing αl-2, αl-3 and αl-6-linked mannose residues at the end of oligosaccharides in both organelles and controls the conversion of high mannose to complex N-glycans, which is the last step of hydrolysis in the N-glycan maturation pathway. This enzyme has been used to screen potential N-glycosylation inhibitors. ManN, but not other hexosamines or their derivatives, showed inhibitory activity at 400 μM, which is much higher than the effective mitogenic concentration in BCEC. The effect of ManN up to 2 mM on α- or β-glucosidase was not detected.

[0184] N-linked glycans were isolated from BCECs by enzymatic cleavage and then purified and characterized using MALDI-TOF-MS. Treatment with 40 μM ManN resulted in a significant time-dependent reduction in the total oligomannosyl N-glycan content of Man6G1cNAc2 (Man-6), Man-8, and Man-9 compared to untreated controls, while significant early accumulation of Man-5 and Man-7 was observed after ManN treatment. ManN has previously been shown to inhibit the synthesis of lipid-linked oligosaccharides (LLOs) to alter protein GPI biosynthesis and the production of hybrid glycans, as well as glycans incorporated into MDCK cells. The accumulation of Man-5 over time suggests that inhibition of mannosidases is unlikely to be the mechanism of pro-angiogenic activity in BCECs.

[0185] Monosaccharide content was measured to analyze the composition of complex N-glycans. Significant decreases in fucose (8 h), mannose (12 h), galactose (24 h), and Neu5Ac (8 h and 24 h) were found in ManN-treated cells compared to untreated control cells, consistent with the inhibitory activity of ManN on global protein N-glycosylation.

[0186] O-glycan modification is another form of post-translational modification of proteins, in which serine or threonine residues are covalently linked to GalNAc residues. GalNAc residues can be further modified by several glycosyl-transferases, which extend branched or linear glycan chains in a sequential manner according to substrate specificity. ppGalNAcT (polypeptidyl GalNAc transferase) catalyzes the transfer of a-GalNAc from UDP-GalNAc to Ser or Thr residues of glycoproteins, thereby producing Tn antigens. When Tn antigens are produced, they may have three different fates: (i) they can be sialylated by ST6GalNAcT enzyme on C6; (ii) they can be replaced by f3-G1cNAc on C3 or C6 to produce core-3 or core-6, respectively; or (iii) they can be galactosylated by C1 GalT1 on C3 to form core-1, which can also be sialylated to produce monosialyl or disialyl core-1 O-glycans.

[0187] O-glycans in BCEC lysates were analyzed by MALDI-Tof mass spectrometry. Since there is no unique enzyme that can cleave all different forms of O-glycans, reductive β elimination was performed to understand the O-glycan backbone. In order to prevent desialylation during mass spectrometry data acquisition, full methylation was performed before MALDI-Tof / Tof mass spectrometry analysis. O-glycosylation was generally reduced after treatment with 40μM ManN. Specifically, it was observed that the ion intensity was decreasing at m / z of 895 (sialylated core, Galf31-3GalNAc-), 1256 (disialyl core 1), 983 [core 2, GlcNAcf31-6(Galf31-3)-GalNAc-] and 1187 (digalactosylated core 2).

[0188] Example 6

[0189] The effect of ManN on activating the UPR by increasing the expression of Bip and CHOP was evaluated. Asparagine-linked N-glycosylation is one of the most common modification reactions in eukaryotic cells and occurs during biosynthesis in proteins that are co-translationally translocated across the ER or integrated into the ER. After the transfer of N-linked oligosaccharides to nascent proteins by OST (oligosaccharyltransferase), ER-resident glycosidases and mannosidases generate a series of glycan-trimmed intermediates that are specifically recognized by ER-localized lectins to direct the nascent protein into protein folding, degradation, or export pathways. One of the consequences of inhibiting protein glycosylation is impaired protein folding, leading to ER stress. The physiological response to the UPR is mediated by changes in gene expression, such as regulation of the ER Hsp70 chaperone BiP (also known as glucose-regulated protein 78, which binds to immunoglobulins) and another multifunctional transcription factor CHOP (CCAAT-enhancer binding protein homologous protein). For example, during aging, impaired UPR function creates a permissive environment for protein aggregation, unresolved ER stress, and chronic inflammation.

[0190] To investigate possible ManN-mediated ER stress, the expression of Bip and CHOP in cells treated with ManN or mannose was investigated by Western blot analysis. The data showed that when growing cells were deprived of growth factor supply, ManN, but not mannose or VEGF, could significantly turn on Bip expression in a concentration-dependent manner, with the accumulation of Bip increasing at 24 h ( Figure 5A and 5B ) and 48 hours ( Figure 5A ). CHOP induction appears to be more rapid, in a dose-dependent manner at approximately 6 hours ( Figure 5A No synergistic effect of ManN and VEGF was observed in promoting the expression of Bip or CHOP ( Figure 5B ).

[0191] Two well-known chemical chaperones, 4-PBA (4-phenylbutyric acid) and TUDCA (tauroursodeoxycholic acid), were tested to alleviate ER stress in ManN-treated BCECs. Both drugs have been previously shown to attenuate tunicamycin-induced UPR and Bip expression of the eIF2a-ATF4-CHOP arm. It was found that 2mM 4-PBA, but not 500μM TUDCA, prevented CHOP expression induced by ManN at 400μM and 5mM, and restored ATF-6 (activating transcription factor-6) expression by ManN at 400μM ( Figure 5C ). Similarly, TUDCA restored ATF-6 expression much weaker than 4-PBA. As a transmembrane ER glycoprotein, ATF-6 is cleaved to release a 50 kDa amino-terminal fragment that translocates to the nucleus, which activates the transcription of ER chaperones and ER-associated degradation components such as Bip and CHOP upon accumulation of improperly folded proteins in the ER. Pretreatment of cells with 1 mM 4-PBA for 4 h effectively reversed the bell-shaped activity of ManN on BCEC proliferation in the presence or absence of VEGF. The additivity between ManN and VEGF was largely abolished ( Figure 5D ).

[0192] Example 7

[0193] The effects of ManN on non-endothelial cells were evaluated. To extend the observations on EC, various non-EC types from different species were examined. These included NIH3T3 fibroblasts and AML12 hepatocytes (mouse), ARPE-19RPE cells (human), and freshly isolated bovine pituitary cells. Several human cell types relevant to the in vivo model, such as skin fibroblasts and keratinocytes, were also tested. In addition, four human or mouse cancer cell lines (A673, U87MG, Calu6, and 4T1) were screened (Figure 6). To examine post-translational modifications of proteins in non-EC, bFGFR1 or f31 integrin was used to monitor molecular mass changes. Similar to BCEC, ManN, but not mannose, could induce molecular mass changes in all these non-ECs (Figure 6 inset). However, in contrast to BCEC ( Figure 1B )、BREC( Figure 1C Unlike hRMVECs, HUVECs, and hDMVECs, no proliferative effects of ManN were observed at μM to mM concentrations, either alone or in combination with other growth stimulants (Figure 6), although efficient ManN uptake and comparable levels of free ManN were detected in all cell types. Cytotoxicity varied between cell types, with AML12 being the most sensitive cell, and human RPE cells and human keratinocytes being the least sensitive to ManN at 5 mM levels (Figure 6). Fig. 6E and 6H ). It has been reported that 25 mM mannose inhibited growth in vitro in several tumor lines with low levels of PMI (phosphomannose isomerase). At 5 mM, ManN but not mannose showed significant toxicity to 4T1 cells ( Fig.6D ), which may be due to the higher PMI level in 4T1 than all reported sensitive tumor lines.

[0194] Example 8

[0195] Similar to ManN, protein N-glycosylation inhibitors stimulated EC growth. To determine whether extensive changes in protein glycosylation could promote cell proliferation, two well-characterized inhibitors, kif and castanespermine (Cas), were tested. BCEC proliferation was stimulated in a dose-dependent manner in the presence or absence of 5 ng / ml VEGF ( Fig. 7A and 7B ). After 24 hours of treatment with Kif or Cas, the molecular weight of VEGFR2 on SDS-PAGE was significantly reduced ( Figure 7C ).

[0196] At 40 μM, Kif can significantly activate BCEC ( Fig. 7E ), ERK and AKT in HUVEC and hDMVEC. Cas activation of ERK was less pronounced in both BCEC and hDMVEC. However, both inhibitors were able to activate the JNK pathway in BCEC ( Fig. 7E Blocking JNK activation with 5 μM SP 600125 significantly reduced the effects of both glycosylation inhibitors on BCEC proliferation ( Figure 7F ). Figure 7C We demonstrate a dose-dependent induction of Bip expression when growing BCECs were switched to growth factor-free medium for 24 hours in the presence of Kif or Cas at concentrations that promote cell proliferation.

[0197] Both Kif and Cas had significant activity in the BCEC "scratch" assay, where each molecule closed the gap faster than the control within 48 hours ( Fig.7D ). Fig.7D The inserts show representative images of assays using Kif or Cas. Quantitative analysis showed that gap closure was significantly accelerated in a dose-dependent manner compared to the control.

[0198] Example 9

[0199] The relationship between the effects of ManN on endothelial cells in vitro and angiogenesis in vivo was studied through the effect of ManN in a mouse splint wound model. In this model, the repair process is completely dependent on epithelialization, cell proliferation and angiogenesis, which is closely related to the biological process of human wound healing. The effects of ManN and VEGF alone or in combination were tested. 20 μg VEGF or 20 μg ManN were applied topically every day for the first 3 days after injury. When VEGF and ManN were combined, a significant acceleration of wound closure was observed during the early stages of healing ( Fig. 8A Compared with VEGF or ManN monotherapy, the combination had significantly faster wound closure starting from day 2 ( Figure 8B On day 4, the mean wound closure rates were 81.5%, 75.6%, 66.9%, and 29.8% for the PBS-, ManN-, VEGF-, and combination-treated groups, respectively. On day 4, the number of small blood vessels surrounding the wound area was quantified. A significant increase in CD31-positive blood vessels was found in the combination group compared to PBS control, VEGF, or ManN alone ( Figure 8C and 8D ).

[0200] Thus, ManN combined with VEGF promotes angiogenesis in the skin injury model. In this acute model, wound closure occurs rapidly without any treatment.

[0201] The stability of ManN was evaluated in wound fluids contaminated with bacteria, a common feature of wounds. ManN was added to wound fluids freshly collected from a mouse model of skin infection with Staphylococcus aureus, a common cause of skin and soft tissue infections in humans. No significant loss of free ManN was detected after incubation with this wound fluid at 37°C for up to 24 hours. Therefore, ManN could be used to treat infected wounds on the hands, possibly in combination with antimicrobial drugs or other agents.

[0202] A known property of VEGF is the rapid induction of vascular permeability following injection into the guinea pig skin. The effect of ManN on inducing vascular permeability was evaluated in the same assay. However, ManN did not elicit a permeability enhancement effect when tested at 1 ng-5 μg, whereas 25 ng VEGF induced vascular permeability.

[0203] Example 10

[0204] The angiogenic effects of ManN and Kif were evaluated in a mouse hindlimb ischemia model. The activity of ManN in a chronic ischemia model, which may more specifically reflect its role as an endothelial cell mitogen and pro-angiogenic factor, was evaluated and a mouse hindlimb ischemia model was considered. Several variants have been described, depending on the vessel occluded. The selected variant consists of ligation and resection of the femoral artery, which results in more severe ischemia compared to simple femoral artery ligation. Occlusion of both vessels produces more severe ischemia, but has the disadvantage of inducing severe pain and distress in mice, as well as frequent ulcers and necrosis.

[0205] Orally administered ManN was tested in this femoral artery ligation excision model. Since Kif has been previously used for intraperitoneal administration in in vivo studies, this approach was adopted. Laser Doppler perfusion imaging (LDPI) was used as a non-invasive method to monitor the timing and extent of blood flow restoration in the ischemic limb. Blood flow was continuously examined using LDPI, and the increase in the perfusion ratio of the ischemic (ligated; left) to non-ischemic (sham; right) hindlimb after ligation was used to indicate the restoration of blood flow. Starting immediately after the surgical procedure, mice were orally fed 20% ManN or 1 mg / ml Kif ip every other day as described in the methods. One week after surgery, the perfusion ratio of the H2O-fed group indicated a blood flow recovery rate of -25%, a value that is very consistent with published data for the same type of lesions in the same strain of mice. However, the blood flow recovery rates in the ManN and Kif-treated groups were approximately 40% and 47%, respectively, which indicates an accelerated blood flow recovery rate compared to the H2O-treated mice ( Fig. 9A and 9B Within 3 weeks after ManN and Kif treatment, the blood perfusion ratio continued to increase to 50% of the sham-treated limb and was significantly higher than that of the control group ( Fig. 9A and 9B ).

[0206] Consistent with the improved blood flow, the ischemic hindlimbs of the ManN- and Kif-treated groups showed increased vascular density compared with the control group, as assessed by CD31 immunostaining of surrounding muscle tissue 3 weeks after ligation. The vascular density of the ManN- and Kif-treated groups was 2.3- and 1.8-fold higher, respectively, compared with the H2O-treated control group ( Fig. 9C and 9D ).

[0207] After oral administration, ManN plasma levels dropped relatively quickly. Plasma free ManN levels reached a peak level of -100nmol / ml plasma at 1 hour. After 3 hours, only about half of that amount could be detected. Muscle samples were collected from ischemic legs 2 hours after oral feeding of 20% ManN. Significant amounts of ManN reached the ischemic legs, with free ManN protein at 0.17+1-0.18nmol / mg and ManN-6p protein at 0.91+1-0.24nmol / mg. At least in BCECs, in the absence of exogenous ManN, the effects of ManN on protein quality lasted for at least 8 hours ( Figure 4E ), suggesting that even relatively brief exposure may be sufficient to elicit pharmacological effects.

[0208] Example 11

[0209] The role of ManN and Kif in inducing retinal neovascularization was evaluated. The findings in cultured ocular ECs were extended to suitable in vivo model systems. Over the past few decades, the mouse retina has been widely used to study both physiological and pathological angiogenesis. In order to obtain a detailed description of the retinal vasculature, the vascular area fraction (the ratio of the area covered by blood vessels to the total retinal area) of images of retinal flat mounts were processed. Using this model, the role of ManN in retinal neovascularization was evaluated. Kif was also tested in this model because it is a water-soluble inhibitor and its glycosylation inhibition mechanism has been well established. In addition, it shares with ManN the ability to activate ERK, AKT and stress pathways in BCECs ( Fig. 7E ).

[0210] Five hundred nanograms of ManN or Kif were injected intravitreally, and the retinal vasculature was examined seven days later. In this model, 200 ng of bFGF was administered intravitreally as a positive control. In the bFGF, ManN, and Kif treated groups, the density of retinal vessels increased by approximately 35%, 30%, and 20%, respectively, compared to the PBS group ( Fig. 10A and 10B ).

[0211] Example 12

[0212] The effect of ManN treatment on cerebral infarction (induced by MCAO) was tested. The experiment was conducted in an acute ischemia-reperfusion-induced brain injury model in mice and represented the combined use of pretreatment and posttreatment of N-glycosylation inhibitors. In this case, ManN was tested as an N-glycosylation inhibitor. The MCAO model of mice is a well-studied and reproducible example of acute ischemic events in a single hemisphere of the brain. Compared with other in vivo ischemic models disclosed herein (e.g., hindlimb ischemia model and blood flow assessment), the MCAO model has an acute ischemic period (2 hours), after which time normal blood flow is restored to the affected area. In this case, the effect of ManN treatment on tissues (tissues that are particularly sensitive to transient blood flow interruptions, such as brain tissue) was evaluated and measured at time points after normal blood flow had been restored.

[0213] On day -1, mice were treated with 40 mg ManN (200 μL of 20% ManN in water), or 200 μL of water was delivered to control animals by oral gavage. The timing of pretreatment of the test group mice with ManN doses was about 16 hours before surgery. This dosing regimen continued to be applied once every 48 hours (e.g., on day 1, day 3, etc.) until the extent of the animal's cerebral infarction area could be assessed. About 16 hours after the first treatment (day 0), the animal had undergone a 2-hour surgical occlusion of the right middle cerebral artery, thereby causing the right brain portion to experience an acute ischemic event. After the 2-hour occlusion period, normal blood flow was restored. Fig. 12A The timing of the ManN administration treatment regimen as well as the timing of the procedures and subsequent assays are shown. Fig. 12B The results of the first experiment are shown, in which 4 animals were treated with ManN and paired with 4 control treated animals for analysis. On the 4th day, the MRI results of the coronal plane showed that there was a clear and measurable infarct area in the animals treated with ManN and the animals treated with the vehicle. Multiple coronal sections of the CNS of a single animal were measured using MRI, and the total infarct volume multiples on the 4th day of each animal were calculated. As shown in the figure, the infarct area of ​​the animals treated with ManN was significantly smaller. Fig. 12C The results of the second experiment are shown, in which 4 animals were treated with ManN and paired with 4 control treated animals for analysis. On day 4, the MRI results of the coronal plane showed that there was a clear and measurable infarct size in the animals treated with ManN and the animals treated with controls. Multiple coronal sections of the CNS of a single animal were measured using MRI, and the total infarct volume multiples on day 4 were calculated for each animal. As shown, the infarct size of the animals treated with ManN was significantly smaller. Fig.12D Shows the Figures 12B-12CA combined analysis of the data from the 2014 study showed that ManN treatment was statistically significant in reducing total infarct size assessed 4 days after an acute ischemic event. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test. These results suggest that a treatment regimen that includes pre- and post-treatment with ManN delivered by oral administration has a measurable benefit in reducing total cerebral infarct size following an acute ischemic event. In additional animal testing, Fig.13A Representative MRI images of the results of ManN-treated animals paired with control-treated animals for analysis are shown. On days 2 and 4, MRI results in the coronal plane showed that ManN-treated animals and vehicle-treated animals had defined and measurable infarct size. Compared with the vehicle (H2O)-treated control group, the infarct size of ManN-treated animals was significantly reduced on both days 2 and 4. Fig. 13B The overall results on day 4 were plotted and analyzed in . Multiple coronal sections of the CNS of individual animals were measured using MRI, and the total infarct volume multiples on day 4 were calculated for each animal. MRI images were quantified using Image J. Each point represents the total calculated CNS infarct volume for each animal as measured using MRI. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test (p<0.001), indicating that in the MCAO model, a multiple-dose treatment regimen comprising administering an effective amount of ManN to mice significantly reduced the CNS infarct volume on day 4 in ManN-treated animals compared to vehicle-treated animals. Fig. 13C In Figure 4, TTC (2,3,5-triphenyltetrazolium chloride) staining is visualized in ManN-treated and vehicle-treated coronal sections of the mouse CNS from representative animals at day 4. TTC staining is commonly used to quickly and reliably visualize hypoxic brain tissue to determine the size and area of ​​cerebral infarction. Viable cells exposed to TTC staining will turn healthy / normal tissue a deep red. In contrast, damaged or dead tissue remains white, indicating the absence or reduction of viable cells, which is indicative of infarct size. The therapeutic benefit of reduced infarct size in ManN-treated animals at day 4 is evident as there are no large areas of white staining in ManN-treated animals compared to the extensive CNS areas lacking TTN staining in vehicle-treated animals.

[0214] follow Fig. 12AThe listed treatment regimens, animals were tested on day 6 to examine the vascular density within the infarct-affected area of ​​the cerebral cortex by examining CD31 expression. CD31 (PECAM-1) is a marker highly expressed on endothelial cells. CD31 fluorescent immunohistochemistry was used to identify and quantify the vascular density within a defined area of ​​the cerebral cortex affected by infarction in the MCAO model of ManN-treated animals and vehicle-treated animals. ManN-treated animals were administered a therapeutically effective dose of ManN (40 mg) by oral gavage on days -1, 1, 3, and 5. Vehicle-treated animals were given water on days -1, 1, 3, and 5. On day 6, brain tissue was collected and fixed. The infarct area was identified, and CD31 immunostaining was performed on coronal sections to mark the vasculature. Representative CD31 stained sections from vehicle (H2O)-treated animals and ManN-treated animals are shown in Fig.14A In. Fig. 14B In the Figure 3, vascular density was quantified using Image J on representative sections and the cumulative results were plotted according to CD31 staining density. Statistical analysis was performed using a two-tailed, two-sample unequal variance t-test (p<0.001), indicating that in the MCAO model, a multiple-dose treatment regimen comprising administration of an effective amount of ManN to mice significantly increased vascular density in the area of ​​the cerebral cortex affected by the infarct in ManN-treated animals compared to vehicle-treated controls.

[0215] follow Fig. 12A The animals were tested on day 4 to examine the histopathological changes in the infarct-affected area of ​​the right hemisphere by examining hematoxylin and eosin (H&E) staining in coronal sections according to the treatment regimen shown. ManN-treated animals were administered a therapeutically effective dose of ManN (40 mg) by oral gavage on days -1, 1, and 3. Vehicle-treated animals were given water on days -1, 1, and 3. On day 4, brain tissue was harvested and fixed, then sectioned and stained with H&E. Fig.15 As shown in the top row, in coronal sections of vehicle-treated mouse brains, the cerebral cortex and striatum regions showed alterations in brain morphology and cytoarchitecture. Fig.15 The bottom row shows coronal sections of the CNS of ManN-treated mice, with embedded areas of the cerebral cortex and striatum shown at higher magnification. Due to increased vacuolization and edema, vehicle-treated animals showed lighter H&E staining of the infarct area compared to the infarct area of ​​normal brain tissue. Compared with the nuclei in the equivalent ManN-treated brain area affected by the infarct, the nuclei in the vehicle-treated infarct area were smaller, appeared atrophic, and stained darker. These results indicate that ManN pretreatment and continued posttreatment after an acute ischemic event improve the histopathological changes in the brain of the mouse MCAO stroke model.

[0216] Materials and methods

[0217] Small molecule library

[0218] MSMLS (Mass Spectrometry Metabolite Standard Library) (IROA Technologies, Bolton, MA; now Sigma) is a collection of 619 high-quality small molecules (>95% purity) spanning a wide range of primary metabolites including carboxylic acids, amino acids, biogenic amines, polyamines, nucleotides, coenzymes, vitamins, lipids, etc. The plates were spun at 300 g after reconstitution according to the manufacturer's instructions.

[0219] Chemical compounds

[0220] D-Mannosamine hydrochloride was obtained from Sigma (M4670) or Spectrum Chemical MFG Corp (M3220). 1-Amino-1-deoxy-D-fructose hydrochloride (D-isoglucosamine) (803278), D-(+)-galactosamine (1287722), D-(+)-glucosamine (1294207), N-acetyl-mannosamine (A8176), N-acetyl-galactosamine (A2795), N-acetyl-glucosamine (A8625), meglumine (M9179), muramic acid (M2503), N-acetylneuraminic acid (A2388), D(+)-glucose (D9434), D-(+)-mannose (1375182), meglumine (M9179), tunicamycin from Streptomyces sp. (T7765), and SP600125 (S5567) were obtained from Sigma. High purity cell culture grade water used to dissolve compounds (endotoxin <0.005EU / ml) was obtained from Hyclone. Axitinib was obtained from Santa Cruz (SC-217679). Tauroursodeoxycholic acid (TUDCA) was from Calbiochem (1180-95-6), and 4-phenylbutyric acid (4-PBA) (P21005, castaneospermine (Cas, C3784), kifunine (K1140), DMSO (D2650) were from Sigma. DMSO (D2650) was used as a solvent for Cas.

[0221] Antibody

[0222] Unless otherwise indicated, antibodies used in the present study were from Cell Signaling Technology, Inc. (Denver, MA). Total: VEGFR2 (2479), ERK (4695), p38 (9212), JNK (9252), mTOR (2983), AKT (4691), CREB (9104), CHOP (2895), ACC (3676), ATF-6 (65880), Bip (3183), AMPK (5832), FGFR1 (9740), eNOS (9586), VE-cadherin (2500), c-Met (3127 or 3148), neuropilin (3725), CD31 (3528), c-Jun (9165). Phosphoantibodies: VEGFR2 (Tyr1175, 2478 or 3770), ERK1 / 2 (Thr202 / Tyr204, 4376), p38 (Thr180 / Tyr182, 4511), JNK (Thr183 / Tyr185, 9251), mTOR (Ser2448, 5536), AKT (Ser473, 4060), CREB (Ser133, 919 1), ACC (Ser79, 3661), eNOS (Ser1177, 9571), AMPKa (Thr172, 50081), c-Jun (Ser73, 9164), (31 integrin (4706 and 34971), av integrin (4711), JNK1 (3708), JNK2 (4672), JNK3 (2305). Anti-(3-actin was from Sigma.

[0223] cell

[0224] Primary human umbilical vein endothelial cells (HUVEC, passage 4-10) were obtained from Lonza (Lonza, C2519AS, lot 234871) and cultured on 0.1% gelatin-coated plates in endothelial cell growth medium (EGM) containing 2% FBS, BBE (bovine brain extract), heparin, human EGF, hydrocortisone, ascorbic acid, GA-1000 (gentamicin, amphotericin B) and VEGF. Bovine retinal microvascular endothelial cells (BREC, #BRMVEC-3) and bovine choroidal microvascular endothelial cells (BCEC, #BCME-4) (both from VEC Technologies (Renssellaer Polytechnic Institute, New York, NY)) were maintained on fibronectin-coated plates (1 μg / cm 2). The growth medium was low glucose DMEM supplemented with 10% calf serum (BCS), 5 ng / ml bFGF, and 10 ng / ml human VEGF165. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. bFGF (233-FB) and VEGF165 (293-VE) were purchased from R&D Systems. Human retinal microvascular endothelial cells (<15 passages) were from Cell Systems Corporation (Kirkland, WA). These cells are grown on 0.1% gelatin coated plates in culture medium 131 containing 5% fetal bovine serum, hydrocortisone (1 μg / ml), human fibroblast growth factor (3ng / ml), heparin (10 μg / ml), human epidermal growth factor (1ng / ml) and dibutyryl cyclic AMP (0.08mM) (MVGS, S005-25, Gibco Invitrogen). Human RPE cell line ARPE-19 is from ATCC. Cells are gently lifted in 0.025% trypsin and plated in RtEGM culture medium (Clonetics company (Clonetics)) containing 2% FBS, L-glutamine, human bFGF, GA-1000. Once cells are attached to the plate, serum-free RtEGM culture medium is used to maintain the culture to obtain the best results. ARPE-19 is obtained from ATCC (CRL-2302) and cultured according to the company's instructions. NIH3T3 cells were obtained from ATCC (CRL-1658). Adult dermal MVEC (CC-2543) was cultured in EGM-2MV (CC-4147, Lonza). Keratinocytes (ATCC, PCS-200-011) were cultured in dermal cell basal medium (PCS-200-030) plus keratinocyte growth kit (PCS-200-040). Human primary dermal fibroblasts (ATCC PCS-201-012) were cultured in fibroblast basal medium (ATCC, PCS-201-030) plus growth kit (ATCC, PCS-201-040). Growth stimulants used in the assay included human EGF (R&D Systems, 236-EG), mouse TGFI3 (R&D Systems, 410-MT), KGF (Sigma, K1757) or 10% FBS growth medium. 4T1 cells were obtained from ATCC (CRL-2539) and cultured in RPMI-1640 containing 10% FBS (Omega Scientific, Tarzana, CA) and antibiotics.A673 (CRL-1598), A549 (CCL-185), U87MG (HTB-14) cells were obtained from ATCC and cultured in high glucose DMEM containing 10% FBS. FBS (S12550) was purchased from R&D Systems. BCS (SH30073.03) was obtained from Hyclone. All cell lines used in the study were negative for mycoplasma contamination from the respective suppliers.

[0225] Cell proliferation assay

[0226] Proliferation assay with BCEC and BREC is performed. BCEC or BREC (<10 generations) of logarithmic phase growth are trypsinized, resuspended and seeded in 96-well plates (uncoated) in low glucose DMEM supplemented with 10% calf serum, 2mM glutamine and antibiotics (growth medium) at a density of 1200-1500 cells per well with a volume of 200 μl. All reagents are added at a specified final concentration. After 3-6 days, cells are incubated with AlamarBlue for 4 hours. Fluorescence is measured at 530nm excitation wavelength and 590nm emission wavelength. The experiment is repeated at least three times. In order to create hypoxic conditions, cells are placed in a hypoxic incubator with a gas mixture consisting of 1% O2, 5% CO2 and 94% N2. In each 96-well plate, holes including untreated and VEGF-treated (10 / ng / ml) are included to monitor the difference between plates. 20% methanol or 0.05% DMSO serves as a negative control. When testing Cas in these cells, 0.05% DMSO served as a negative control. Human RMVECs and adult DMVECs were isolated into gelatin-coated 96-wells (2000 cells per well) in low glucose DMEM containing 10% FBS. 1200 cells / well were set up in low glucose medium containing 0.5% FBS for proliferation assays. Data were collected on day 4 or day 5. HUVEC (p7-10) were grown on gelatin-coated plates until 70-80% confluence was reached.

[0227] On the day of the assay, cells were dissociated with 0.05% trypsin, which was neutralized with 0.5% EBM containing FBS. Cells were briefly spun and then resuspended in 0.5% FBS medium. Cells were counted and plated at 1000 cells / well in 96-well plates. Triplicate wells were used for each treatment. Data were collected on day 3, and cells were then fixed in 4% paraformaldehyde for 15 minutes before adding crystal violet. The area covered by cells was quantified after taking pictures by Image J software.

[0228] In the low glucose DMEM containing 1% FBS, with or without 10ng / ml bFGF or 100ng / ml human EGF, the proliferation assay of fibroblasts was carried out, and the assay was terminated at the 3rd day. ARPE-19 cells were gently lifted with 0.025% trypsin, and plated in the RtEGM culture medium (Clonetics company) containing 2% FBS, L-glutamine, human bFGF, GA-1000. Once cells are attached to the plate, serum-free RtEGM culture medium is used to maintain the culture. For proliferation assay, 1500 human RPE cells are plated in the 96-well plates in the low glucose DMEM containing 1% FBS. A673, U87MG, Calu6 and AML12 cells are grown until confluence, and then collected and resuspended in suitable assay culture medium. For proliferation assay, cells are plated in the low glucose DMEM containing 5% FBS with a density of 1000-2000 cells / well, or otherwise described. As previously described, isolate bovine pituitary cells (pituitary follicular astrocytes). For the proliferation assay of human epidermal keratinocytes, human DMVEC and human dermal fibroblasts, 1000 cells / well plates were inoculated in low glucose DMEM containing 1% FBS (with or without various growth factors). The assay of bovine pituitary cells was terminated on the 3rd day, and the assay of all other cell types was terminated on the 4th day. For 4T1, 1000 cells were plated in RPMI-1640 with 2% basement membrane extract (BME) and 2% FBS on 96 wells coated with BME, and processed after 4 hours. Four days later, tumor cell growth was measured by MTS assay (Promega, Madison, WI, Madison, Wisconsin), which is a colorimetric assay for measuring the metabolic activity of living cells. Recombinant human transferrin was obtained from EMD Millipore (Temecula, CA, California). Recombinant mouse apo-transferrin was obtained from Sigma.

[0229] SiRNA knockdown

[0230] BCECs were plated at a density of 1.5×10 5 cells / well in 6-well culture plates and cultured overnight. The old culture medium was replaced with 2 ml of antibiotic-free culture medium. According to the manufacturer's instructions, siRNAs including si negative (Ambion, AM4611), siRNAs against JNK1#2 (Invitrogen, NM_001192974.2_siRNA266), siRNAs against JNK1#4 (Invitrogen, NM_001192974.2_siRNA_485), siRNAs against JNK2#2 (Invitrogen, XM_005208371.4_siRNA_1240), and siRNAs against JNK2#4 (Invitrogen, XM_005208371.4_siRNA_696) were mixed with Lipofectamine RNAiMAX reagent (ThermoFisher Scientific, 13778150) in Opti-MEM I reduced serum medium (Gibco, 31985062). In brief, the cells in each well were transfected with a mixture containing 25pmol siRNA, 7.5lμL RNAiMAX reagent and 125μL Opti-MEM culture medium to reach a final siRNA concentration of 12.5nM. The mixture of RNAiMAX and Opti-MEM was used as a no siRNA control. The cells were incubated with siRNA. After 8 hours, the culture medium containing siRNA was replaced with fresh culture medium. After 24 hours and / or 48 hours of transfection with siRNA, the cells were used for proliferation assays and protein extraction.

[0231] PNGase F processing

[0232] PNGase F without glycerol was obtained from New England Biolabs (Ipswich, MA). In short, BCEC was lysed with NP-40 (Thermo Fisher Scientific, Waltham, MA) containing protease inhibitors. Lysate was clarified at 5000g for 25 minutes at 4°C. Total protein content was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific). 20 mg of protein was mixed with 10X denaturing buffer and H2O to a total volume of 10 ml. Glycoprotein was denatured at 100°C for 10 minutes, followed by addition of sugar buffer and PNGase F. The reaction was carried out at 37°C for 2 hours.

[0233] Western blotting

[0234] Cells are allowed to reach 80% confluence in 12-well plates. With or without VEGF, cells are pretreated with ManN, Kif or Cas for different durations, with H2O as the solvent control for ManN. At different time points, the plate is taken out from the incubator and kept on ice. First, the cell monolayer is washed once with ice-cold PBS, then with 250 μl Pierce RIPA buffer (Thermo Fisher Scientific, Rockford, IL) or with 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 1% NP-40 (100 times) containing a mixture of protease / phosphatase inhibitors (Cell Signaling, #5872) lysis. Lysates were collected and mixed with 4X Bolt LDS sample buffer (Novex, Carlsbad, CA) in the presence of Halt protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, #NP0007). Samples were subjected to SDS-PAGE (Bolt 4-12% Bis-Tris Plus, Invitrogen) using Bolt MESSDS running buffer or NuPAGE 3-8% Tris-acetate gels using Tris-acetate SDS running buffer (Novex). HUVEC (passages 6-8) were plated in EBM-2 basal medium (Lonza) containing 0.2% FBS. After overnight culture, cells were serum starved for 4 hours in EBM-2 medium before treatment with 50ng / ml VEGF165 or different lengths of vehicle control. Equal amounts of protein lysates were analyzed by SDS-PAGE and blotted with the specified antibodies. Proteins were transferred using Tris-glycine buffer (proteomics grade) containing 20% ​​methanol (Apex BioResearch Products). The membrane was first incubated with 5% milk in TBST, pH 7.6 (TEKnova, Hollister, CA), followed by blotting with primary and secondary antibodies. ECL anti-rabbit IgG, horseradish peroxidase-linked whole antibodies from donkey or goat anti-mouse were obtained from General Electric Healthcare Group (UK Ltd.). SuperSignal West Dura Persistent Substrate was from Thermo Fisher Scientific. In some cases, the same PVDF membrane was stripped by incubation in Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) for 8 minutes to reveal total specific protein expression, followed by a secondary strip for beta-actin expression.

[0235] Migration assay

[0236] HUVEC (generation 6-8) was cultured as described in "Western Blot" and serum starved. Then 10,000 cells in 150 mL EBM-2 culture medium were added to the upper chamber of an 8 gm pore cell culture insert (Falcon) coated with 0.1% gelatin. The lower compartment was filled with 600 mL EBM-2 culture medium containing various agents. The plate was incubated at 37 ° C to allow migration. After 4 hours, the cells were fixed with 4% PFA for 20 minutes and then stained with crystal violet (Sigma-Aldrich) at room temperature for 20 minutes. The migrating cells on the bottom side of the insert membrane were quantified by counting the entire area of ​​the insert at 40 times magnification. The experiment was performed in triplicate and repeated three times. BCEC migration was similarly set, except that the wells were coated with FN, the cells were suspended in 1% serum culture medium, and the migration time was 18-24 hours.

[0237] Scratch assay

[0238] BCEC (6-10th generation) and HUVEC (6-8th generation) are used for this determination. Cells are grown in 6-well plates until about 80% confluence, washed twice with PBS, and then starved for 5 hours in serum-free DMEM (low glucose, Hyclone), and then "scratch" is made with 1ml tip. Cell monolayer is simply washed once with serum-free medium, and then various treatments are carried out in the medium containing 1% FBS. After 48 hours, the determination is stopped by adding 2ml 4% paraformaldehyde. After 20 minutes, the fixed cells are stained with 1ml crystal violet (Sigma). Before taking pictures, the plate is gently cleaned and air-dried under running tap water. The image is collected by a ZEISS Discovery V8 stereomicroscope equipped with a PixeLINK million-pixel FireWire camera. Wound closure is quantified using AxioVision LE Re1.4.4 software. Six images were taken for each sample and six measurements (in pixels) were performed for each image using AxioVision LE Re1.4.4 software.

[0239] N-glycan, monosaccharide, sialic acid, O-glycan analysis

[0240] Once BCECs reached approximately 80% confluence, they were washed twice with phosphate buffered saline (PBS, Sigma) and harvested by scraping. Cells were pelleted by centrifugation at 300 g for 3 minutes and washed once with cold PBS. Cells were homogenized and total protein was measured. All subsequent analyses were based on known protein content.

[0241] N-linked glycans were removed from glycoprotein samples using PNGase-F kit (New England Biolabs, P0705S). In short, 300 g protein samples were reconstructed in 180 μl ultrapure water. 20 μl 10X denaturation buffer was added and boiled in a 100 °C water bath for 14 minutes. The sample was cooled to room temperature and centrifuged at 2700 g for 1 minute. Subsequently, 50 μl 10XNP-40 was added, and the sample was kept at room temperature for 30 minutes, vortexed every 5 minutes, and then 25 μL 10X reaction buffer was added and mixed thoroughly. 5 μl PNGaseF (2500U) was then added to the sample, and gently mixed. The sample was incubated at 37 °C for 16 hours. The released N-glycans were purified using solid phase extraction. In brief, N-glycans were purified by passing the reaction mixture sequentially through a pretreated Sep-Pak C18 1cc cartridge (Waters) and a HyperSep PGC (polygraphitized charcoal) cartridge (25 mg, 1 ml Thermo Scientific). The cartridge was washed with 4 ml of water, and the separate PGC was washed with another 1 ml of water. The N-glycans bound to PGC were eluted with a 30% acetonitrile aqueous solution containing 0.1% TFA. Finally, the purified N-glycans were lyophilized and labeled with 2-AB. In brief, the sample was dissolved in 0.44M 2-AB (2-aminobenzamide) in 10 μl of a solution containing 1M sodium cyanoborohydride in DMSO containing 35% acetic acid. The sample was incubated at 65°C for 2.5 hours. GlycoClean S cartridges (GLYKO) were used to purify 2-AB labeled glycans according to their glycan purification scheme. Excess reagents were removed from the samples using Glycoclean S-cartridges (Prozyme) and labeled glycans were dried using SpeedVac and stored at -20°C. Analysis of 2-AB labeled glycans was obtained using a Dionex CarboPac PA1 (4X 250 mm) anion exchange column and a guard column (4X50 mm) at a flow rate of 1 ml / min. Glycans were separated using a 0-250 mM sodium acetate gradient in 100 mM sodium hydroxide over 0-75 minutes. Data were collected using a Dionex ICS-3000 HPLC system with an Ultimate 3000 fluorescence detector (Dionex) set at Aex 330 nm and Atm 420 nm with a sensitivity of 7. Data were processed using Chromeleon software (Thermo Scientific).

[0242] HPAEC-PAD (Thermo-Dionex ICS3000) is used to carry out monosaccharide composition analysis, and the nanomolar amount of each monosaccharide present in 25 μg protein is calculated. Samples are hydrolyzed for 4 hours using 2N trifluoroacetic acid (TFA) at 100 ℃. Dry nitrogen is subsequently used to flush and remove acid. In order to ensure complete removal of acid, the sample is co-evaporated twice with 100 μl 50% isopropanol (IPA). Finally, the sample is dissolved in Milli-Q water and injected on HPAEC-PAD. Monosaccharide analysis is carried out using Dionex CarboPae PA1 column (250mm x 4mm; with 50mm x 4mm guard column). The isocratic solvent mixture of 19mM sodium hydroxide and 0.95mM sodium acetate is used for 25 minutes at a flow rate of 1ml per minute. The standard Quad waveform for carbohydrates supplied by the manufacturer is used to obtain data. All neutral and amino sugars are identified and quantified by comparison to an authentic monosaccharide standard mixture consisting of L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose, and D-mannose.

[0243] Mild acid hydrolysis was used to release sialic acid. In brief, the sample was treated with 2M acetic acid at 80°C for 3 hours, followed by rapid vacuum removal of excess acid. Sialic acid was then labeled with DMB reagent and analyzed using a RP-UPLC-FL (Waters Acquity UPLC) system. A known amount of standard Neu5Ac was used to quantify the amount of sialic acid in the sample.

[0244] For O-glycan analysis, homogenized cell samples were treated with 50 mM NaOH for 16 hours at 45 °C in the presence of 1 M NaBH4. The reaction mixture was slowly neutralized using ice-cold 30% acetic acid. The neutralized reaction mixture was then passed through Dowex 50-X cation exchange resin to remove sodium ions and lyophilized. Excess boric acid produced during neutralization was then removed by co-evaporation using acidified methanol and methanol, respectively. Finally, the O-glycans were purified by a C18 cartridge. The dried and purified O-glycans were then methylated and used for O-glycan analysis after permethylation. The permethylated samples were then dissolved in anhydrous methanol and mixed with SDHB (Super-DHB) MALDI matrix at a 1:1 v / v ratio and spotted on a maldi plate. Mass spectral data were acquired using a Bruker AutoFlex mass spectrometer in positive reflection mode. The mass spectral data were analyzed and annotated using GlycoWork Bench software, and the masses that matched the proposed structure were annotated. Monoisotopic ion intensities were used for calculations.

[0245] To measure the cellular uptake of ManN and the subsequent conversion to ManN-6P, BCEC were grown in 60 mm culture dishes to a density of approximately -6X 105 cells per dish. ManN was added to the culture at a final concentration of 400 μM. The cells were then incubated for 2 hours. The monolayer cells were washed three times with PBS at room temperature, and the cells were lifted in 10 ml PBS by a cell scraper on ice. The cell pellet was obtained by centrifugation at 400 g for 5 minutes and stored at -80 ° C for further use. In the presence of 1 μl of protease inhibitors, the cell pellet was suspended in 200 μl of ultrapure ice-cold water. The cells were sonicated for 1 minute with 30-second pulses and vortexed to form a homogenous solution. 2.5 μl of homogenate was used for protein evaluation in triplicate using the BCA assay. A standard curve of BSA with a concentration between 0-800 μg / ml was used to quantify the total protein amount. The cell homogenate was filtered through a pre-washed 3K filter, and the filtrate was dried with a speed-vac. The dried sample was reconstituted in 100 μl ultrapure water, and the sample containing 200 μg equivalent protein was injected onto the HPAEC-PAD. Known amounts (1 nmol) of ManN, glucose, mannose and ManN-6P standards were used to quantify the sugars present in the sample. Except for ManNH2-6P, all standards were obtained from Sigma Aldrich. ManNH2-6P was obtained from Omicron Biochemicals, Inc (South Bend, IN, Indiana). The amount of monosaccharides present in different cells was expressed as nmol / mg of total protein. All analyses were performed in Thermo-Dionex ICS systems using CarboPac-PA-1 columns with 100 mM NaOH and 250 mM NaOAc as HPLC running buffer.

[0246] Biotinylation of surface proteins

[0247] 3 days before cell surface protein separation, BCEC plates were inoculated in 10cm cell culture dishes. The cells were washed three times with Dulbecco's PBS containing CaCl2 and MgCl2, and then incubated on ice for 30 minutes with EZ-Link Sulfo-NHS-SS-biotin (Pierce, Rockford, IL, USA; 0.5mg / ml in Dulbecco). The cells were washed twice with Dulbecco's, and unreacted biotin was blocked with 20mM glycine for 15 minutes. In order to prevent the reduction of disulfide bonds in biotin molecules during the cell lysis process, 100 μM oxidized glutathione (Sigma Aldrich, St. Louis, MO) was added in the last washing solution. For cell lysis, 500 μL of lysis buffer (2% NP-40, 1% Triton X-100, 10% glycerol, 100 μM oxidized glutathione, EDTA-free protease inhibitor tablets) (Roche, Mannheim, Germany) in PBS was added to the cells. The lysed cell extracts were scraped off the plate and transferred to Eppendorf tubes and then incubated on ice for 30 minutes on a shaker. The cell extracts were incubated with 30U DNase (50 minutes at 22°C, Roche, Mannheim, Germany) and centrifuged for 20 minutes (20,800xg at 4°C) to precipitate insoluble material. The protein concentration of the supernatant was determined. Equal amounts of protein (-2 mg) from each extract were used for cell surface protein isolation. The supernatant was pre-clarified using biotin agarose beads (Pierce ImmunoPure immobilized D-biotin, Thermo Fisher Scientific, 20221), and the pre-clarified solution was used for cell surface protein separation using streptavidin beads. The beads were washed four times with lysis buffer, four times with lysis buffer containing 300mM NaCl, and twice with 50mM Tris-HCl at pH 7.8. The protein was eluted twice at 30°C with elution buffer (50mM Tris-HCl at pH 7.8 containing 50mM DTT), and the eluates were then combined. Three biological replicates and one non-biotinylated control were used in the study.

[0248] Gene expression analysis by real-time Q-PCR

[0249] RNA was prepared using the RNeasy mini kit (Qiagen). Real-time PCR (Taqman) analysis was performed using 50 ng of total RNA per reaction. The reaction was set in a MicroAmp fast optical 96-well reaction plate, sealed with a MicroAmp optical adhesive film, and run on a ViiA7 real-time PCR system (Applied Biosystems), and absolute quantification was performed using a standard curve using a sequence detection system (SDS) software. The expression level of each gene was further quantified relative to the housekeeping gene RPL19 in the same sample. Taqman primer and probe mixtures were obtained from Thermo Fisher Scientific. Bovine VEGF-A (Bt03213282), bovine RPL19 (Bt03229687) and bovine specific VEGFR2 (Bt03258877), GLUT1 (Bt03215313) and GLUT4 (Bt03215316).

[0250] α-Mannosidase, α- and β-glucosidase activity assays

[0251] The α-mannosidase activity was measured using the substrate p-nitrophenyl α-mannopyranoside (1 mM). The enzyme from Canavalia juncea (M7257) (final concentration of 0.077 U) was incubated at 37°C in a final volume of 50 μl of 50 mM potassium phosphate buffer at pH 7.5. The α-glucosidase was assayed using the substrate p-nitrophenyl α-glucoside (7 mM). The enzyme from Saccharomyces cerevisiae type 1 (Sigma, G5003) (final concentration of 0.1 U) was incubated at 37°C in a final volume of 50 μl of PBS at pH 7.5. The β-glucosidase was assayed using the substrate 4-nitrophenyl β-D-pyranoside (Roche). The enzyme from almond (Sigma, G0395) (final concentration 0.002U) was incubated at 37°C in a final volume of 50 μl PBS pH 7.5 containing 1% SDS. The incubation was stopped by adding an equal volume of acid-based stop solution (R&D Systems, 895032). The enzyme activity of α-glucosidase from Saccharomyces cerevisiae type I (G5003) was measured at 405 nm using p-nitrophenyl α-D-pyranoglucopyranoside (Sigma, N1377) as substrate.

[0252] Measurement of ManN in wound fluid of mice infected with Staphylococcus aureus (S. aureus)

[0253] In order to test the ManN stability in wound fluid collected from mice suffering from skin infection as described below, 1.5 μl 5% ManN solution was added to each 200 μl wound fluid, which was first diluted with PBS at 1:1 (v / v). At each time point, samples were taken out from 37°C incubators and stored at -80°C. Plasma proteins were precipitated by adding ice-cold acetonitrile to 1:3 (v / v) ratio of plasma:acetonitrile. The samples were stored on ice for 1 hour, and then centrifuged at 12000g for 10 minutes at 7°C to form a precipitate. The supernatant was transferred to other tubes, dried on Speed ​​Vac, and then reconstituted in ultrapure distilled water, and filtered by pre-washed Nanosep 3K Omega filter (Pall Corporation). The filtrate was dried on Speed ​​Vac. The dried sample was dissolved in 100 μl water, and HPLC analysis was performed on 2 μl plasma or wound fluid samples. Neutral sugars and amino sugars are separated on a Dionex CarboPae PA1 column of 4mm x250mm with a 4mm x 50mm guard column. An isocratic gradient of 19mM sodium hydroxide and 0.95mM sodium acetate is run for 20 minutes at a flow rate of 1 ml / min. A Dionex ICS-3000HPLC system with a pulsed amperometric detector is used to collect data using a standard Quad waveform. ManN is identified and quantified by comparing it with monosaccharide standards using Thermo Scientific Chromeleon software. No ManN sample serves as a negative control.

[0254] Skin wound healing model

[0255] All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego and performed in an ethical manner and in accordance with the guiding principles of the Animal Care Program (ACP).

[0256] The model has been described previously. In brief, C57BL / 6 female mice (8-10 weeks old) were obtained from Jackson Labs (Sacramento, California). In a Class II biological safety cabinet, under the influence of isoflurane, a fresh full-thickness perforation wound (4mm diameter) was produced on the back of the animal using a sterile neoprene ring (6-mm outer diameter and 4-mm inner diameter) splint and a perforator (Acu perforator, Acuderm, Ft. Lauderdale, FL) fastened with 5-6 sutures (4-0 nylon). All surgical procedures follow aseptic techniques. Buprenorphine was given subcutaneously before waking up from anesthesia for expected pain. Mice were monitored until they were fully awake and housed separately to minimize damage / bites / fighting to the surgical site. Recombinant human VEGF was a gift from Roche-Genentech (Telbermin, recombinant human VEGF165). The treatment was prepared in PBS, sterile filtered, and 25 μl of solution was applied directly to the wound bed daily under the influence of isoflurane for the first 4-5 days and then observed daily. Wound closure was monitored by conventional imaging, and wound area was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).

[0257] On the 4th day after injury, the wound with 2mm margin of surrounding tissue was excised and placed in 10% formalin for up to 24 hours. The wound was then divided into two from the center, and 5 μm paraffin sections were processed for hematoxylin and eosin (H&E) and Masson's Trichrome staining. The epithelial gap was measured for histomorphometry using AxioVision LE Re1.4.4 software. The skin tissue was fixed in 10% formalin for 24 hours. Paraffin embedding and sectioning were performed by the Histology Center of UCSD Moores Cancer Center. Before performing heat-induced antigen retrieval in 10mM citrate buffer (pH6.0), 5 μm paraffin sections were deparaffinized and rehydrated. Immunostaining was performed as previously described. Anti-CD31 (SZ31, rat IgG2a) (Dianova, Warburgstrasse 45, 20354 Hamburg, Germany) was used at 2 μg / ml. Small blood vessels positive for CD31 were counted microscopically in 10 fields (20X) taken around the wound.

[0258] Vascular permeability assay

[0259] Vascular permeability was assessed using a modified Miles assay. Hairless male guinea pigs (Crl:HA-Hrhr / IAF, 75 days old, 450-500 g, Charles River Laboratories) were anesthetized by intraperitoneal (ip) administration of xylazine (5 mg / kg) and ketamine (75 mg / kg). The animals then received an intravenous injection (penile vein) of 1 ml 1% Evans blue dye. After 15 minutes, different doses of ManN were intradermally injected (0.05 ml / each site) into the trunk area behind the shoulder. All reagents were diluted in PBS for intradermal administration. 25 ng of VEGF165 per site was used as a positive control. 30 minutes after the intradermal injection, the animals were euthanized by ip injection of pentobarbital (200 mg / kg). Skin tissue was dissected out from the connective tissue and photographed.

[0260] Mouse skin infection model

[0261] Skin infection model of mice was established. In brief, mid-logarithmic Staphylococcus aureus subcultured from overnight cultures in Todd Hewitt broth was used in this study. 6-8 week-old C57BL / 6 mice were obtained from Charles River Laboratories. Before infection, mice were shaved and depilated with Nair cream. 5x 107CFU of Staphylococcus aureus was injected intradermally into the left groin of mice. After 3 days, the abscess was removed surgically and homogenized on ice. Fluid was collected and rotated at 14000rpm. The clarified supernatant was diluted with PBS 1:1 for further use. Animals were housed in clean cages, and the experimental procedures thereafter were carried out under pathogen-free conditions. Todd Hewitt broth (THB) plates were used to confirm the presence of bacteria in wound fluid.

[0262] Hindlimb ischemia model and blood flow assessment

[0263] C57BL / 6 male mice (6-8 weeks old) received unilateral hind limb surgery under anesthesia with a ketamine / xylazine mixture. In short, the left femoral artery was separated from the vein and nerve, ligated proximally, and excised. The right hind limb served as a control. Blood flow was measured using a laser Doppler perfusion imager (PeriScan PSI; Perimed). Ischemic limb perfusion and non-ischemic limb perfusion were measured before and after surgery and 1 week, 2 weeks, and 3 weeks after surgery. After surgery, mice were randomly assigned to different groups (8 mice per group). Starting from the 3rd day after surgery, 200 μl of 20% ManN was orally administered every other day. 200 μl 1 mg / nil Kif was administered every other day by ip injection. H2O was used as a vehicle control. The final blood flow value was expressed as the ratio of ischemic hind limb perfusion to non-ischemic hind limb perfusion of the same animal. Vascular area was quantified as described.

[0264] Retinal neovascularization

[0265] Retinal angiogenesis after intravitreal administration was evaluated. Briefly, 6-8 week old C57BL / 6 male mice were randomly assigned to different groups and anesthetized with a ketamine / xylazine mixture. 1 μl of ManN, Kif or bFGF (R&D Systems, AF-233-NA) indicated in PBS and PBS vehicle control were injected intravitreally with a 33-gauge Hamilton syringe. Seven days after injection, the animals were euthanized. The eyes were then removed and fixed in 4% paraformaldehyde (PFA) for 30 minutes. The retina was separated and stained with anti-CD31 immunofluorescence (IF) to confirm the vasculature. The evaluation was performed by a researcher who was blind to the treatment. For CD31 IF, rat anti-mouse antibody (BD Biosciences, catalog number 550274) was diluted 1:100 and incubated overnight at 4°C. After incubation for 4 hours with Alexa Fluor-488 conjugated anti-rat antibody (Life Technologies, A11006), intact scaffolds were imaged using an A1R confocal STORM super-resolution system (Nikon) through the 488 nm channel. Vascular density in the choroid and retina was quantified by Image J. Each experiment was repeated three times with similar results, and each treatment group consisted of 5 individual samples.

[0266] Middle cerebral artery occlusion stroke model

[0267] The extent of cerebral infarct size as regulated by ManN treatment was assessed. A mouse model of acute stroke was selected to study the effects of ManN pretreatment and sustained posttreatment on pathogenic ischemic events. This experiment was performed in duplicate. Adult C57BL / 6J mice weighing between 20-25 g (Jackson Laboratory, Bar Harbor, ME) were randomly assigned to each group. One day before surgery (day -1), hexosamine D-mannosamine (ManN) treatment was started. The drug (ManN) was administered by oral gavage; 40 mg (200 μL 20% ManN aqueous solution) was taken to each mouse. In the control group of mice, water was given each time as a vehicle control. This treatment regimen was repeated every 48 hours (e.g., on the 1st day, the 3rd day, etc.) until the extent of the infarct area of ​​the animal could be examined. Surgery was performed as previously described. In brief, on day 0, mice were anesthetized with a ketamine / xylazine mixture. Then the mouse is placed in the supine position and prepared in an aseptic manner. Make a midline incision in the neck, and gently retract the soft tissue on the trachea. Carefully expose and separate the left common carotid artery (CCA). After the proximal double ligation of CCA, the external carotid artery (ECA) is ligated to prevent bleeding, and the internal carotid artery (ICA) is temporarily occluded with a micro-artery clamp. A small incision is made at 2mm away from the distal side of the ECA-CCA branch, and a 6-0 silicone-coated round tip monofilament nylon suture (6022910PK10Re, Doccol Corporation, MA, USA) is gently inserted through the CCA. After removing the micro-artery clamp on the ICA, the filament is advanced into the ICA until it blocks the starting point (approximately 9-10mm) of the middle cerebral artery. After occlusion for 2 hours, the suture is removed to restore the blood supply in the MCA region. During the whole procedure, from the beginning of the surgical operation until the animal wakes up from anesthesia, the heating pad on the surgical table is used to maintain the body temperature at 36.5-37.5°C. For sham-operated animals, vascular separation was performed without inserting sutures. One day after the operation, the neurological score was assessed according to the behavior of the mice. MRI imaging of the brain was performed 24 hours after reperfusion and every other day until a definite endpoint was reached. T2-weighted (T2w) imaging was performed to cerebral infarction. Ten slices with a thickness of 1 mm and a field of view of 2 x 2 cm were placed above the brain. Alternatively, the brain of the mouse was collected 24 hours after reperfusion to perform TTC staining. Brain slices were taken at intervals of 1 mm on the coronal plane. Fresh brain slices were placed in 1% TTC solution to determine the infarct area.

[0268] Statistics and reproducibility

[0269] Statistical parameters including n values ​​are indicated in the legend. According to the advice of the Biostatistics and Bioinformatics Department of Moores Cancer Center, the sample size was determined to ensure sufficient power. Two-tailed, two-sample unequal variance t-tests were used. Since the method does not require normal assumptions for variables, the statistical significance between the concerned treatment groups of some in vitro data sets was further confirmed using the Wilcoxon rank sum test. Statistical inference is based on the p-value of each comparison using the R function "wilcox.test". The wound area (percentage) between the three treatment groups (ManN, VEGF, VEGF+ManN) and the PBS group was investigated using a linear mixed effects (LME) model. Two LME models were fitted. In the first LME model, the control group was considered as a reference group. The day effect (treating the day as a categorical variable rather than a continuous variable) and its interaction with treatment were included as fixed effects, and the subject id was included as a random effect to involve the correlation between the measurements of the same subject on different days. There was no difference between the different groups at baseline. In the second LME model, the VEGF+ManN group was used as a reference group to study the comparison between a single treatment group and a combined treatment. For each LME model, different treatment effects and their corresponding p-values ​​were explored relative to the reference group at day 3, day 5, and day 8. Data were considered significant when p < 0.05. Significant p-values ​​are indicated in the figure as follows: *** p<0.001, ** p<0.01, * p<0.05. For each experiment, representative experimental results from 2-5 independent studies are shown.

[0270] Although the foregoing disclosure has been described in some detail for the purpose of clarity and understanding, it will be clear to those skilled in the art, through reading this disclosure, that various changes in form and detail may be made without departing from the true scope of the invention. For example, all techniques and equipment described above may be used in various combinations. All disclosures, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes, to the same extent as if each individual disclosure, patent, patent application, and / or other document were individually and separately indicated to be incorporated by reference for all purposes.

[0271] aspect

[0272] Aspect 1: A pharmaceutical composition for treating stroke, comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof.

[0273] Aspect 2: The pharmaceutical composition according to aspect 1, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas) or a combination thereof.

[0274] Aspect 3: The pharmaceutical composition according to Aspect 1, wherein the N-glycosylation inhibitor comprises ManN.

[0275] Aspect 4: The pharmaceutical composition according to any one of Aspects 1 to 3, wherein the subject has experienced ischemic stroke.

[0276] Aspect 5: The pharmaceutical composition according to Aspect 4, wherein the ischemic stroke comprises thrombotic stroke or embolic stroke.

[0277] Aspect 6: The pharmaceutical composition according to any one of Aspects 1 to 3, wherein the subject has experienced a hemorrhagic stroke.

[0278] Aspect 7: The pharmaceutical composition according to Aspect 6, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

[0279] Aspect 8: The pharmaceutical composition according to any one of Aspects 1 to 3, wherein the subject has experienced a brainstem stroke.

[0280] Aspect 9: The pharmaceutical composition according to any one of Aspects 1 to 3, wherein the subject has experienced a transient ischemic attack.

[0281] Aspect 10: The pharmaceutical composition according to any one of Aspects 1 to 3, wherein the subject has experienced a cryptogenic stroke.

[0282] Aspect 11: The pharmaceutical composition according to any one of Aspects 1 to 10, wherein the effective amount of the N-glycosylation inhibitor comprises a single dosing treatment regimen.

[0283] Aspect 12: The pharmaceutical composition according to Aspect 11, wherein the single-dose treatment regimen comprises pre-treatment, concurrent treatment and / or post-treatment associated with an ischemic event affecting the brain.

[0284] Aspect 13: The pharmaceutical composition according to any one of Aspects 1 to 10, wherein the effective amount of the N-glycosylation inhibitor comprises a multiple administration treatment regimen.

[0285] Aspect 14: The pharmaceutical composition according to Aspect 13, wherein the multiple dosing treatment regimen comprises pre-treatment, concurrent treatment and / or post-treatment associated with an ischemic event affecting the brain.

[0286] Aspect 15: The pharmaceutical composition according to any one of Aspects 1 to 14, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally.

[0287] Aspect 16: The pharmaceutical composition according to any one of Aspects 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0288] Aspect 17: The pharmaceutical composition according to any one of Aspects 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0289] Aspect 18: The pharmaceutical composition according to any one of Aspects 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 100 g of the N-glycosylation inhibitor.

[0290] Aspect 19: The pharmaceutical composition according to any one of Aspects 1 to 18, wherein the pharmaceutical composition further comprises an effective amount of a pro-angiogenic factor.

[0291] Aspect 20: The pharmaceutical composition according to Aspect 19, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

[0292] Aspect 21: The pharmaceutical composition according to Aspect 20, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF) or a combination thereof.

[0293] Aspect 22: The pharmaceutical composition according to Aspect 20 or Aspect 21, wherein the VEGF is recombinant VEGF.

[0294] Aspect 23: The pharmaceutical composition according to any one of Aspects 20 to 22, wherein the VEGF is administered locally near the site of the brain of the subject affected by the ischemic event.

[0295] Aspect 24: A method of treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof.

[0296] Aspect 25: The method according to aspect 24, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas) or a combination thereof.

[0297] Aspect 26: The method according to aspect 25, wherein the N-glycosylation inhibitor comprises ManN.

[0298] Aspect 27: The method according to any one of Aspects 24 to 26, wherein the subject has experienced an ischemic stroke.

[0299] Aspect 28: The method according to Aspect 27, wherein the ischemic stroke is a thrombotic stroke or an embolic stroke.

[0300] Aspect 29: The method according to any one of Aspects 24 to 26, wherein the subject has experienced a hemorrhagic stroke.

[0301] Aspect 30: The method according to Aspect 29, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

[0302] Aspect 31: The method according to any one of Aspects 24 to 26, wherein the subject has experienced a brainstem stroke.

[0303] Aspect 32: The method according to any one of Aspects 24 to 26, wherein the subject has experienced a transient ischemic attack.

[0304] Aspect 33: The method according to any one of Aspects 24 to 26, wherein the subject has experienced a cryptogenic stroke.

[0305] Aspect 34: The method according to any one of aspects 24 to 33, wherein the effective amount of the N-glycosylation inhibitor comprises a single dosing treatment regimen.

[0306] Aspect 35: The method according to Aspect 34, wherein the single-dose treatment regimen comprises pre-treatment, concurrent treatment and / or post-treatment associated with an ischemic event affecting the brain.

[0307] Aspect 36: The method according to any one of aspects 24 to 35, wherein the effective amount of the N-glycosylation inhibitor comprises a multiple administration treatment regimen.

[0308] Aspect 37: The method according to Aspect 36, wherein the multiple dosing treatment regimen comprises pre-treatment, concurrent treatment and / or post-treatment associated with an ischemic event affecting the brain.

[0309] Aspect 38: The method according to any one of aspects 24 to 37, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally.

[0310] Aspect 39: The method according to any one of aspects 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0311] Aspect 40: The method according to any one of aspects 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0312] Aspect 41: The method according to any one of aspects 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 100 g of the N-glycosylation inhibitor.

[0313] Aspect 42: The method according to any one of aspects 34 to 41, wherein the single dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 20 mg to about 900 mg / kg body weight of the subject.

[0314] Aspect 43: The method according to any one of aspects 34 to 41, wherein the single dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 200 mg to about 700 mg / kg body weight of the subject.

[0315] Aspect 44: The method according to any one of aspects 36 to 41, wherein the multiple dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 10 mg to about 900 mg / kg body weight of the subject.

[0316] Aspect 45: The method according to any one of aspects 36 to 41, wherein the multiple dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 200 mg to about 700 mg / kg body weight of the subject.

[0317] Aspect 46: The method according to any one of aspects 24 to 45, wherein said administering to the subject further comprises administering an effective amount of a pro-angiogenic factor.

[0318] Aspect 47: The method according to aspect 46, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

[0319] Aspect 48: The method according to aspect 47, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF) or a combination thereof.

[0320] Aspect 49: The method according to aspect 47 or aspect 48, wherein the VEGF is recombinant VEGF.

[0321] Aspect 50: The method according to any one of aspects 47 to 49, wherein the VEGF is administered locally near the site of the brain of the subject affected by the ischemic event.

[0322] Aspect 51: The method according to any one of aspects 24 to 50, wherein N-linked protein glycosylation is inhibited in endothelial cells of the subject.

[0323] Aspect 52: The method according to any one of aspects 24 to 51, wherein the administration is effective to stimulate endothelial cell proliferation and angiogenesis in blood vessels near the brain.

[0324] Aspect 53: The method according to any one of aspects 24 to 52, wherein the administration is effective to stimulate brain endothelial cell proliferation and angiogenesis.

[0325] Aspect 54: The method according to any one of aspects 24 to 53, wherein the administration is effective to activate JNK signaling and upregulate the unfolded protein response caused by endoplasmic reticulum stress.

[0326] Aspect 55: A method of preventing stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof.

[0327] Aspect 56: The method according to aspect 55, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas) or a combination thereof.

[0328] Aspect 57: The method according to aspect 56, wherein the N-glycosylation inhibitor comprises ManN.

[0329] Aspect 58: The method according to any one of aspects 55 to 57, wherein the effective amount of the N-glycosylation inhibitor comprises a single dosing treatment regimen.

[0330] Aspect 59: The method according to any one of aspects 55 to 57, wherein the effective amount of the N-glycosylation inhibitor comprises a multiple administration regimen.

[0331] Aspect 60: The method according to any one of aspects 55 to 59, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally.

[0332] Aspect 61: The method according to any one of aspects 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0333] Aspect 62: The method according to any one of aspects 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0334] Aspect 63: The method according to any one of aspects 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 100 g of the N-glycosylation inhibitor.

[0335] Aspect 64: The method according to any one of Aspects 55 to 63, wherein the risk of recurrent stroke in the subject is reduced.

[0336] Aspect 65: The method according to Aspect 64, wherein the subject has previously experienced an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke.

[0337] Aspect 66: The method according to Aspect 64 or Aspect 65, wherein the risk of recurrent stroke comprises the risk of ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack or cryptogenic stroke.

[0338] Aspect 67: The method according to any one of aspects 55 to 66, wherein the measurement of C-reactive protein (CRP) in the subject's blood is reduced compared to a previous measurement of CRP levels in the subject's blood.

[0339] Aspect 68: The method according to aspect 67, wherein the measurement of CRP in the subject's blood is reduced to below about 10 mg / L.

[0340] Aspect 69: A method of reducing a sustained ischemic event affecting the brain, the method comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor.

[0341] Aspect 70: The method according to aspect 69, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas) or a combination thereof.

[0342] Aspect 71: The method according to aspect 70, wherein the N-glycosylation inhibitor comprises ManN.

[0343] Aspect 72: The method according to any one of Aspects 69 to 71, wherein the subject is experiencing an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke.

[0344] Aspect 73: The method according to any one of aspects 69 to 72, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally.

[0345] Aspect 74: The method according to any one of aspects 69 to 73, wherein the effective amount of the N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0346] Aspect 75: The method according to any one of aspects 69 to 73, wherein the effective amount of the N-glycosylation inhibitor comprises about 5 g to about 100 g of the N-glycosylation inhibitor.

[0347] Aspect 76: The method according to any one of Aspects 69 to 75, wherein the subject's neurological assessment of acute stroke is improved compared to the subject's previous assessment.

[0348] Aspect 77: The method according to any one of Aspects 69 to 76, wherein blood flow to the subject's brain is improved.

[0349] Aspect 78: The method according to Aspect 77, wherein the blood flow of the brain is assessed by transcranial Doppler ultrasound.

[0350] Aspect 79: A method of treating stroke in a subject in need thereof, the method comprising:

[0351] a) identifying subjects who have experienced a stroke event;

[0352] b) administering to the subject a therapeutically effective amount of one or more N-glycosylation inhibitors;

[0353] c) determining whether the subject has suffered an ischemic stroke or a hemorrhagic stroke;

[0354] d) administering a therapeutically effective amount of an anticoagulant if the subject is determined to have an ischemic stroke, or refraining from administering a therapeutically effective amount of an anticoagulant if the subject is determined to have a hemorrhagic stroke; and

[0355] e) administering at least one N-glycosylation inhibitor for the duration of the treatment; wherein the N-glycosylation inhibitors of step b) and step e) are the same or different.

[0356] Aspect 80: The method according to Aspect 79, wherein step c) comprises performing a CT scan or an MRI scan on the subject.

[0357] Aspect 81: The method according to aspect 79 or 80, wherein in step e), the N-glycosylation inhibitor is administered 1 to 7 times per week.

[0358] Aspect 82: The method according to any one of aspects 79 to 81, wherein the duration of treatment in step e) is in the range of about 1 day to about 12 weeks.

[0359] Aspect 83: The method according to any one of Aspects 79 to 82, wherein the subject has a reduction in infarct size compared to anticoagulation therapy alone, TPA therapy alone, or a combination of anticoagulation alone and TPA therapy.

[0360] Aspect 84: The method according to any one of Aspects 79 to 83, wherein the subject has reduced disruption of the blood-brain barrier compared to anticoagulation therapy alone, TPA therapy alone, or a combination of anticoagulation alone and TPA therapy.

[0361] Aspect 85: The method according to any one of Aspects 79 to 84, wherein the subject has reduced hemorrhagic injury caused by ischemic stroke compared to anticoagulation therapy alone, TPA therapy alone, or a combination of anticoagulation alone and TPA therapy.

[0362] Aspect 86: The method according to any one of Aspects 79 to 85, wherein the subject's mortality rate is reduced compared to anticoagulation therapy alone, TPA therapy alone, or a combination of anticoagulation alone and TPA therapy.

[0363] Aspect 87: A method of treating degenerative (pathological) myopia in a subject in need thereof, the method comprising administering an effective amount of one or more N-glycosylation inhibitors into the vitreous of the subject, wherein after administration, one or more symptoms of degenerative (pathological) myopia are improved.

[0364] Aspect 88: The method according to aspect 88, wherein the one or more N-glycosylation inhibitors comprise ManN, Kif, Cas or any combination thereof.

[0365] Aspect 89: The method according to aspect 87 or 88, wherein the one or more N-glycosylation inhibitors comprise ManN.

[0366] Aspect 90: The method according to any one of aspects 87 to 89, wherein said administering of said effective amount of one or more N-glycosylation inhibitors induces retinal angiogenesis in the eye of said subject.

[0367] Aspect 91: The method according to any one of aspects 87 to 90, wherein said administering said effective amount of one or more N-glycosylation inhibitors induces choroidal angiogenesis in the eye of said subject.

[0368] Aspect 92: The method according to aspect 90 or 91, wherein the induced retinal angiogenesis or induced choroidal angiogenesis restores vascular perfusion in the eye of the subject.

[0369] Aspect 93: The method according to any one of Aspects 87 to 92, wherein myopic choroidal neovascularization (CNV) is reduced in the eye of the subject.

[0370] Aspect 94: The method according to any one of aspects 87 to 93, wherein after said administering, the presence or progression of chorioretinal atrophy (CRA) proximal to the area of ​​myopic CNV in the eye of said subject is reduced.

[0371] Aspect 95: The method according to any one of Aspects 87 to 94, wherein after said administration, progression of an additional visual disorder in said subject is reduced or halted.

[0372] Aspect 96: A method according to any one of Aspects 87 to 95, wherein the subject exhibits a reduction in disease progression of one or more symptoms selected from the group consisting of diffuse chorioretinal atrophy, patchy chorioretinal atrophy, lacquer cracks, myopic CNV, CNV-related macular atrophy, progressive visual loss, foveal fissure, metamorphopsia, blur, dark spots, reading difficulties, ocular floaters or flashes, loss of central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and poor contrast sensitivity.

[0373] Aspect 97: A method according to any one of Aspects 87 to 96, wherein the subject exhibits cessation of disease progression of one or more symptoms selected from the group consisting of diffuse chorioretinal atrophy, patchy chorioretinal atrophy, lacquer cracks, myopic CNV, CNV-related macular atrophy, progressive visual loss, foveal fissure, metamorphopsia, blur, dark spots, reading difficulties, ocular floaters or flashes, loss of central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and poor contrast sensitivity.

[0374] Aspect 98: A method according to any one of Aspects 87 to 97, wherein the subject exhibits improvement in one or more symptoms selected from the group consisting of diffuse chorioretinal atrophy, patchy chorioretinal atrophy, lacquer cracks, myopic CNV, CNV-related macular atrophy, progressive visual loss, foveal fissure, metamorphopsia, blur, dark spots, reading difficulties, ocular floaters or flashes, loss of central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and poor contrast sensitivity.

[0375] Aspect 99: The method according to any one of aspects 87 to 98, wherein spectral domain optical coherence tomography is used to monitor disease progression and treatment effectiveness.

[0376] Aspect 100: A method according to Aspect 99, wherein the use of spectral domain optical coherence tomography in monitoring disease progression is used in part to determine the frequency of administration of the N-glycosylation inhibitor as part of a treatment regimen, the therapeutic dose of the N-glycosylation inhibitor to be administered to the subject, or to stop administering the N-glycosylation inhibitor to the subject after one or more symptoms of degenerative (pathological) myopia improve.

Claims

1. A method for treating stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof.

2. The method of claim 1, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, or castanospermine (Cas), or a combination thereof.

3. The method of claim 1, wherein the N-glycosylation inhibitor comprises ManN.

4. The method of claim 2, wherein the subject has experienced an ischemic stroke.

5. The method of claim 4, wherein the ischemic stroke is a thrombotic stroke or an embolic stroke.

6. The method of claim 2, wherein the subject has experienced a hemorrhagic stroke.

7. The method of claim 6, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

8. The method of claim 2, wherein the subject has experienced a brainstem stroke.

9. The method of claim 2, wherein the subject has experienced a transient ischemic attack.

10. The method of claim 2, wherein the subject has experienced a cryptogenic stroke.

11. The method of claim 2, wherein the effective amount of the N-glycosylation inhibitor is delivered to the subject via a single dosing regimen.

12. The method of claim 11, wherein the single-dosing treatment regimen comprises pre-treatment, concurrent treatment, or post-treatment associated with an ischemic event affecting the subject's brain.

13. The method of any one of claims 2, wherein the effective amount of the N-glycosylation inhibitor is delivered to the subject via a multiple dosing regimen.

14. The method of claim 13, wherein the multiple dosing treatment regimen comprises pre-treatment, concurrent treatment, and / or post-treatment associated with an ischemic event affecting the subject's brain.

15. The method of claim 2, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

16. The method of claim 3, wherein the effective amount of ManN comprises about 10 mg to about 300 g per administration.

17. The method of claim 3, wherein the effective amount of ManN comprises about 200 mg to about 100 g per administration.

18. The method of claim 3, wherein the effective amount of ManN comprises about 5 g to about 40 g per administration.

19. The method of claim 12, wherein the single-dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 20 mg to about 2000 mg / kg of subject body weight.

20. The method of claim 12, wherein the single dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 100 mg to about 300 mg / kg body weight of the subject.

21. The method of claim 14, wherein the multiple dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 10 mg to about 2000 mg / kg body weight of the subject.

22. The method of claim 14, wherein the multiple dosing treatment regimen comprises administering the formulation of the N-glycosylation inhibitor at a dose of about 100 mg to about 300 mg / kg body weight of the subject.

23. The method of claim 2, wherein the administering to the subject further comprises administering an effective amount of a pro-angiogenic factor.

24. The method of claim 22, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

25. The method of claim 23, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF), or a combination thereof.

26. The method of claim 23, wherein the VEGF is recombinant VEGF.

27. The method of claim 23, wherein the VEGF is administered locally near a site in the brain of a subject affected by an ischemic event.

28. The method of claim 3, wherein N-linked protein glycosylation is inhibited in endothelial cells of the subject.

29. The method of claim 3, wherein the administration is effective to stimulate endothelial cell proliferation and angiogenesis in blood vessels proximal to the brain.

30. The method of claim 3, wherein the administration is effective to stimulate brain endothelial cell proliferation and angiogenesis.

31. The method of claim 20, wherein the administration is effective to activate JNK signaling and upregulate an unfolded protein response caused by endoplasmic reticulum stress.

32. The method of claim 3, wherein the subject exhibits improvement in one or more symptoms of stroke following the administering.

33. The method of claim 31, wherein the one or more symptoms are selected from the group consisting of muscle weakness, facial numbness, facial paralysis, limb paralysis, paralysis on one side of the body, slurred speech, disorganized speech, difficulty understanding others, sudden blindness in one or both eyes, double vision, vertigo, confusion, lack of mental alertness, loss of balance, loss of coordination, stroke-induced death, anoxic damage to one or more areas of the brain, stroke-induced memory impairment, stroke-induced autonomic movement disorders, stroke-induced language disorders, stroke-induced cognitive decline, stroke-induced decreased mobility.

34. The method of claim 3, wherein the subject exhibits a significant increase in blood vessel density in or near an area of ​​stroke-induced brain damage following the administration.

35. A pharmaceutical composition for treating stroke, comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof.

36. The pharmaceutical composition of claim 34, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas), or a combination thereof.

37. The pharmaceutical composition of claim 34, wherein the N-glycosylation inhibitor comprises ManN.

38. The pharmaceutical composition of any one of claims 34 to 36, wherein the subject has experienced an ischemic stroke.

39. The pharmaceutical composition of claim 37, wherein the ischemic stroke comprises thrombotic stroke or embolic stroke.

40. The pharmaceutical composition of any one of claims 34 to 36, wherein the subject has experienced a hemorrhagic stroke.

41. The pharmaceutical composition of claim 39, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

42. The pharmaceutical composition of any one of claims 34 to 36, wherein the subject has experienced a brainstem stroke.

43. A pharmaceutical composition according to any one of claims 34 to 36, wherein the subject has experienced a transient ischemic attack.

44. The pharmaceutical composition of any one of claims 34 to 36, wherein the subject has experienced a cryptogenic stroke.

45. The pharmaceutical composition of any one of claims 34 to 43, wherein the effective amount of the N-glycosylation inhibitor is delivered to the subject via a single dosing regimen.

46. ​​The pharmaceutical composition of claim 44, wherein the single-dose treatment regimen comprises pre-treatment, concurrent treatment, or post-treatment associated with an ischemic event affecting the brain.

47. The pharmaceutical composition of any one of claims 34 to 43, wherein the effective amount of the N-glycosylation inhibitor is delivered to the subject via a multiple dosing regimen.

48. The pharmaceutical composition of claim 46, wherein the multiple dosing treatment regimen comprises pre-treatment, concurrent treatment and / or post-treatment associated with an ischemic event affecting the brain.

49. The pharmaceutical composition according to any one of claims 34 to 47, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally or intraperitoneally.

50. The pharmaceutical composition of any one of claims 34 to 48, wherein the effective amount of an N-glycosylation inhibitor comprises about 10 mg to about 100 g of the N-glycosylation inhibitor.

51. The pharmaceutical composition of any one of claims 34 to 49, wherein the effective amount of an N-glycosylation inhibitor comprises about 200 mg to about 40 g of the N-glycosylation inhibitor.

52. The pharmaceutical composition of any one of claims 34 to 50, wherein the effective amount of an N-glycosylation inhibitor comprises about 5 g to about 20 g of the N-glycosylation inhibitor.

53. The pharmaceutical composition according to any one of claims 34 to 51, wherein the pharmaceutical composition further comprises an effective amount of a pro-angiogenic factor.

54. The pharmaceutical composition of claim 52, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

55. The pharmaceutical composition of claim 53, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF), or a combination thereof.

56. The pharmaceutical composition of claim 53 or 54, wherein the VEGF is recombinant VEGF.

57. A pharmaceutical composition according to any one of claims 53 to 55, wherein the VEGF is administered locally near a site in the brain of a subject affected by an ischemic event.

58. A method for preventing stroke, the method comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof.

59. The method of claim 57, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas), or a combination thereof.

60. The method of claim 57, wherein the N-glycosylation inhibitor comprises ManN.

61. The method of any one of claims 57 to 59, wherein the effective amount of the N-glycosylation inhibitor is delivered to the subject via a single dosing regimen.

62. The method of any one of claims 57 to 59, wherein the effective amount of an N-glycosylation inhibitor is delivered to the subject via a multiple dosing regimen.

63. The method of any one of claims 57 to 61, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

64. The method of any one of claims 57 to 62, wherein the effective amount of an N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

65. The method of any one of claims 57 to 63, wherein the effective amount of an N-glycosylation inhibitor comprises about 200 mg to about 100 g of the N-glycosylation inhibitor.

66. The method of any one of claims 57 to 64, wherein the effective amount of an N-glycosylation inhibitor comprises about 5 g to about 40 g of the N-glycosylation inhibitor.

67. The method of any one of claims 57 to 65, wherein the risk of recurrent stroke in the subject is reduced.

68. The method of claim 66, wherein the subject has previously experienced an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke.

69. The method of claim 66 or claim 67, wherein the risk of recurrent stroke comprises a risk of ischemic stroke, hemorrhagic stroke, brainstem stroke, transient ischemic attack, or cryptogenic stroke.

70. The method of any one of claims 57 to 68, wherein the measurement of C-reactive protein (CRP) in the subject's blood is reduced compared to a previous measurement of CRP levels in the subject's blood.

71. The method of claim 69, wherein the measurement of the CRP in the subject's blood is reduced to below about 10 mg / L.

72. A method of reducing a sustained ischemic event affecting the brain, the method comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor.

73. The method of claim 71, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kif, castanospermine (Cas), or a combination thereof.

74. The method of claim 72, wherein the N-glycosylation inhibitor comprises ManN.

75. The method of any one of claims 71 to 73, wherein the subject is experiencing an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke.

76. The method of any one of claims 71 to 74, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

77. The method of any one of claims 71 to 75, wherein the effective amount of an N-glycosylation inhibitor comprises about 10 mg to about 300 g of the N-glycosylation inhibitor.

78. The method of any one of claims 71 to 76, wherein the effective amount of an N-glycosylation inhibitor comprises about 5 g to about 40 g of the N-glycosylation inhibitor.

79. The method of any one of claims 71 to 77, wherein the subject's neurological assessment of acute stroke improves compared to the subject's previous assessment.

80. The method of any one of claims 71 to 78, wherein blood flow to the subject's brain is improved.

81. The method of claim 79, wherein the blood flow to the subject's brain is assessed by transcranial Doppler ultrasound.

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