Glycosylation inhibitors as therapeutic agents for stroke

Abstract

A pharmaceutical composition for treating an ischemic condition in a subject may include an inhibitor of protein glycosylation. The method may include administering an effective amount of an inhibitor of protein glycosylation to a subject in need thereof. The administration may be effective to promote endothelial cell proliferation and angiogenesis in the subject. The subject may be in need of induction of angiogenesis due to an ischemic condition caused by stroke. A pharmaceutical composition for inhibiting protein glycosylation in a cell may include ManN. The method for inhibiting protein glycosylation in a cell may include administering an effective amount of ManN to the cell.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 332,932, filed April 20, 2022, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference 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. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material. [Background technology]

[0003] Angiogenesis is a complex process involving the growth of new blood vessels from pre-existing vasculature and occurs in both physiological and pathological situations. In tumors, angiogenesis facilitates rapid growth and metastasis through the delivery of nutrients and oxygen and the removal of metabolic waste products. Vasculature development requires coordinated activation of multiple signaling pathways, including VEGF / VEGFR, angiopoietin (Ang) / Tie2, Notch, ephrin / Eph, and PDGF / PDGFR. Stimulating angiogenesis may facilitate the treatment of several conditions characterized by reduced perfusion, including diabetic ulcers, myocardial ischemia, and limb ischemia. Conversely, blocking angiogenesis is a clinically validated strategy for treating malignant tumors and intraocular neovascular disorders.

[0004] Endothelial cell (EC) metabolism has been hypothesized to play a key role in regulating angiogenesis in normal and pathological situations. It has been reported that metabolic switches in ECs, such as fatty acid, glucose, and glutamine metabolism, induce angiogenesis. ECs in tumor vasculature are known to depend on glycolysis for ATP production, for example, by enhanced expression of glucose transporter GLUT1. Reduced glycolysis in tumor ECs blocks their proliferation. Furthermore, aberrant glycosylation patterns have been identified in oncogenic transformation and cancer progression, and it has been proposed that inhibition of glycosylation may result in the suppression of key angiogenic pathways, including VEGF / VEGFR2 and Notch. Emerging evidence has shown glycans as novel regulators of angiogenesis through alterations in protein glycosylation. For example, it has been reported that the glycan-binding protein galectin-1 interacts with VEGFR2 and activates the receptor in a ligand-independent manner, which may contribute to tumor resistance to anti-VEGF therapy. Therefore, EC metabolism has been identified as a new target for antiangiogenic therapy, particularly through the inhibition of energy metabolism and glycosylation. Summary of the Invention

[0005] There remains a need for effective medications and methods for treating stroke. Stroke remains a debilitating condition that can result in lengthy rehabilitation periods, impaired ability to perform activities of daily living, social and economic difficulties, and can be fatal. Preventing first strokes, preventing recurrent strokes, reducing the effects of ongoing strokes, or improving stroke treatment and recovery would all greatly benefit society and improve the quality of life for many individuals.

[0006] The present disclosure provides pharmaceutical compositions and methods for treating an ischemic condition in a subject caused by stroke. In some embodiments, the pharmaceutical composition for treating stroke comprises 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), kifunensine (Kif), castanospermine (Cas), or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. 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 brain stem stroke. In some embodiments, the subject has experienced a transient ischemic attack. In some embodiments, the subject has experienced a cryptogenic stroke.

[0007] Described herein are pharmaceutical compositions that provide an effective amount of an N-glycosylation inhibitor for treating stroke. In some embodiments, the effective amount of an N-glycosylation inhibitor comprises a single dose treatment regimen. In some embodiments, the single dose treatment regimen comprises pre-treatment, concomitant treatment, or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of an N-glycosylation inhibitor comprises a multiple dose treatment regimen. In some embodiments, the multiple dose treatment regimen comprises pre-treatment, concomitant treatment, and / or post-treatment associated with an ischemic event affecting the brain. In some embodiments, the effective amount of an N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally. In some embodiments, the effective amount of an N-glycosylation inhibitor comprises about 10 mg to about 100 g of an N-glycosylation inhibitor. In some embodiments, the effective amount of an N-glycosylation inhibitor comprises about 200 mg to about 40 g of an N-glycosylation inhibitor. In some embodiments, the effective amount of an N-glycosylation inhibitor comprises from about 5 g to about 20 g of an N-glycosylation inhibitor.

[0008] Described herein are pharmaceutical compositions that provide an effective amount of an N-glycosylation inhibitor for treating stroke, which can be combined with other factors that regulate angiogenesis. In some embodiments, the pharmaceutical compositions described herein further comprise an effective amount of a pro-angiogenic factor. In some embodiments, the pro-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 site of the subject's brain affected by the ischemic event.

[0009] Described herein are methods for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the subject has experienced an ischemic stroke. In some embodiments, the ischemic stroke is 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 brain stem stroke. In some embodiments, the subject has experienced a transient ischemic attack. In some embodiments, the subject has experienced a cryptogenic stroke.

[0010] Described herein are methods for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. 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, concomitant 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, concomitant 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 N-glycosylation inhibitor comprises about 5 g to about 40 g of N-glycosylation inhibitor. In some embodiments, the single dose treatment regimen comprises administering the N-glycosylation inhibitor formulation at a dosage of about 20 mg to about 2000 mg / kg of subject body weight. In some embodiments, the single dose treatment regimen comprises administering the N-glycosylation inhibitor formulation at a dosage of about 100 mg to about 300 mg / kg of subject body weight. In some embodiments, the multiple dose treatment regimen comprises administering the N-glycosylation inhibitor formulation at a dosage of about 10 mg to about 2000 mg / kg of subject body weight. In some embodiments, the multiple dose treatment regimen comprises administering the N-glycosylation inhibitor formulation at a dosage of about 100 mg to about 300 mg / kg of subject body weight.

[0011] Described herein are methods of treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor, which may be combined with other factors that modulate angiogenesis. In some embodiments, administering to the subject further comprises administering an effective amount of a pro-angiogenic factor. In some embodiments, the pro-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 site of the subject's brain affected by the ischemic event.

[0012] Described herein is a method for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor, whereby N-linked glycosylation of proteins is inhibited in endothelial cells of the subject. Also described herein is a method for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor, whereby the administration is effective to stimulate endothelial cell proliferation and angiogenesis of blood vessels near the brain. Also described herein is a method for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor, whereby the administration is effective to stimulate endothelial cell proliferation and angiogenesis of the brain. Also described herein is a method for treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor, whereby the administration is effective to activate JNK signaling and enhance the unfolded protein response caused by endoplasmic reticulum stress. Also described herein is a method of treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof, wherein the subject shows improvement in one or more symptoms of stroke after administration. In some embodiments, the one or more symptoms are selected from the group consisting of muscle weakness, facial numbness, facial paralysis, paralysis of limbs, paralysis on one side of the body, slurred speech, incoherent speech, difficulty understanding others, sudden loss of vision in one or both eyes, double vision, dizziness, confusion, lack of mental alertness, loss of balance, impaired coordination, death due to stroke, hypoxic damage to one or more regions of the brain, memory impairment due to stroke, impaired voluntary movement due to stroke, speech impairment due to stroke, cognitive impairment due to stroke, and impaired mobility due to stroke. In some embodiments of the method of treating stroke described herein, the subject shows a significant increase in vascular density in or near the area of ​​brain damage due to stroke after administration.

[0013] In some embodiments, a pharmaceutical composition for treating an ischemic condition in a subject caused by stroke comprises an inhibitor of N-glycosylation. In some embodiments, a pharmaceutical composition for treating an ischemic condition in a subject caused by stroke comprises hexosamine D-mannosamine (ManN). In some embodiments, a method for treating an ischemic condition in a subject caused by stroke comprises administering to a subject in need thereof an effective amount of hexosamine D-mannosamine (ManN). The present disclosure also provides pharmaceutical compositions and methods for preventing the occurrence of stroke in a subject. The present disclosure also provides pharmaceutical compositions and methods for mitigating the effects of an ongoing ischemic event affecting the brain.

[0014] 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 to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of VEGF.

[0015] Described herein is a method for treating stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, N-linked glycosylation of proteins 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 endothelial cell proliferation and angiogenesis of the brain. In some embodiments, the administration is effective to activate JNK signaling and enhance the unfolded protein response caused by endoplasmic reticulum stress.

[0016] Described herein are methods of preventing stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (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 an N-glycosylation inhibitor comprises from about 5 g to about 40 g of an N-glycosylation inhibitor.

[0017] Described herein is a method of preventing stroke, comprising administering an effective amount of an N-glycosylation inhibitor to a subject in need thereof. In some embodiments, the risk of recurrent stroke is reduced in the 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. In some embodiments, the measurement of C-reactive protein (CRP) in the blood of the subject is reduced compared to the previous measurement of the blood CRP level in the subject. In some embodiments, the measurement of CRP in the blood of the subject is reduced to less than about 10 mg / L.

[0018] Described herein are methods of mitigating an ongoing ischemic event affecting the brain, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof. In some embodiments, the N-glycosylation inhibitor comprises ManN. In some embodiments, the subject is experiencing an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a 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 improves compared to a previous assessment. In some embodiments, blood flow to the brain of the subject is improved. In some embodiments, blood flow to the brain is assessed by transcranial Doppler ultrasound.

[0019] In some embodiments, the ischemic condition affecting the brain is caused by disease or trauma. In some embodiments, the subject is in need of induction of angiogenesis due to an ischemic condition caused by disease or trauma. In some embodiments, the administration is effective to reduce ischemia in the subject. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of VEGF. In some embodiments, the administration is oral, intravenous, intrathecal, or intraperitoneal.

[0020] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, comprising administering to a subject in need thereof an effective amount of hexosamine D-mannosamine (ManN). In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, comprising administering to a subject in need thereof an effective amount of kifunensine (Kif). In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, comprising administering to a subject in need thereof an effective amount of castanospermine (Cas).

[0021] In some embodiments, the administration is in vivo. In some embodiments, the subject in need of administration of the N-glycosylation inhibitor 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 musculus. 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 is effective to stimulate EC proliferation and angiogenesis. In some embodiments, the administration is effective to activate JNK signaling and the unfolded protein response triggered by ER stress. In some embodiments, the administration is effective to induce changes in N-glycan and O-glycan profiles in endothelial cells. [Brief description of the drawings]

[0022] This application for patent contains at least one drawing executed in color. Copies of this patent or patent application containing color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0023] 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 can be obtained by reference to the following detailed description that sets forth illustrative embodiments.

[0024] [Fig. 1A-1F]Examples are shown showing the effect of mannosamine (ManN) on bovine choroidal microvascular endothelial cell (BCEC) proliferation. Figure 1A is an image showing crystal violet stained BCEC samples treated with ManN in the presence or absence of VEGF. BCECs were treated with various concentrations of ManN ranging from 0.5 μM to 1 mM for 5-6 days with or without 5 ng / ml VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. The cell covered area of ​​the various treatment groups was quantified by ImageJ software. Figure 1B is a chart showing the effect of ManN in the presence or absence of VEGF on cell number. Cell number was quantified by adding AlamarBlue and fluorescence was measured at 530 nm / 590 nm. n=3 independent samples were used. Figure 1C is a chart showing the effect of ManN on bovine retinal microvascular endothelial cell (BREC) proliferation. n=3 biologically independent samples were used. FIG. 1D is an image showing the effect of hexosamines other than ManN on BCEC proliferation in samples. Each treatment group was tested in duplicate. FIG. 1E is a chart and image showing the effect of ManN on wounded BCEC samples. BCEC confluent monolayers were scratched with a 1 ml pipette tip, washed, and then incubated in low glucose DMEM containing 1% FBS for 40 hours. n=3 independent samples were used. Scale bar=400 μm. Images were taken and the gap between the apical wound fronts was quantified using AxioVision LE Rel.4.4 software. A representative image from crystal violet staining is shown. FIG. 1F is a chart showing the effect of ManN in a BCEC transwell migration assay. n=4 independent samples were used. Asterisks indicate significant differences compared to controls. Lines were used between specific groups when statistical analysis was performed using different controls. A representative experiment from two independent studies is shown. Data are mean + / - SD and statistical analysis was performed by two-tailed two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 2A-2C]Western blot images showing activation of ERK, AKT, mTOR, AMPKα, CREB, ACC, and eNOS, not specific to ManN. Enhanced activation of ERK (Thr202 / Tyr204), AKT (Ser473), and CREB (Ser133) in BCECs after treatment with ManN with VEGF for various times (Figure 2A) or pretreatment with ManN for 8 hours followed by 15 minutes of VEGF stimulation (Figure 2B). For samples shown in Figure 2C, BCECs were treated with 40 μM ManN, ManNAc, or mannose for various times. Total mTOR, ACC, eNOS, AMPKα, ERK, AKT, CREB, and phosphorylation of mTOR (Ser2448), ACC (Ser79), eNOS (Ser1177), AMPKα (Thr172), ERK (Thr202 / Tyr204), AKT (Ser473), and CREB (Ser133) were examined by Western blot analysis. f3-actin served as a loading control. Molecular weights (KDa) are labeled on the right. A representative experiment from two independent studies is shown. [Figure 3A-3E]We show an example showing that ManN specifically activates the JNK pathway in BCECs. Figure 3A is a Western blot image. BCECs grown in growth medium (GM: low glucose DMEM containing 10% bovine calf serum (BCS), 10 ng / ml VEGF, and 5 ng / ml bFGF) were switched to growth factor-free medium and subsequently treated with 4 μM to 4 mM ManN or mannose. After 4 h, cell lysates were collected and subjected to Western blot analysis for phosphorylated JNK (Thr183 / Tyr185), p38 (Thr180 / Tyr182), and ERK (Thr202 / Tyr204), as well as total JNK, p38, and ERK. Figure 3B is a Western blot image showing that ManN activated JNK and its downstream c-Jun, but mannose did not. f3-actin served as a loading control. For each study, a representative experiment from 2-3 independent studies is shown. Figure 3C is a chart showing the effect of ManN on pretreated samples. BCECs plated in 96-well plates were allowed to attach and pretreated with the specific JNK inhibitor SP600125 (5 μM) for 2 h, followed by treatment with either 40 μM or 2 mM ManN with or without 5 ng / ml VEGF. After 6 days, cell proliferation was quantified using AlamarBlue. n=3 independent samples were used. Figure 3D is a Western blot image showing screening of siRNAs against JNK1 and JNK2. 24 h after siRNA transfection, BCECs were lysed and proteins were subjected to Western blot analysis. f3-actin served as a loading control. Quantification of target knockdown is shown. Figure 3E is a chart showing exemplary results showing that approximately 80% knockdown of JNK1 and / or JNK2 by two independent siRNAs was 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 control. Lines were used between specific groups when statistical analysis was performed using different controls. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01. [Figure 4A-4G] Examples are shown showing that ManN affects protein glycosylation. Figure 4A is a Western blot image showing the reduction in VEGFR2 molecular weight after ManN treatment. BCECs were treated with 40 μM of various hexosamines, their derivatives, and monosaccharides, or with 5 ng / ml VEGF for 24 hours. VEGFR2 Western blot analysis was performed. Figure 4B is an imaging of a Western blot showing the dose-dependent effect of ManN on VEGFR2 molecular weight in BCECs. Figure 4C is a Western blot image showing that mannose can dose-dependently reverse the effect of 2 mM ManN on VEGFR2 molecular weight change, whereas mannose alone had no effect even at 10 mM. Figure 4D is a chart 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 plated in 96-well plates were allowed to attach, followed by the addition of ManN. After 2 hours, cells were treated with different concentrations of mannose with or without VEGF. After 6 days, 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 24 h treatment with 40 μM ManN, BCECs were washed three times with low glucose DMEM. Cells were kept in low glucose DMEM for an additional 8 h or 24 h. VEGFR2 Western blot analysis was performed. Figure 4F is a Western blot image showing the reduction in molecular weight of VEGFR2, neuropilin-1, CD31, and c-met in HUVECs after various concentrations of ManN treatment. Figure 4G is a Western blot image showing the reduction in molecular weight of VEGFR2, f31 integrin, and bFGFR1 in hDMVECs by various concentrations of ManN. f3-actin served as a loading control. Data are mean + / - SD, and asterisks indicate significant differences compared to the control. For each study, a representative experiment from 2 to 5 independent studies is shown. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01. [Figure 5A-5D]Examples are shown showing that ManN specifically induces the expression of unfolded protein response (UPR)-responsive proteins. Figure 5A is a Western blot image. BCECs were grown in growth medium (GM) to approximately 80% confluency. The medium was changed to growth factor-free medium containing 10% BCS in the presence or absence of 40 or 400 μM ManN or mannose for various 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 is a Western blot image of cells treated with various 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. FIG. 5C is a Western blot image showing that 4-PBA, but not TUDCA, effectively blocks induction of CHOP in BCECs, accompanied by recovery of expression of transcription factor ATF-6 upon 400 μM ManN treatment. BCECs were pretreated with two chemical chaperones, 2 mM 4-PBA or 500 μM TUDCA. After 16 h, cells were switched to growth factor-free medium in the presence of ManN for 4 h. GM: growth medium. FIG. 5D 4-PBA significantly blocked the bell-shaped effect of ManN on BCEC proliferation. Pretreatment of cells with 1 mM 4-PBA for 8 h abrogated the additive effect of 40 μM ManN and 5 ng / ml VEGF and protected cells from the toxic effects induced by 2 mM ManN. n=3 independent samples were used. For each study, a representative experiment from 2-3 independent studies is shown. Data are mean + / - SD, asterisks indicate significant differences compared to control. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01. [Figures 6A-6J]6 is a chart showing the effect of ManN on non-endothelial cells of bovine, murine, or human origin. ManN did not promote the growth of Calu6 (FIG. 6A), A673 (FIG. 6B), U87MG (FIG. 6C), and 4T1 (FIG. 6D) tumor cells. 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, no increase in proliferation was induced by ManN alone or in combination with growth factors on AML12 (FIG. 6E), bovine pituitary cells (FIG. 6F), NIH3T3 cells (FIG. 6G), human RPE (FIG. 6H), human dermal fibroblasts (FIG. 6I), and 10 human keratinocytes (FIG. 6J). Quantification of proliferation was performed using AlamarBlue or MTS (for 4T1 cells). n=3 independent samples were used. Inserted in the charts of Figures 6A-6J are representative Western blot analyses showing the dose-dependent effect of ManN and mannose at 400 μM (2, 4) and 2 mM (3, 5) on bFGFR1 or f31 integrin (for 4T1, AML12, NIH3T3 cells, human skeletal muscle cells, human skin fibroblasts, and human keratinocytes) compared to the untreated control (1). f3-actin served as a loading control. GM: growth medium. For Western blot analysis, proteins were separated on NuPAGE 3-8% Tris-Acetate gels. For each study, a representative experiment from two independent studies is shown. Asterisks indicate significant differences compared to the control. When statistical analysis was performed using different controls, brackets were used between specific groups. Data were the mean or mean + / - SD when n=2. Statistical analysis was performed by two-tailed two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 7A-7F]Examples showing the effect of protein glycosylation inhibitors on BCEC proliferation are shown. Figure 7A includes images of samples showing dose-dependent stimulation of BCEC proliferation by various glycosylation inhibitors. Inhibitors were added with or without 5 ng / ml VEGF for 3 days at concentrations ranging from 0.01 to 100 μM. At the end of the experiment, cells were fixed and stained with crystal violet. A representative experiment is shown. ER α-1,2-mannosidase I and Golgi α-mannosidase I inhibitor kifunesine (Kif), a-glucosidase inhibitor castanospermine (Cas). Cell coverage area of ​​the various treatment groups was quantified by ImageJ software. Figure 7B is a chart showing the dose-dependent effect of Kif and Cas in promoting BCEC proliferation with or without 5 ng / ml VEGF. n=3 independent samples were used. Figure 7C includes Western blot images showing that both inhibitors reduced VEGFR2 molecular weight and induced Bip expression in a dose-dependent manner, as assessed by Western blot analysis. Proteins from total cell lysates were separated using 3-8% Tris-Acetate gels. BCECs were treated with various inhibitors for 24 hours. Quantification of Western blots was performed by densitometry. β-actin was a loading control. Figure 7D is a chart showing acceleration of monolayer gap closure by Kif and Cas in a BCEC scratch assay, with Kif (HO) and Cas (DMSO) controls. Gaps were quantified using AxioVision LE Rel.4.4 software. n=3 independent samples were used. Scale bar=400 μm. Figure 7E is a Western blot image showing activation of AKT and JNK in BCECs by 40 μM glycosylation inhibitors and 10 ng / ml VEGF. However, Cas did not activate ERK. Quantification of phosphorylated AKT, JNK, and ERK was performed by densitometric analysis on total protein. Figure 7F is a chart showing that pretreatment of BCECs with 5 μM SP600125 for 2 h significantly blocked the effect of both glycosylation inhibitors on BCEC proliferation. n=3 independent samples were used. A representative experiment is shown from 2-4 independent studies.Data shown are mean + / - SD. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01. [Figure 8A-8D] An example is shown in which topical application of ManN and VEGF in mice stimulates angiogenesis and accelerates wound healing. Figure 8A is a chart showing the effect of ManN on wounds. Wounds were created on the dorsal skin of mice with a 6 mm punch. VEGF and ManN were each administered daily at 20 μg per wound in 25 μL of PBS for the first 4 days, with PBS as the control. A 10-day wound-healing study with 5 mice per group. Wound closure rate (%) was quantified by Image J software in two independent studies. Asterisks indicate significant differences compared to the control at each time point. Figure 8B includes images from a 4-day wound-healing study with images of the wound-healing process on days 1, 2, and 4. n=5 animals / treatment group were used. Figure 8C includes representative images of immunohistochemical staining of CD31 in the PBS control group and the VEGF and ManN combination group (scale bar=200 μm). FIG. 8D is a chart showing quantification of CD31 positive blood vessel (red dotted circle) density around the wound area by eye under a microscope (20x magnification). Data are mean + / - SD. Statistical significance was further confirmed using Wilcoxon rank sum test between treatment groups of interest. Asterisks indicated significant differences compared to PBS control. For each study, a representative experiment is shown. n=3 animals / treatment group were used. Lines were used between specific groups when statistical analysis was performed using different controls. Statistical analysis was performed by two-tailed two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 9A-9D]An example is shown showing that ManN accelerates blood perfusion recovery in a mouse ischemic hindlimb model. Figure 9A includes images obtained from continuous laser Doppler analysis of blood perfusion in the hindlimb of ManN-treated, Kif-treated, and control mice. Different colors were used to indicate blood perfusion in the ischemic limb (ligated, left side) versus the non-ischemic limb (sham, right side). Representative images are shown at week 0 and week 1. Figure 9B is a chart showing quantification of blood perfusion ratio between area 2 (ischemic, left limb) and area 1 (non-ischemic, right limb), n=8 animals / treatment group. Figure 9C includes images of sample tissues. Three weeks after surgery, skeletal muscle tissues were harvested and fixed. CD31 immunostaining on these tissue sections was performed to label the vasculature. H&E staining was also performed. Representative CD31 staining and H&E histological images of the ischemic hindlimb 21 days after surgery are shown. Scale bar=50 μm. Figure 9D is a chart showing quantification of vascular density by CD31 immunostaining performed using ImageJ software, n=8 animals / treatment group, 3 independent experiments; data are mean + / - SEM. Statistical analysis was performed by two-tailed, unequal variance t-test. *p<0.05, **p<0.01. [Figure 10A-10B] Examples showing ManN promoting retinal neovascularization in mice are shown. Figure 10A includes images of tissues. Intravitreal injection of ManN increases blood vessel density. Adult mice received a single intravitreal injection of 500 ng ManN, Kif, or 200 ng 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 blood vessels are shown. n=10 animals / treatment group, 3 independent experiments, scale bar=50 μm. Figure 10B is a chart showing blood vessel density as determined with ImageJ software, n=10 animals / treatment group, 3 independent experiments. Data were mean + / - SEM. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01, ***p<0.001. [Figures 11A-11D]Examples are shown showing that ManN, but not structurally related molecules, stimulates endothelial cell proliferation. Figure 11A is a chart showing the additive effect of ManN and bFGF on BCEC proliferation. Bell-shaped effect of ManN on BCEC proliferation. BCECs were treated with ManN ranging from 0.4 to 400 μM with or without 20 ng / ml bFGF for 5-6 days. At the end of the experiment, proliferation was quantified using AlamarBlue. Figure 11B is a chart showing that the additive effect of VEGF and ManN on BCEC proliferation is dependent on the glycolysis pathway. Proliferation assays were performed in low glucose DMEM medium without growth factors or DMEM medium without glucose and pyruvate. Asterisks indicate significant differences compared to no treatment controls. Statistical analysis was also performed to compare VEGF alone and VEGF+ManN treated groups for cells grown in the two different assay media. Figures 11C and 11D are charts showing the effect of various drugs from 0.04 μM to 5 mM on BCECs in the absence (Figure 11C) or presence (Figure 11D) of 5 ng / ml VEGF. n=3 independent samples were used. For each study, a representative experiment from two independent studies is shown. Data are mean + / - SD. Statistical analysis was performed by two-tailed, two-sample, unequal variance t-test. *p<0.05, **p<0.01. [Figures 12A-12D] The experimental setup and analysis of the effect of ManN treatment on cerebral infarction are shown. Figure 12A is a summary of the timing of ManN treatment regimen, as well as the timing of neuroischemia induction and subsequent infarction assessment. Figure 12B shows the results of the first experiment of acute middle cerebral artery occlusion (MCAO) stroke model in mice testing the effect of ManN treatment. Figure 12C shows the results of the second experiment of acute middle cerebral artery occlusion (MCAO) stroke model in mice testing the effect of ManN treatment. Figure 12D shows a graph and analysis combining the results of the experiments in Figure 12B and Figure 12C. [Figures 13A-13C]Figure 13 shows an analysis of the effect of ManN treatment on cerebral infarction. Figure 13A shows representative MRI coronal slices of ManN-treated and vehicle-treated mouse brains showing the infarct area in the MCAO stroke model on days 2 and 4. Figure 13B shows a graph combining the results and analysis of the infarct volume on day 4 of the MCAO stroke model in ManN-treated and vehicle-treated mouse brains. Figure 13C shows TTC staining in coronal sections of ManN-treated and vehicle-treated mouse brains on day 4 of the MCAO stroke model. Figures 14A-14B show an analysis of CD31 staining and vascular density in the MCAO stroke model. Figure 14A shows CD31 immunostaining that labeled endothelial cells of ManN-treated and vehicle-treated mouse brains in the cerebral cortex region affected by infarction on day 6. FIG. 14B shows a graph and analysis of CD31 staining density of representative sections of the cerebral cortex on day 6 of infarct-affected vehicle-treated and ManN-treated mice, demonstrating increased vascular density in the ischemic region of the brain after ManN administration. [Figure 15] 1 shows histopathological analysis of the cerebral cortex and striatum in vehicle- and ManN-treated mice on day 4 in an MCAO stroke model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] overview 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.

[0026] Pharmaceutical Compositions Compositions for preventing, mitigating, and / or treating stroke Described herein is a composition for preventing, mitigating, and / or treating stroke, 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), kifunensine (Kif), castanospermine (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.

[0027] 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 belongs to the genus Rattus. In some embodiments, the subject belongs to the genus Mus musculus. 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.

[0028] In some embodiments, the pharmaceutical composition for treating stroke is for use after a stroke occurs in a subject. In some embodiments, the subject has experienced an ischemic stroke. In some embodiments, the ischemic stroke includes a thrombotic stroke or an embolic stroke. In some embodiments, the subject has experienced a hemorrhagic stroke. In some embodiments, the hemorrhagic stroke includes a subarachnoid hemorrhage or an intracerebral hemorrhage. In some embodiments, the subject has experienced a brain stem stroke. In some embodiments, the subject has experienced a 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 occur prior to the stroke event for which the subject is treated. The TIA may last for a short period of time, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 minutes or less. A TIA may temporarily cut off the blood supply to an area of ​​the subject's brain. In some embodiments, the subject suddenly experiences or has experienced an onset of TIA symptoms. In some embodiments, the symptoms of TIA in the subject disappear after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes or more. In some embodiments, the TIA does not cause permanent damage to the subject's brain. In some embodiments, the TIA causes permanent damage to the subject's brain. In some embodiments, one or more TIAs in a subject indicate a warning that the subject is at increased risk of stroke. In some embodiments, the subject is administered a pharmaceutical composition comprising an N-glycosylation inhibitor based on the occurrence of one or more prior TIAs. In some embodiments, the subject has experienced a cryptogenic stroke. In some embodiments, the subject has been diagnosed with a stroke. In some embodiments, the subject is presumed to have had a stroke. In some embodiments, the subject is suspected to have had a stroke. In some embodiments, the subject is not known to have had a stroke, hi some embodiments, the subject is not known to have had a stroke but has experienced one or more TIAs.In some embodiments, a subject is administered a pharmaceutical composition comprising one or more N-glycosylation inhibitors based on an increased risk of stroke after one or more TIAs. In some embodiments, a subject is administered a pharmaceutical composition comprising one or more N-glycosylation inhibitors based on an increased risk of stroke recurrence after one or more TIAs. In some embodiments, a subject is administered a pharmaceutical composition comprising one or more N-glycosylation inhibitors based on an increased risk of stroke recurrence after one or more previous strokes.

[0029] 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 into the cerebrospinal fluid. In some embodiments, the pharmaceutical composition for treating stroke is administered into the choroid plexus. In some embodiments, the pharmaceutical composition for treating stroke is administered locally near the site of ischemia. In some embodiments, the pharmaceutical composition for treating stroke is administered into a blood vessel that directly supplies blood to the brain. In some embodiments, the pharmaceutical composition for treating stroke is administered into the carotid artery.

[0030] Therapeutically Effective Amount of the Pharmaceutical Composition In some embodiments, the pharmaceutical composition for preventing, mitigating, 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), kifunensine (Kif), or castanospermine (Cas). In some embodiments, the N-glycosylation inhibitor is hexosamine D-mannosamine (ManN), kifunensine (Kif), or castanospermine (Cas). In some embodiments, the one or more N-glycosylation inhibitors are hexosamine D-mannosamine (ManN), kifunensine (Kif), or castanospermine (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, an effective amount of an 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, 110mg, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350m g, 375mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 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, 32 g, 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, 1 20g, 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 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 20 mg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 mg. In some embodiments, 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, an effective amount of an N-glycosylation inhibitor is 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, g, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 37 5mg, 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, 3 4g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120 g, 125 g, 130 g, 135 g, 140 g, 145 g, 150 g, 155 g, 160 g, 165 g, 170 g, 175 g, 180 g, 185 g, 190 g, 195 g, 200 g, 215 g, 230 g, 245 g, 260 g, 275 g, 300 g, 325 g, 350 g, 375 g, 400 g, 430 g, 460 g, 500 g, 530 g, 560 g, 600 g, 630 g, 660 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g, or 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 50 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 45 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 g or less.In some embodiments, the effective amount of the N-glycosylation inhibitor is about 35 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 30 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 25 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 20 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 16 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 14 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 12 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 8 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 6 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 4 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 2 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 100 mg or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 50 mg or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 mg or less.In some embodiments, an effective amount of an N-glycosylation inhibitor is about 1 mg to about 10 mg, about 3 mg to about 10 mg, about 5 mg to about 15 mg, about 10 mg to about 20 mg, about 15 mg to about 45 mg, about 20 mg to about 55 mg, about 25 mg to about 70 mg, about 35 mg to about 75 mg, about 40 mg to about 105 mg, about 45 mg to about 85 mg, about 50 mg to about 80 mg, about 55 mg to about 125 mg, about 60 mg to about 150 mg, about 65 mg to about 185 mg, about 70 mg to about 125 mg, about 75 mg to about 200 mg, about 80 mg to about 225 mg, or about 85 mg to about 150 mg. 5mg to about 300mg, about 90mg to about 180mg, about 100mg to about 150mg, about 100mg to about 225mg, about 100mg to about 350mg, about 100mg to about 500mg, about 110mg to about 200mg, about 110mg to about 275mg, about 150mg to about 37 5mg, about 175mg to about 500mg, about 190mg to about 315mg, about 225mg to about 600mg, about 250mg to about 700mg, about 250mg to about 500mg, about 260mg to about 800mg, about 275mg to about 600mg, about 300mg to about 900mg, about 3 50mg to about 750mg, about 400mg to about 2g, about 425mg to about 1.1g, about 450mg to about 1.5g, about 500mg to about 850mg, about 525mg to about 1.3g, about 600mg to about 2g, about 700mg to about 3g, about 800mg to about 4g, about 900mg to about 5 g, about 1g to about 3g, about 1.1g to about 6g, about 1.2g to about 8g, about 1.4g to about 10g, about 2.5g to about 7.5g, about 3g to about 1 2g, about 4g to about 15g, about 4.5g to about 16g, about 5g to about 20g, about 6g to about 18g, about 7g to about 21g, about 8g to about 23g, Approximately 9g to approximately 25g, approximately 10g to approximately 30g, approximately 12g to approximately 35g, approximately 14g to approximately 28g, approximately 15g to approximately 45g, approximately 18g to approximately 36g, Approximately 20g to approximately 50g, approximately 25g to approximately 65g, approximately 30g to approximately 70g, approximately 35g to approximately 80g, approximately 40g to approximately 100g, approximately 45g to approximately 11 5g, about 50g to about 135g, about 20g to about 300g, about 20g to about 500g, about 25g to about 800g, about 70g to about 385g, about 75g to about 450g, about 85g to about 600g, about 100g to about 800g, about 200g to about 1kg, or about 475g to about 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 20 mg to about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 1 g to about 60 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 1 g to about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 1 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 3 ...1 g to about 15 g. An effective amount of the N-glycosylation inhibitor is about 3 g to about 15 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 5 g to about 50 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 5 g to about 30 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 5 g to about 20 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 5 g to about 15 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 5.8 g to about 7.3 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 7.8 g to about 10 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 11.8 g to about 14.8 g. In some embodiments, an effective amount of the N-glycosylation inhibitor is about 23.7 g to about 29.7 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 31.7 g to about 39.6 g.

[0031] Pro-angiogenic factors In some embodiments, described herein are pharmaceutical compositions comprising a pro-angiogenic factor that, when combined with a therapeutically effective amount of an N-glycosylation inhibitor, results in a synergistic increase in angiogenic growth. In some embodiments, a pro-angiogenic factor is combined with a therapeutically effective amount of an N-glycosylation inhibitor to synergistically increase endothelial growth. In some embodiments, a pro-angiogenic factor is combined with a therapeutically effective amount of an N-glycosylation inhibitor to synergistically increase angiogenic growth without a significant amount of vascular leakage or vascular edema. In some embodiments, the pro-angiogenic factor comprises VEGF. In some embodiments, the VEGF comprises VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PLGF), or a combination thereof. In some embodiments, the VEGF comprises recombinant VEGF. In some embodiments, the VEGF is administered locally near the site of the subject's brain affected by the ischemic event. In some embodiments, the VEGF is administered systemically. In some embodiments, the 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 is formulated in a pharmaceutical composition separately from the N-glycosylation inhibitor. In some embodiments, the angiogenic factor is formulated in a pharmaceutical composition together with the N-glycosylation inhibitor.

[0032] Pharmaceutical preparations Described herein is a pharmaceutical composition for preventing, mitigating, 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, mitigating, 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 similar chemical structure to ManN. In some embodiments, the one or more N-glycosylation inhibitors having a similar chemical structure to ManN are listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises ManN. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises ManN and one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to the subject consists essentially of ManN. In some embodiments, the N-glycosylation inhibitor administered to the 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 formulation, in which one or more N-glycosylation inhibitors and one or more pharma- ceutically acceptable excipients are dissolved in a selected solvent system. In some embodiments, the pharma-ceutically acceptable excipient is purified water. In some embodiments, the vehicle solution is purified water. In some embodiments, the pharmaceutical solution comprises a co-solvent. In some embodiments, the co-solvent is propylene glycol, glycerol, propylene glycol, glycerin, poly(ethylene glycol), or ethanol.In some embodiments, the pharmaceutical solution includes one or more agents for specifically increasing the solubility of the N-glycosylation inhibitor in the vehicle. In some embodiments, the one or more agents for specifically increasing the solubility of the N-glycosylation inhibitor in the vehicle are surfactants. In some embodiments, the pharmaceutical solution includes one or more preservatives. In some embodiments, the one or more preservatives are paraoxybenzoic acid esters (methyl and propyl oxybenzoates), boric acid and borate salts, sorbic acid and sorbate salts, phenol, 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 paraoxybenzoic acid (0.001-0.2%) are used as preservatives. In some embodiments, methyl and propyl paraoxybenzoates (in a 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, hydroxyanisole butyrate, hydroxytoluene butyrate, 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 includes one or more buffer systems to adjust the pH of the formulation. In some embodiments, the pH of the pharmaceutical solution is about pH 5.0-8.0. In some embodiments, the pH of the pharmaceutical solution is 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 includes a therapeutically effective amount of one or more N-glycosylation inhibitors formulated as a pharmaceutical oral suspension including a suspending agent and one or more of a vehicle, a solvent, a co-solvent, a preservative, a sweetener, an antifoaming agent, a wetting agent, a buffering agent, and a flavoring agent. In some embodiments, the choice of formulation of the pharmaceutical composition is determined by the route of administration by which the one or more N-glycosylation inhibitors are administered to the subject.

[0033] In some embodiments, a pharmaceutical composition for preventing, mitigating, and / or treating stroke comprising an effective amount of one or more N-glycosylation inhibitors administered to a subject in need thereof is formulated by dissolving the one or more N-glycosylation inhibitors in water for oral administration. In some embodiments, the one or more N-glycosylation inhibitors are formulated 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, a pharmaceutical composition for preventing, mitigating, 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, mitigating, 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, the pharmaceutical composition for preventing, mitigating, and / or treating stroke is used in a method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical solution comprising about 20% ManN dissolved in water. In some embodiments, the pharmaceutical composition for preventing, mitigating, and / or treating stroke is used in a method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical solution comprising about 20% ManN dissolved in water.In some embodiments, a pharmaceutical composition for preventing, mitigating, and / or treating stroke is used in a method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical solution, the pharmaceutical solution consisting of about 20% ManN dissolved in water. In some embodiments, a pharmaceutical solution for use in a method for preventing, mitigating, 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 for use in a method for preventing, mitigating, and / or treating stroke is dissolved in water for oral administration. In some embodiments, a therapeutically effective amount of ManN for use in a method for preventing, mitigating, and / or treating stroke is dissolved in milk for oral administration. In some embodiments, a therapeutically effective amount of ManN for use in a method for preventing, mitigating, and / or treating stroke is dissolved in a protein nutritional shake (e.g., Ensure®) for oral administration. In some embodiments, a therapeutically effective amount of ManN is dissolved in a balanced electrolyte drink (e.g., Pedialyte® or Pedialyte AdvancedCare® Plus) for oral administration for use in methods for preventing, mitigating, and / or treating stroke.

[0034] method How to Treat a Stroke Described herein are methods of treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (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 of treating stroke comprises administering to a subject in need thereof an effective amount of one or more N-glycosylation inhibitors. In some embodiments, the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), or castanospermine (Cas). In some embodiments, the N-glycosylation inhibitor is hexosamine D-mannosamine (ManN), kifunensine (Kif), or castanospermine (Cas). In some embodiments, the one or more N-glycosylation inhibitors are hexosamine D-mannosamine (ManN), kifunensine (Kif), or castanospermine (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 similar chemical structure to ManN. In some embodiments, the one or more N-glycosylation inhibitors having a similar chemical structure to ManN are listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises ManN. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises ManN and one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor administered to the subject comprises one or more compounds listed in Table 1. In some embodiments, the N-glycosylation inhibitor is formulated into a pharmaceutical composition.

[0035] 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 belongs to the genus Rattus. In some embodiments, the subject belongs to the genus Mus musculus. 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.

[0036] In some embodiments, the method of treating stroke is for use after a stroke has occurred in a subject. 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 brain stem 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, the subject is suspected of having had 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 occur prior to the stroke event for which the subject is being treated. A TIA may last for a short period of time, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 minutes or less. A TIA may temporarily block blood supply to an area of ​​the subject's brain. In some embodiments, the subject suddenly experiences or has experienced an onset of TIA symptoms. In some embodiments, the symptoms of TIA in the subject disappear after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes or more than 30 minutes. In some embodiments, a TIA does not cause permanent damage to the subject's brain. In some embodiments, a TIA causes permanent damage to the subject's brain. In some embodiments, one or more TIAs in a subject indicate a warning that the subject is at increased risk of stroke. In some embodiments, a subject is administered a pharmaceutical composition comprising an N-glycosylation inhibitor based on the occurrence of one or more previous TIAs.

[0037] In some embodiments, methods for treating stroke are described herein, in which 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 into the cerebrospinal fluid. In some embodiments, the pharmaceutical composition for treating stroke is administered into the choroid plexus. In some embodiments, the pharmaceutical composition for treating stroke is administered locally near the site of ischemia. In some embodiments, the pharmaceutical composition for treating stroke is administered into a blood vessel that directly supplies blood to the brain. In some embodiments, the pharmaceutical composition for treating stroke is administered into the carotid artery.

[0038] In some embodiments, methods for treating stroke are described herein, in which 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, the single dose treatment regimen comprises a pre-treatment regimen. In some embodiments, the single dose treatment regimen comprises a concomitant treatment regimen. In some embodiments, the single dose treatment regimen comprises a post-treatment regimen. In some embodiments, the treatment regimen comprises a multiple dose treatment regimen. In some embodiments, the multiple dose treatment regimen comprises a pre-treatment regimen. In some embodiments, the multiple dose treatment regimen comprises a concomitant treatment regimen. In some embodiments, the multiple dose treatment regimen comprises a post-treatment regimen. In some embodiments, the multiple dose treatment regimen comprises a pre-treatment and a post-treatment regimen. In some embodiments, the multiple dose treatment regimen comprises a pre-treatment and a concomitant regimen. In some embodiments, the multiple dose treatment regimen comprises a concomitant and a post-treatment regimen. In some embodiments, multiple dose treatment regimens include pre-treatment, concurrent, and post-treatment regimens, hi some embodiments, multiple treatment regimens include 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 may include 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, 3-20 treatments, 3-20 treatments, 3-3 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 ... including 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 includes one or more treatments comprising administering a pharmaceutical composition described herein to the subject, and the pretreatment is initiated after the subject is determined to be at high risk of suffering from a stroke. In some embodiments, the pretreatment regimen includes one or more treatments comprising administering a pharmaceutical composition described herein to the subject, and the pretreatment is initiated after the subject is determined to be at high risk of recurrent stroke. In some embodiments, the pretreatment regimen includes one or more treatments comprising administering a pharmaceutical composition described herein to the subject, and the pretreatment is initiated after one or more TIAs in the subject.In some embodiments, the pretreatment regimen includes one or more pretreatments comprising administering a pharmaceutical composition described herein to the subject, wherein the one or more pretreatments are initiated at least about 48, 44, 40, 36, 32, 28, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, or 1 hour prior to the stroke event. In some embodiments, the concomitant regimen includes one or more concurrent therapies comprising administering a pharmaceutical composition described herein to a subject, wherein the one or more concurrent therapies are initiated within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 24, 28, 32, 36, 40, 44, or 48 hours of the estimated onset of the onset time of a stroke event in the subject. In some embodiments, the post-treatment regimen includes one or more post-treatments comprising administering a pharmaceutical composition described herein to the subject, wherein the one or more post-treatments are initiated about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 24, 28, 32, 36, 40, 44, 46, 48, 52, 56, 60, 64, 68, 72, 84, 96, 108, or 120 hours after blood flood to the area of ​​the subject's brain affected by the recent stroke event has returned to near normal. In some embodiments, the method includes the subject undergoing a pre-treatment regimen and a post-treatment regimen. In some embodiments, the method comprises the subject receiving a pre-treatment regimen and a concomitant treatment regimen. In some embodiments, the method comprises the subject receiving a pre-treatment regimen, a concomitant treatment regimen, and a post-treatment regimen. In some embodiments, the method comprises the subject receiving a concomitant 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, co-treatment regimen, or post-treatment regimen comprises administering a pharmaceutical composition described herein for 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, 28 hours, 30 hours, 32 hours, The post-treatment regimen comprises administering to the subject a dosage determined at an administration frequency of about once every other day, for 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 18 days, 20 days, 21 days, 24 days, 28 days, or 30 days. In some embodiments, the post-treatment regimen comprises administering to the subject a pharmaceutical composition described herein at a dosage determined at an administration 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 dosage determined at an administration frequency of about once every other day, until one or more symptoms of stroke in the subject show improvement or are completely eliminated. In some embodiments, the method includes initiating administration of a pharmaceutical composition described herein to a subject within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 48, 52, 56, 60, 64, 68, 72, 84, 96, or 120 hours of the ischemic event. In some embodiments, the method includes initiating administration of a pharmaceutical composition described herein to a subject within about 1 hour to about 12 hours of the ischemic event. In some embodiments, the method includes initiating administration of a pharmaceutical composition described herein to a subject within about 2 hours to about 6 hours of the ischemic event. In some embodiments, the method includes initiating administration of a pharmaceutical composition described herein to a subject within about 2 hours to about 48 hours of the ischemic event. In some embodiments, the methods include administering to the subject a pharmaceutical composition described herein beginning within about 2 hours to about 24 hours of the ischemic event.

[0039] In some embodiments described herein, the method of 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, an effective amount of an 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, 110mg, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350m g, 375mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 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, 32 g, 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, 1 20g, 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 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 mg. In some embodiments, 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 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, an effective amount of an N-glycosylation inhibitor is 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, g, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 37 5mg, 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, 3 4g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120 g, 125 g, 130 g, 135 g, 140 g, 145 g, 150 g, 155 g, 160 g, 165 g, 170 g, 175 g, 180 g, 185 g, 190 g, 195 g, 200 g, 215 g, 230 g, 245 g, 260 g, 275 g, 300 g, 325 g, 350 g, 375 g, 400 g, 430 g, 460 g, 500 g, 530 g, 560 g, 600 g, 630 g, 660 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g, or 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 35 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 30 g or less.In some embodiments, the effective amount of the N-glycosylation inhibitor is about 25 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 20 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 18 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 15 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 12 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 8 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 6 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 4 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 2 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 mg or less.In some embodiments, an effective amount of an N-glycosylation inhibitor is about 1 mg to about 10 mg, about 3 mg to about 10 mg, about 5 mg to about 15 mg, about 10 mg to about 20 mg, about 15 mg to about 45 mg, about 20 mg to about 55 mg, about 25 mg to about 70 mg, about 35 mg to about 75 mg, about 40 mg to about 105 mg, about 45 mg to about 85 mg, about 50 mg to about 80 mg, about 55 mg to about 125 mg, about 60 mg to about 150 mg, about 65 mg to about 185 mg, about 70 mg to about 125 mg, about 75 mg to about 200 mg, about 80 mg to about 225 mg, or about 85 mg to about 150 mg. 5mg to about 300mg, about 90mg to about 180mg, about 100mg to about 150mg, about 100mg to about 225mg, about 100mg to about 350mg, about 100mg to about 500mg, about 110mg to about 200mg, about 110mg to about 275mg, about 150mg to about 37 5mg, about 175mg to about 500mg, about 190mg to about 315mg, about 225mg to about 600mg, about 250mg to about 700mg, about 250mg to about 500mg, about 260mg to about 800mg, about 275mg to about 600mg, about 300mg to about 900mg, about 3 50mg to about 750mg, about 400mg to about 2g, about 425mg to about 1.1g, about 450mg to about 1.5g, about 500mg to about 850mg, about 525mg to about 1.3g, about 600mg to about 2g, about 700mg to about 3g, about 800mg to about 4g, about 900mg to about 5 g, about 1g to about 3g, about 1.1g to about 6g, about 1.2g to about 8g, about 1.4g to about 10g, about 2.5g to about 7.5g, about 3g to about 1 2g, about 4g to about 15g, about 4.5g to about 16g, about 5g to about 20g, about 6g to about 18g, about 7g to about 21g, about 8g to about 23g, Approximately 9g to approximately 25g, approximately 10g to approximately 30g, approximately 12g to approximately 35g, approximately 14g to approximately 28g, approximately 15g to approximately 45g, approximately 18g to approximately 36g, Approximately 20g to approximately 50g, approximately 25g to approximately 65g, approximately 30g to approximately 70g, approximately 35g to approximately 80g, approximately 40g to approximately 100g, approximately 45g to approximately 11 5g, about 50g to about 135g, about 20g to about 300g, about 20g to about 500g, about 25g to about 800g, about 70g to about 385g, about 75g to about 450g, about 85g to about 600g, about 100g to about 800g, about 200g to about 1kg, or about 475g to about 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 20 mg to about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 40 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 3 g to about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 3 g to about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 9 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 40 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor administered to a subject for use in the method of treating stroke in a single treatment regimen. The effective amount of N-glycosylation inhibitor administered to subject for use in the method of alleviating one or more stroke symptoms in a single treatment regimen is listed in this paragraph.In some embodiments, the effective amount of N-glycosylation inhibitor administered to subject for use in the method of preventing stroke in a single treatment regimen is listed in this paragraph.

[0040] In some embodiments described herein, the method of multiple dose treatment regimen comprises administering a therapeutically effective amount of an N-glycosylation inhibitor. In some embodiments, the effective amount of an 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, 110mg, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350m g, 375mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 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, 32 g, 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, 1 20g, 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 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is at least about 40 mg. In some embodiments, 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 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, an effective amount of an N-glycosylation inhibitor is 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, g, 115mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 37 5mg, 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, 3 4g, 36g, 38g, 40g, 42g, 44g, 46g, 48g, 50g, 53g, 56g, 60g, 63g, 66g, 70g, 73g, 76g, 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, 120 g, 125 g, 130 g, 135 g, 140 g, 145 g, 150 g, 155 g, 160 g, 165 g, 170 g, 175 g, 180 g, 185 g, 190 g, 195 g, 200 g, 215 g, 230 g, 245 g, 260 g, 275 g, 300 g, 325 g, 350 g, 375 g, 400 g, 430 g, 460 g, 500 g, 530 g, 560 g, 600 g, 630 g, 660 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g, or 1 kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 35 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 30 g or less.In some embodiments, the effective amount of the N-glycosylation inhibitor is about 25 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 20 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 18 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 15 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 12 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 8 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 6 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 4 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 2 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g or less. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 40 mg or less.In some embodiments, an effective amount of an N-glycosylation inhibitor is about 1 mg to about 10 mg, about 3 mg to about 10 mg, about 5 mg to about 15 mg, about 10 mg to about 20 mg, about 15 mg to about 45 mg, about 20 mg to about 55 mg, about 25 mg to about 70 mg, about 35 mg to about 75 mg, about 40 mg to about 105 mg, about 45 mg to about 85 mg, about 50 mg to about 80 mg, about 55 mg to about 125 mg, about 60 mg to about 150 mg, about 65 mg to about 185 mg, about 70 mg to about 125 mg, about 75 mg to about 200 mg, about 80 mg to about 225 mg, or about 85 mg to about 150 mg. 5mg to about 300mg, about 90mg to about 180mg, about 100mg to about 150mg, about 100mg to about 225mg, about 100mg to about 350mg, about 100mg to about 500mg, about 110mg to about 200mg, about 110mg to about 275mg, about 150mg to about 37 5mg, about 175mg to about 500mg, about 190mg to about 315mg, about 225mg to about 600mg, about 250mg to about 700mg, about 250mg to about 500mg, about 260mg to about 800mg, about 275mg to about 600mg, about 300mg to about 900mg, about 3 50mg to about 750mg, about 400mg to about 2g, about 425mg to about 1.1g, about 450mg to about 1.5g, about 500mg to about 850mg, about 525mg to about 1.3g, about 600mg to about 2g, about 700mg to about 3g, about 800mg to about 4g, about 900mg to about 5 g, about 1g to about 3g, about 1.1g to about 6g, about 1.2g to about 8g, about 1.4g to about 10g, about 2.5g to about 7.5g, about 3g to about 1 2g, about 4g to about 15g, about 4.5g to about 16g, about 5g to about 20g, about 6g to about 18g, about 7g to about 21g, about 8g to about 23g, Approximately 9g to approximately 25g, approximately 10g to approximately 30g, approximately 12g to approximately 35g, approximately 14g to approximately 28g, approximately 15g to approximately 45g, approximately 18g to approximately 36g, Approximately 20g to approximately 50g, approximately 25g to approximately 65g, approximately 30g to approximately 70g, approximately 35g to approximately 80g, approximately 40g to approximately 100g, approximately 45g to approximately 11 5g, about 50g to about 135g, about 20g to about 300g, about 20g to about 500g, about 25g to about 800g, about 70g to about 385g, about 75g to about 450g, about 85g to about 600g, about 100g to about 800g, about 200g to about 1kg, or about 475g to about 1kg. In some embodiments, the effective amount of the N-glycosylation inhibitor is from about 20 mg to about 60 mg.In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 40 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 1 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 3 g to about 30 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 3 g to about 20 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 5 g to about 9 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 40 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 25 g. In some embodiments, the effective amount of the N-glycosylation inhibitor is about 10 g to about 15 g. In some embodiments, the effective amount of the N-glycosylation inhibitor administered to a subject for use in a method for treating stroke in a multiple treatment regimen is listed in this paragraph. In some embodiments, the effective amount of the N-glycosylation inhibitor administered to a subject for use in a method for alleviating one or more stroke symptoms in a multiple treatment regimen is listed in this paragraph. In some embodiments, the effective amount of the N-glycosylation inhibitor administered to a subject for use in a method for preventing stroke in a multiple treatment regimen is listed in this paragraph.

[0041] In some embodiments, methods are described herein that include administering a formulation of an N-glycosylation inhibitor at a particular dosage per subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 10 mg to about 100 mg / kg of subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 20 mg to about 200 mg / kg of subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 25 mg to about 250 mg / kg of subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 35 mg to about 300 mg / kg of subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 20 mg to about 400 mg / kg of subject's body weight. In some embodiments, the formulation comprises an N-glycosylation inhibitor dosage of about 40 mg to about 500 / kg of subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 50 mg to about 200 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 55 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 85 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 125 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 215 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 325 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 425 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor from about 250 mg to about 700 mg / kg of body weight of the subject. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor from about 315 mg to about 750 mg / kg of body weight of the subject. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor from about 355 mg to about 800 mg / kg of body weight of the subject.In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 435 mg to about 620 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 1.5 mg to about 2.0 mg / gram of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 50 mg to about 600 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 75 mg to about 300 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 100 mg to about 200 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 130 mg to about 163 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of the N-glycosylation inhibitor of about 130 mg to about 300 mg / kg of the subject's body weight. In some embodiments, the formulation comprises a dosage of about 130 mg to about 660 mg / kg of the subject's body weight of the N-glycosylation inhibitor. Described herein are methods of preventing, mitigating, or treating stroke in a subject, comprising administering a pharmaceutical composition described herein at a particular dose of one or more N-glycosylation inhibitors in a single treatment regimen or a multiple treatment regimen. 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, a rat, a rabbit, a guinea pig, a cat, a dog, or a pig. In some embodiments, the dose selection is determined by calculating a human equivalent dose (HED). In some embodiments, the dose administered to the subject is a human equivalent dose (HED). In some embodiments, the HED is calculated according to the dose by factor method. In some embodiments, the HED is calculated based on a comparison of body surface area between species. In some embodiments, the HED is calculated based on a comparison of body surface area between mice and humans.In some embodiments, the HED is calculated based on a comparison of body surface area between mice and humans, and for the animal equivalent dose (AED) in mg / kg body weight (HED in mg / kg body weight), the AED is divided by a factor of 12.3. In some embodiments, the dose conversion factor method uses the no observed adverse effect level (NOAEL) of the drug from preclinical toxicology studies to estimate the HED. In some embodiments, the dose selection is based on minimal risk of toxicity in humans. In some embodiments, the dose selection is based on minimal pharmacological activity in humans. In some embodiments, the dose selection is based on existing pharmacokinetic data for another drug of the same pharmacological class. In some embodiments, the other drug of the same pharmacological class is a hexosamine. In some embodiments, the hexosamine is a mannosamine. In some embodiments, the hexosamine is a fructosamine. In some embodiments, the hexosamine is a galactosamine. In some embodiments, the hexosamine is a glucosamine. In some embodiments, the mannosamine is N-acetyl-D-mannosamine (ManNAc), diazirine-derivatized mannosamine (ManNDAz), tetraacetylated N-azidoacetyl-d-mannosamine (ManNAcAz), N-glycolyl-D-mannosamine, 2-N-acetyl-6-O-acetyl-D-mannosamine, N-levulinoyl mannosamine (Man2NLev), N-azido-acetyl mannosamine (Man2NAz), or L-mannosamine. In some embodiments, the 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 interspecies dose scaling. In some embodiments, the dose administered to a subject is determined by one or more guidelines set forth in Nair AB, Jacob SA simple practice guide for dose conversion between animals and humans. J Basic Clin Pharm. 2016, Mar; 7(2): 27-31, the contents of which are incorporated herein by reference.

[0042] Stroke prevention Described herein are methods of preventing stroke, comprising administering to a subject in need thereof an effective amount of one or more N-glycosylation inhibitors. In some embodiments, the one or more N-glycosylation inhibitors comprise hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a 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.

[0043] 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 belongs to the genus Rattus. In some embodiments, the subject belongs to the genus Mus musculus. 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.

[0044] 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.

[0045] 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.

[0046] Stroke risk markers In some embodiments described herein, the method of preventing stroke comprises measuring C-reactive protein (CRP) in blood in a subject.In some embodiments, the measurement of C-reactive protein (CRP) in blood in a subject is reduced compared to the previous measurement of CRP level in blood in this subject.In some embodiments, the measurement of CRP in blood in a subject is reduced to less than about 10mg / L.

[0047] Symptoms of Stroke In some embodiments, a method of preventing, mitigating, or treating stroke in a subject in need thereof, wherein the subject is selected for administration of a pharmaceutical composition described herein. In some embodiments, the subject is selected because he or she has previously suffered one or more TIAs. In some embodiments, the subject is selected to begin treatment after diagnosis that he or she has experienced one or more TIAs. In some embodiments, the subject is diagnosed as having experienced one or more TIAs using a subject experiencing one or more symptoms commonly seen during a TIA. In some embodiments, the one or more symptoms commonly seen during a TIA include muscle weakness, facial numbness, facial paralysis, paralysis of the limbs, paralysis on one side of the body, slurred speech, incoherent speech, difficulty understanding others, sudden loss of vision in one or both eyes, double vision, dizziness, confusion, lack of mental alertness, loss of balance, or impaired coordination, or any combination thereof. In some embodiments, the subject is selected after the subject is observed to show signs of carotid artery disease, a common cause of ischemic stroke. In some embodiments, the subject is selected after diagnosis of stroke. In some embodiments, a computed tomography (CT) scan is used to identify bleeding in the brain, which is indicative of a diagnosis of stroke. In some embodiments, a CT scan is used to identify damage in the brain, which is indicative of a diagnosis of stroke. In some embodiments, a magnetic resonance imaging (MRI) scan is used to identify damage in the brain, which is indicative of a diagnosis of stroke. In some embodiments, imaging tests that reveal narrowing of blood vessels in the neck, aneurysms, or tangles in blood vessels in the brain are used to diagnose stroke. In some embodiments, a subject experiencing one or more symptoms commonly seen in stroke is selected as a subject for the treatment methods described herein. In some embodiments, the one or more symptoms commonly seen in stroke include muscle weakness, facial numbness, facial paralysis, paralysis of the limbs, paralysis on one side of the body, slurred speech, incoherent speech, difficulty understanding others, sudden loss of vision in one or both eyes, double vision, dizziness, confusion, lack of mental alertness, loss of balance, or impaired coordination, or any combination thereof. In some embodiments, the method includes reducing mortality in the subject following administration.In some embodiments, the method comprises reducing mortality in the subject compared to administration of an anticoagulant as a monotherapy, tissue plasminogen activator (TPA) as a monotherapy, an antiplatelet drug as a monotherapy, or an antihypertensive drug as a monotherapy in a control subject. In some embodiments, the method comprises reducing mortality in the subject compared to administration of an anticoagulant, tissue plasminogen activator, antiplatelet drug, or antihypertensive drug, or any combination thereof, in a control subject. In some embodiments, the method comprises reducing mortality in the subject compared to a control subject that either does not receive the treatment or receives a vehicle control therapy. In some embodiments, the method comprises improving one or more symptoms of stroke in the subject following administration. In some embodiments, the method comprises reducing the area or size of damage to neural tissue in the brain of the subject following administration. In some embodiments, the method comprises rescuing hypoxic damage to one or more regions of the brain of the subject following administration. In some embodiments, the method comprises reversing stroke brain damage in the subject following administration. In some embodiments, the method comprises reversing stroke memory impairment in the subject following administration. In some embodiments, the method comprises reversing stroke-induced voluntary movement impairment in the subject after administration. In some embodiments, the method comprises reversing stroke-induced language impairment in the subject after administration. In some embodiments, the method comprises improving cognitive performance 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 impairment in the subject after administration.In some embodiments, the method comprises reversing infarct brain damage in the subject after administration. In some embodiments, the method comprises reducing hemorrhagic transformation of an ischemic event in the subject after administration. In some embodiments, the reduction in hemorrhagic transformation is determined by measuring the hemoglobin level of the subject compared to administering an anticoagulant alone as monotherapy.

[0048] Mitigating ongoing ischemic events Described herein is a method of alleviating an ongoing ischemic event affecting the brain. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor simultaneously with the ischemic event. In some embodiments, the ischemic event comprises the subject experiencing an ischemic stroke, a hemorrhagic stroke, a brainstem stroke, a transient ischemic attack, or a cryptogenic stroke. In some embodiments described herein, the effective amount of an N-glycosylation inhibitor is administered orally, intravenously, intrathecally, and / or intraperitoneally.

[0049] Neurological assessment and blood flow restoration Described herein are methods of mitigating an ongoing ischemic event affecting the brain. In some embodiments, a neurological assessment of a subject is performed during an ischemic event affecting the brain. In some embodiments, a neurological assessment of a subject is performed after an ischemic event affects the brain. In some embodiments, a plurality of neurological assessments of a subject are performed during an ischemic event affecting the brain. In some embodiments, a plurality of neurological assessments of a subject are performed after an ischemic event affects the brain. In some embodiments, neurological assessments performed at different times are compared. In some embodiments described herein, a neurological assessment of an acute stroke in a subject improves compared to a previous assessment. 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 evaluated by transcranial Doppler ultrasound.

[0050] Compounds with a chemical structure similar to ManN In some embodiments of the methods of preventing, mitigating, or treating stroke described herein, one or more compounds comprising a chemical structure similar to ManN are administered to a subject in need thereof. In some embodiments, one or more compounds comprising a chemical structure similar to ManN are administered in combination with ManN. In some embodiments, one or more compounds comprising a chemical structure similar to ManN are administered in combination with Kif. In some embodiments, one or more compounds comprising a chemical structure similar to ManN are administered in combination with Cas. In some embodiments, one or more compounds comprising a chemical structure similar to ManN administered to a subject function as N-glycosylation inhibitors. In some embodiments, one or more compounds comprising a chemical structure similar to ManN are listed in Table 1. In some embodiments, a compound listed in Table 1 is administered to a subject in the methods of preventing, mitigating, or treating stroke described herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

[0051] Combination with known stroke medications In some embodiments, the method of preventing, mitigating, or treating stroke in a subject in need thereof, comprising administering a pharmaceutical composition described herein, further comprises administering to the subject one or more known stroke therapeutics. In some embodiments, the one or more known stroke therapeutics are an anticoagulant, a tissue plasminogen activator drug, an antiplatelet drug, or any combination thereof. In some embodiments, the anticoagulant is rivaroxaban (Xarelto®), dabigatran (Pradaxa®), apixaban (Eliquis®), or edoxaban (Lixiana®). In some embodiments, the tissue plasminogen activator drug is alteplase (Activase®), reteplase (Retavase®), or tenecteplase (TNKase®). In some embodiments, the antiplatelet agent is acetylsalicylic acid (ASA), clopidogrel, ASA and extended release dipyridamole, or ticagrelor administered at 75-325 mg / day.

[0052] 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.

[0053] 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.

[0054] The present disclosure provides pharmaceutical compositions and methods for treating an ischemic condition in a subject, comprising administering to a subject in need thereof an effective amount of the hexosamine D-mannosamine (ManN). Since ManN is converted in vivo to ManN-6-phosphate (ManN-6p), the present disclosure provides for the use of such metabolic precursors and derivatives.

[0055] ManN is a hexosamine with the ability to inhibit protein post-translational modifications, activate stress pathways, and exhibit additivity with VEGF in promoting endothelial cell (EC) proliferation and angiogenesis. The effects of ManN on EC and angiogenesis have not been reported previously. Without being bound by theory, the use of known glycosylation inhibitors together with ManN may provide a link between changes in glycosylation patterns in mammalian EC and angiogenesis. The effects of ManN on endothelial cells may be independent of VEGFR2 activation.

[0056] ManN was discovered in the 1960s as a component of the bacterial wall and accounts for 5–10% of capsular polysaccharides. The related N-acetylmannosamine is thought to be an intermediate in the biosynthesis of sialic acid. Over the years, multiple effects of ManN on enzymes, growth factor-mediated signaling, protein stability, and cell viability have been described. Most of these effects are not specific to ManN and can be induced by other hexosamines. Moreover, they required high concentrations. ManN was reported to have antitumor properties, stimulate osteogenic differentiation, and protect articular cartilage. More recently, ManN was used as an intermediate to facilitate high-throughput screening in the synthesis of unnatural ManNAc analogs for the modification of various molecules / nanoparticles and the expression of thiols on cell surface sialic acids. However, so far, the effect of ManN on ECs has not been described.

[0057] ManN was previously reported to affect the formation of lipid-linked oligosaccharides (LLO) in MDCK cells, possibly by inhibiting a-1,2-mannosyltransferase. Upon ManN treatment, the major oligosaccharides associated with dolichol were Man5G1cNAc2 and Man6GlcNAc2, rather than Glc3Man9G1cNAc2, which is normally found in MDCK cells. Furthermore, ManN was reported to alter protein GPI biosynthesis and hybrid glycan production in the ER. However, none of the angiogenesis-related proteins previously examined are GPI-anchored. Without being bound by theory, the decrease in Man-9 may be a direct result of inhibiting LLO donor synthesis, i.e., Glc3Man9G1cNAc20PP-Dol formation, and then transferring it from the dolichol donor to the polypeptide. In some embodiments, Man-5 may be significantly increased over 24 hours by treating cells with 40 μM ManN. ManN may not affect ER a-mannosidase.

[0058] Activation of PI3K-AKT, PLCy-ERK and p38 has been associated with VEGFR2-mediated EC survival, proliferation and migration. Other cellular metabolic stress sensors such as AMPK (AMP-activated protein kinase) may also confer stress adaptation and promote EC survival via eNOS. Without being bound by theory, activation of ERK, AKT, mTOR, AMPKa, eNOS and ACC is a general phenomenon of hexosamines and mannose. However, activation of JNK / c-Jun and UPR pathways in BCECs is specific to ManN as well as glycosylation inhibitors. Glycosylation is required for correct protein folding in the ER. A link between LLO inhibition and activation of the UPR has been reported. Indeed, despite the complexity of ManN action, LLO inhibition followed by UPR activation appears to be a plausible explanation for the reported ManN effect.

[0059] ECs can cope with acute / minor ER stress due to glycosylation inhibition by activating the UPR pathway. The UPR detects misfolded proteins accumulated in the ER and initiates a response to maintain cellular homeostasis via induction of Bip, a major ER chaperone protein. BiP binds to exposed hydrophobic patches on nascent or incompletely folded proteins that are often non-glycosylated. ManN shows a stronger induction of Bip expression compared to hexosamines. Similar effects on stress pathway activation may result from the glycosylation inhibitors Kif and Cas.

[0060] Glycosylation inhibition is considered to be a new pharmacological strategy that targets metabolic pathways essential for excessive angiogenesis in various pathologies, and glycosylation inhibitors are expected to have antiangiogenic and anti-metastatic properties. Glycosylation has been shown to be involved in cellular stress response and compensatory angiogenesis in response to VEGF-VEGFR2 signaling blockade. Stress-induced O-G1cNAcylation was previously reported to promote survival in response to DNA damage, ER stress, glucose deprivation, and hypoxia in various cell types. Without being bound by theory, glycosylation inhibition may be associated with angiogenesis promotion, and inhibiting glycosylation within the tumor microenvironment may result in stimulation, rather than suppression, of tumor angiogenesis.

[0061] In some embodiments, ManN can be used to promote angiogenesis in a mouse skin injury model with accelerated wound closure. In some embodiments, ManN can be used to stimulate angiogenesis and blood flow recovery in the ischemic hindlimb of mice. The combination of VEGF-A with ManN, or other glycosylation inhibitors, may have advantages over monotherapy for the treatment of ischemic injury. The lack of a direct permeability enhancing effect of ManN may result in less edematous tissue. In this context, damage to the pulmonary endothelium is a central pathogenic event in respiratory failure associated with various infections, including SARS-CoV-2. An endothelial cell mitogen such as ManN, which lacks a permeability effect, may help protect and stabilize blood vessels and thus limit tissue damage.

[0062] In some embodiments, intravitreal administration of ManN may be used to enhance retinal neovascularization, for example, in therapeutic applications in ocular diseases. While 10-15% of patients with moderate AMD progress to neovascular form, the remaining patients may present with geographic atrophy (GA). Previous studies have shown that loss of choriocapillaris is frequently detected in GA, and choriocapillaris regeneration / protection may be a strategy for GA treatment. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN may be used in methods of 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 may be used in a method for treating dry age-related macular degeneration (AMD), diabetic macular edema, macular edema due to 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 may 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 may be used in a method for treating wet age-related macular degeneration (AMD), choroidal neovascularization (CNV), polypoidal choroidal vasculopathy, degenerative (pathological) myopia, giant cell arteriopathy, 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 ocular diseases. In some embodiments, intravitreal administration of a therapeutically effective amount of ManN stops the progression of ocular diseases. The halting or slowing of disease progression can be due to an increase in retinal perfusion with no or minimal leakage and / or a non-leaky or non-leaky retinal revascularization. The halting or slowing of disease progression can be due to an increase in choroidal perfusion with no or minimal leakage and / or a non-leaky or non-leaky choroidal revascularization.

[0063] 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 to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of VEGF.

[0064] In some embodiments, the ischemic condition is caused by disease or trauma. The present disclosure provides treatment for a number of conditions characterized by reduced perfusion, including, but not limited to, diabetic ulcers, macular degeneration, peripheral artery disease (PAD), limb ischemia, cerebral or cerebral ischemia, and coronary ischemia.

[0065] In some embodiments, administration is oral, intravenous, intrathecal, or intraperitoneal.

[0066] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, comprising administering to a subject in need thereof an effective amount of hexosamine D-mannosamine (ManN).

[0067] In some embodiments, the administration is effective to reduce ischemia in the subject. In some embodiments, the ischemia can include cerebral ischemia. The administration can be effective to prevent, reduce, or treat conditions associated with cerebral ischemia, such as edema, ischemic stroke, or infarction. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor. In some embodiments, the method further comprises administering to a subject in need thereof an effective amount of VEGF.

[0068] In some embodiments, the subject is in need of induction of angiogenesis due to an ischemic condition caused by disease or trauma.

[0069] In some embodiments, administration is oral, intravenous, intrathecal, or intraperitoneal.

[0070] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in a cell, comprising administering to the cell an effective amount of hexosamine D-mannosamine (ManN).

[0071] In some embodiments, the administration is in vivo. In some embodiments, the administration is ex vivo. In some embodiments, the administration is effective to stimulate EC proliferation and angiogenesis. In some embodiments, the administration is effective to activate JNK and the unfolded protein response triggered by ER stress.

[0072] In some embodiments, the administration is effective to induce changes in N-glycan and O-glycan profiles, hi embodiments, the administration is effective to induce a reduction in Man6GlcNAc2 (Man-6), Man-8, and Man-9 in total oligomannose N-glycan content, an accumulation of Man-5 and Man-7, and a decrease in O-glycosylation following treatment with ManN, compared to untreated controls.

[0073] Conversely, in some embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting angiogenesis, including, but not limited to, methods for treating malignant tumors and intraocular neovascular disorders in a subject comprising administering to a subject in need thereof an effective amount of an inhibitor of hexosamine D-mannosamine (ManN) or reducing the amount of ManN available to the subject.

[0074] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Usubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); 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 University of the Sciences 2012).

[0075] As used herein, the words "comprise," "comprising," "include," "including," "has," "having," "contain," "containing," "characterized by," or any other variation thereof, are intended to encompass a non-exclusive inclusion of the recited components, subject to any limitations expressly indicated otherwise. For example, a pharmaceutical composition and / or method that "comprises" 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 recited or inherent to the pharmaceutical composition and / or method.

[0076] It is understood that aspects and embodiments of the disclosure described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments. As used herein, the transitional phrases "consist of" and "consisting of" exclude any elements, steps, or components not specified. For example, "consist of" or "consisting of" used in a claim limits the claim to the components, materials, or steps specifically recited in the claim, excluding impurities normally associated with them (i.e., impurities within a given component). When the phrase "consists of" or "consisting of" appears in a clause in the body of a claim, rather than immediately following the preamble, the phrase "consists of" or "consisting of" limits only the elements (or components or steps) recited in that clause, and does not exclude other elements (or components) from the claim as a whole.

[0077] 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 these 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" is intermediate between "comprising" and "consisting of."

[0078] 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.

[0079] The term "and / or," when used in the context of a list of two or more items, means that any one of the listed items may be used by itself or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean one or both of A and B, i.e., A only, B only, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A only, B only, C only, 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.

[0080] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Also, herein, values ​​or ranges may be expressed as "about," "about" from one particular value, and / or "about" to another particular value. When such values ​​or ranges are expressed, other embodiments disclosed include the particular values ​​recited, from the one particular value, and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It is further understood that there are several values ​​disclosed herein, and that 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 recited value, within 5% of the recited value, or within 2% of the recited value.

[0081] As used herein, "patient" or "subject" means a human or other mammalian subject being treated.

[0082] As used herein, the term "pharmaceutical composition" refers to a pharma- ceutically acceptable composition that includes a pharma- ceutically active agent and, in some embodiments, further includes a pharma- ceutically acceptable carrier. In some embodiments, a pharmaceutical composition may be a combination of a pharma- ceutically active agent and a carrier.

[0083] The term "combination" refers to either a fixed combination in one dosage unit form or a kit of parts for combined administration, where one or more active compounds and combination partners (e.g., another drug described below, also referred to as "therapeutic agent" or "adjuvant") can be administered simultaneously or separately within a time interval. In some situations, combination partners exhibit a cooperative effect, e.g., a synergistic effect. As used herein, terms such as "co-administration" or "combined administration" are meant to encompass administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which agents are not necessarily administered by the same route of administration or at the same time.

[0084] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining two or more active ingredients, including both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that both active ingredients, e.g., both compounds and combination partners, are administered to a patient at the same time in the form of a single entity or dosage. The term "non-fixed combination" means that both active ingredients, e.g., both compounds and combination partners, are administered to a patient simultaneously, concurrently, or sequentially as separate entities without specific time constraints, such that such administration provides a therapeutically effective concentration of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0085] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or State government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, in addition to other formulations that are safe for use in animals, and more specifically, humans and / or non-human mammals.

[0086] As used herein, the term "pharmaceutical acceptable carrier" refers to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or vehicles administered with the demethylenated compound. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents. Antibacterial agents, such as benzyl alcohol or methylparabens, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity, such as sodium chloride or dextrose, may also be carriers. Methods of combining carriers to prepare pharmaceutical compositions are known to those skilled in the art. In some embodiments, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical 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 present pharmaceutical compositions is contemplated.

[0087] As used herein, "therapeutically effective amount" refers to an amount of a pharma- ceutically active compound that is sufficient to treat or ameliorate, or in some way reduce, symptoms associated with a disease or condition. When used in relation to a method, the method is effective enough to treat or ameliorate, or in some way reduce, symptoms associated with a disease or condition. For example, with respect to a disease, an effective amount is an amount that is sufficient to arrest or prevent its onset, or, if disease symptoms have begun, to alleviate, ameliorate, stabilize, reverse, or delay the progression of the disease, or reduce the pathological consequences of the disease. In either case, the effective amount may be given in a single dose or in divided doses. The phrase "therapeutically effective amount" also refers to an amount of an N-glycosylation inhibitor described herein that results in a measurable therapeutic response. The therapeutic response may be any response that the user of the method (e.g., a clinician) recognizes as an effective response to treatment, including improvement in one or more symptoms (e.g., one or more symptoms of stroke or other ischemic disorder) and surrogate clinical markers (e.g., stroke biomarkers). The therapeutic effect is generally an improvement or inhibition of one or more symptoms of a disease or condition (e.g., ischemic stroke, hemorrhagic stroke, or ischemic condition). A measurable therapeutic response also includes the finding that a therapeutic agent (e.g., an N-glycosylation inhibitor) described herein prevents, delays the onset of, or otherwise alleviates one or more symptoms of a disease or disorder. 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, elevated hemoglobin levels, and death.

[0088] As used herein, the terms "treat", "treatment", or "treating" encompass at least ameliorating symptoms associated with a disease in a patient, where amelioration is used in a broad sense to refer to at least a decrease in the severity of a parameter, such as a symptom, associated with the disease or condition being treated. Thus, "treatment" includes situations in which a disease, disorder, or pathological condition, or at least a symptom associated therewith, is completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated), rendering the patient no longer susceptible to the condition, or at least the symptoms that characterize the condition.

[0089] As used herein, unless otherwise specified, the terms "prevent", "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or one or more symptoms thereof. In some embodiments, these terms refer to treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agents, prior to the onset of symptoms, particularly to subjects at risk of a disease or disorder provided herein. These terms encompass the inhibition or reduction of symptoms of a particular disease. In some embodiments, subjects with a family history of a disease are potential candidates for a preventive regimen. In some embodiments, subjects with a medical history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be used interchangeably with the term "prophylactic treatment".

[0090] As used herein, unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder or prevent its recurrence. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent that alone or in combination with one or more other agents provides a prophylactic benefit in the prevention of a disease. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent.

[0091] As used herein, unless otherwise specified, the compounds described herein are intended to encompass all possible stereoisomers unless a specific stereochemistry is specified. When structural isomers of a compound are interchangeable via a low energy barrier, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism, or so-called valence tautomerism in compounds containing, for example, aromatic moieties. The term "derivative" refers to a chemical that is structurally related to another substance, or that can be made from another substance (i.e., the substance from which it is derived), for example, by chemical or enzymatic modification.

[0092] As used herein, the term "pharmaceutically acceptable salt" refers to an acid or 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 impart any harmful or undesirable effects on the subject to which it is administered and in the context in which it is administered. Pharmaceutically acceptable salts can be derived from amino acids, including but not limited to cysteine. Methods for making compounds as salts are known to those of skill in the art (see, for example, 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 salt" is intended to mean a free acid or base salt of an agent or compound represented herein that is non-toxic, biologically acceptable, or otherwise biologically suitable for administration to a subject. See generally, Berge, et al., J.Pharm.Sci., 1977,66,1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic response. The agents or compounds described herein have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and therefore can react with some inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0093] Examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-l,4-dione, hexyne-1,6 ... These include phosphate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [gamma]-hydroxybutyrate, glycolate, tartrate, and mandelate salts.

[0094] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "then," "before," "after," "lastly," and "finally," is intended to be illustrative and not limiting of the scope of the embodiments disclosed herein.

[0095] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0096] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended terms that are both conjunctive and disjunctive in function. For example, each of the terms "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 A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together.

[0097] As used herein, "or" may refer to "and," "or," or "and / or," and may be used both exclusive and inclusive. For example, the term "A or B" may refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0098] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0099] The term "about" when referring to a number or numerical range means that the number or numerical range referred to is approximate within experimental variation (or within statistical experimental error) and that the number or numerical range may vary, for example, by 1% to 15% from the stated number or numerical range. In the examples, the term "about" refers to ±10% of the stated number or value.

[0100] The terms "increased," "increasing," or "increase" are generally used herein to mean an increase by a statically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, for example, 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 an increase up to and including 100%, or any increase between 10-100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0101] The terms "reduced," "reducing," or "reducing" are generally used herein to mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reducing" means a reduction of at least 10% compared to a reference level, e.g., a reduction of 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 a reduction up to and including 100% (e.g., nonexistent or undetectable levels compared to a reference level), or any reduction between 10 and 100%. In the case of a marker or symptom, these terms refer to a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to a level that is accepted as within the normal range in individuals without a particular disease.

[0102] While 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. It is not intended that the present invention be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein which depend upon a variety of conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the present invention. It is therefore contemplated that the present invention also covers any such alternatives, modifications, variations, or equivalents. It is intended that the claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered. EXAMPLES

[0103] The following illustrative examples are representative of embodiments of the stimuli, systems, and methods described herein, and are not intended to be limiting in any way.

[0104] As disclosed in the Examples below, the hexosamine mannosamine (hereafter 2-amino-2-deoxy-D-mannose or ManN) inhibits protein glycosylation but stimulates EC proliferation in vitro. The biological effects of ManN in other in vitro and in vivo models, and its possible mechanism of action, have been investigated. ManN is an EC mitogen and survival factor for bovine and human microvascular ECs, with additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response triggered by ER stress. ManN leads to enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, accelerating limb blood flow recovery compared to controls. Furthermore, intraocular injection of ManN induces retinal neovascularization. Thus, inhibition of protein glycosylation followed by activation of stress pathways can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treating ischemic injury.

[0105] Example 1 The effect of ManN on EC proliferation was evaluated. A library of 619 highly purified metabolites, covering a wide range of chemicals, was screened for their ability to affect the growth of bovine choroidal microvascular ECs (BCECs) in the presence or absence of VEGF. This and similar assays have been used previously to identify and characterize angiogenesis stimulators and inhibitors. Under the conditions tested, little or no proliferation was detected in the absence of VEGF.

[0106] An initial screen was performed in which each compound was tested at concentrations of approximately 1 and 10 μM (assuming a molecular weight of 100 Da for each compound) with or without 5 ng / ml VEGF, which could induce an approximately 4-5 fold increase in cell proliferation. Six compounds of various chemical natures showed some inhibitory or stimulatory activity. The analysis focused on one of these, ManN, a hexosamine originally identified as a constituent of the bacterial cell wall, because it showed the most potent and consistent effect. ManN had a significant stimulatory effect in the dose range of 5-500 μM and was also additive with VEGF in promoting the proliferation of BCECs. The dose-dependent effect of ManN on BCEC proliferation in the absence or presence of VEGF is shown in Figures 1A and 1B. When cells were treated with 50 μM ManN and 5 ng / ml VEGF, we obtained a maximum of about 6.5-fold increase in EC-coated surface area with 50 μM ManN alone (Figure 1A) or a maximum of about 2.5-3-fold increase in fluorescence units upon addition of AlamarBlue (Figure 1B) compared to VEGF alone. AlamarBlue detects mitochondrial activity as an indicator of cell viability that correlates with cell number in a certain range. The effect of ManN had a bell-shaped dose-response curve, with inhibition at higher concentrations (Figures 1A and 1B). An additive effect of ManN in promoting BCEC proliferation was also observed with bFGF (Figure 11A) and bovine retinal ECs (BRECs) (Figures 11C and 11D).

[0107] Various hexosamines (galactosamine, glucosamine, and their N-acetyl derivatives) were tested together with ManN in the BCEC proliferation assay. However, none of these hexosamines showed a significant stimulatory effect (Fig. 1D). Several structurally related molecules such as D-isoglucosamine (fructosamine), meglumine, muramic acid, N-acetyl-neuraminic acid (a sialic acid present in all mammalian cells), glucose, and mannose were also tested. None of these molecules stimulated BCEC proliferation with or without VEGF (Fig. 11C and D).

[0108] ManN entered and accumulated in cells in a concentration-dependent manner. When BCECs were treated with 400 μM ManN for 2 h, 0.66 nmol ManN was detected in 1 mg of cell lysate. Once inside the cells, ManN was rapidly converted to ManN6-phosphate (ManN-6p), but not mannose. No incorporation of ManN was detected in N-glycans. Efficient incorporation of ManNAc and mannose has been reported.

[0109] The effect of ManN on BCEC proliferation was dependent on cellular glycolysis. When glucose-free medium was used, the additivity between ManN and VEGF disappeared, whereas the activity of VEGF was independent of the glycolytic pathway (Fig. 11B). However, in glucose-free medium, even in the presence of VEGF, there was significant cytotoxicity at as low as 4 μM ManN.

[0110] To further characterize the effect of ManN on EC survival, proliferation, and migration, confluent BCEC monolayers were mechanically wounded. Figure 1E shows that 40 μM ManN or 50 ng / ml VEGF significantly accelerated BCEC migration and / or proliferation, as reflected by more complete closure of the "scratched" area, compared to the control group after 48 h. Similar to the proliferation assay, additivity was observed when cells were treated with both ManN and VEGF (Figure 1E). In addition, 40 μM ManN showed significant additivity with VEGF in promoting BCEC migration (Figure 1F).

[0111] The observations were extended to human retinal microvascular ECs (hRMECs), HUVECs, and dermal microvascular endothelial cells (hDMVECs). ManN itself stimulated the growth of HUVECs and hDMVECs. Furthermore, in all EC types tested, there was dose-dependent additivity with VEGF, with minimal toxicity even at 5 mM. Similarly, stimulation of migration and wound closure was observed in HUVECs treated with 40 μM ManN alone (migration assay) and / or in combination with 50 ng / ml VEGF (scratch assay).

[0112] Example 2 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 STAT 3. Together, they affect glycolysis, EC sprouting, proliferation, and migration. The effect of ManN and / or VEGF on activation of key signaling pathways known to promote proliferation in BCECs, such as ERK, AKT, mTOR, and CREB (cAMP response element binding protein), was evaluated. ManN activated ERK, AKT, mTOR, and CREB at 40 μM. Stimulation of ERK, AKT, and CREB was rapid, occurring within 10 to 30 min after adding ManN (Figures 2A-2C). Furthermore, enhanced activation of ERK, AKT, and CREB was observed when both ManN and VEGF were present compared to ManN or VEGF alone (Figures 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 of which exerts a bell-shaped effect on EC proliferation. Both eNOS and ACC were significantly activated by 40 μM ManN within 10–30 min (Figure 2D). However, activation of ERK, AKT, mTOR, CREB, ACC, and eNOS was not unique to ManN. Indeed, other hexosamines such as ManNAc and mannose induced similar activation of these signaling pathways (Figure 2C). Although activation of these common proliferation pathways likely contributed, without being bound by theory, several unique mechanisms may be involved in the EC mitogenic effect of ManN.

[0113] Example 3 Using a series of specific pharmacological inhibitors, we identified JNK / c-jun as a signaling pathway uniquely activated by ManN among the hexosamines. Western blot analysis revealed that among the three MAPK family members (ERK, p38, and 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 (Figures 3A and 3B). ManN activated the JNK pathway, but not the other hexosamines tested. Treatment of BCECs with the JNK-specific inhibitor SP600125 (5 μM) abolished the effect of ManN on BCEC proliferation (Figure 3C).

[0114] The effect of ManN on BCECs was assessed after transfection with siRNA against JNKs (i.e., JNK1 and JNK2, since JNK3 is not expressed in BCECs). Knocking down approximately 80% of either JNK1 and / or JNK2 by two independent siRNAs against JNK1 or JNK2 abolished the mitogenic effect of ManN on BCECs at μM concentrations (Figures 3D and 3E), indicating that both JNK1 and JNK2 are important in transducing stress signals.

[0115] Example 4 ManN affects protein glycosylation in endothelial cells. Additivity of ManN with VEGF could potentially occur at the transcriptional and / or translational level or through the VEGF-VEGFR2 mediated signaling pathway. However, when cells were treated with various concentrations of ManN for 4 hours (for gene expression level) or 24 hours (for protein expression level), neither the transcription of VEGF, VEGFR2, and GLUT4, nor total VEGFR2 protein expression was significantly altered in BCECs (Figures 4A-4C, Figures 5A and 5B, Figures 7C and 7E). The same was true for BRECs and hRMVECs. Biotinylation studies showed no change in the amount of VEGFR2 on the cell surface. However, VEGFR2 phosphorylation in response to VEGF was decreased in ManN pretreated cells, suggesting that VEGFR2 activation was hindered rather than enhanced in BCECs. Ligand-independent VEGFR2 activation did not occur after ManN addition in BCECs. The same was true for HUVEC (SFIG .10a) and hDMVEC.

[0116] The apparent molecular weight of VEGFR2 shifted significantly from 40 μM after ManN treatment in both BCECs (Figures 4A-4C and 4E) and BRECs. A new lower molecular weight band (approximately 170-200 KDa) appeared in BCECs treated with ManN in a dose-dependent manner compared to controls (major band at approximately 230 KDa and minor band at approximately 210 KDa) (Figures 4B, C, and E; Figures 5A and 5B; and Figure 7C). This shift was specific to ManN among hexosamines and their derivatives (Figure 4A). VEGF alone had no effect on molecular weight. Addition of VEGF to ManN did not cause an additional shift (Figure 4A). The lower molecular weight VEGFR2 band is likely not a degradation product, since removal of ManN completely reversed the effect of ManN on molecular weight after 24 h (Figure 4E). However, based on PNGase F treatment, it appears that not all glycosylation on VEGFR2 was abolished by ManN, at least at μM concentrations. Experiments with the small molecule tyrosine kinase inhibitor axitinib, a potent VEGFR2 inhibitor, indicate that reduction of VEGFR2 molecular weight and stimulation of BCEC proliferation by ManN are independent of VEGFR2 signaling.

[0117] Significant changes in VEGFR2 protein mass were also observed in hRMVEC, HUVEC (Fig. 4F), and hDMVEC (Fig. 4G) at 40 μM ManN, although additive effects of ManN and VEGF on proliferation of these cells occurred at mM levels.

[0118] To better understand how ManN may affect VEGFR2 post-translational modifications, cells were treated with ManN in the presence of one of four monosaccharides (mannose, glucose, galactose, or fucose) at up to 1:10 molar ratios. These monosaccharides are known to be important in protein N-glycosylation. Our results suggest that mannose can block the ManN effect on VEGFR2 molecular weight and BCEC proliferation in a dose-dependent manner (Figures 4C and 4D). The effect of mannose may not be limited to preventing the entry of ManN into cells via the same transporter, since the effect was seen when BCECs were first treated with ManN for 2 hours to ensure good cellular uptake. Glucose had a similar effect as mannose, but not galactose or fucose.

[0119] The reduction in protein abundance after ManN treatment in BCECs was not limited to VEGFR2: other N-glycosylated growth factor receptors / coreceptors or adhesion molecules, including av integrins, neuropilin-1, VE-cadherin, and bFGFR1, were similarly affected.

[0120] Example 5 The effect of ManN on general protein glycosylation profiles was evaluated. N-glycosylation is a complex process that depends on multiple enzymes that act sequentially on glycoproteins to generate hybrid and high mannose glycan structures as they pass through the secretory pathway from the ER to the Golgi apparatus. It plays a key role in determining the fate of newly synthesized glycoproteins in the ER, their correct folding, cellular destination, and proper function.

[0121] Several key enzymes involved in protein N-glycosylation in both the ER and Golgi were evaluated. α-mannosidase from jack bean is an exoglycosidase with broad specificity that catalyzes the hydrolysis of terminal non-reducing αl-2, αl-3, and αl-6 linked mannose residues of oligosaccharides in both organelles and controls the conversion of high-mannose to complex N-glycans, the final hydrolytic step 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, considerably higher than the effective mitogenic concentration in BCECs. No effect of up to 2 mM ManN on α- or β-glucosidases was detected.

[0122] N-linked glycans from BCECs were isolated by enzymatic cleavage and subsequently purified and characterized using MALDI-TOF-MS. Treatment with 40 μM ManN resulted in a significant time-dependent reduction of Man6G1cNAc2 (Man-6), Man-8, and Man-9 in the total oligomannose N-glycan content compared to untreated controls, whereas a significant early-stage accumulation of Man-5 and Man-7 was observed after ManN treatment. ManN has previously been shown to inhibit lipid-linked oligosaccharide (LLO) synthesis, alter protein GPI biosynthesis and hybrid glycan production, and incorporate into glycans in MDCK cells. The accumulation of Man-5 over time suggested that inhibition of mannosidases is unlikely to be the mechanism of pro-angiogenic activity in BCECs.

[0123] Monosaccharide content was measured to profile the composition of complex N-glycans. Compared to untreated control cells, a significant decrease in fucose (8 h), mannose (12 h), galactose (24 h), and Neu5Ac (8 h and 24 h) was found in ManN-treated cells, consistent with the inhibitory activity of ManN on global protein N-glycosylation.

[0124] O-glycan modification is another form of post-translational modification of proteins, in which a serine or threonine residue is covalently linked to a GalNAc residue. The GalNAc residue can be further modified by several glycosyltransferases that act sequentially to elongate the glycan chain in either a branched or linear 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 to produce the Tn antigen. When the Tn antigen is generated, it can have three different fates: (i) it can be sialylated on C6 by the enzyme ST6GalNAcT, (ii) it can be substituted on C3 or C6 by f3-G1cNAc giving rise to core-3 or core-6, respectively, or (iii) it can be galactosylated on C3 by ClGalT1 to form core-1, which can also be sialylated to produce mono- or di-sialyl core-1 O-glycans.

[0125] O-glycan analysis was performed in BCEC lysates by MALDI-Tof mass spectrometry. As a unique enzyme to cleave all different forms of O-glycans is not available, reductive beta-elimination was performed to understand the O-glycan backbone. Permethylation was performed before MALDI-Tof / Tof mass spectrometry to protect against desialylation during mass spectral data acquisition. Overall reduction of O-glycosylation after treatment with 40 μM ManN. In particular, we observed a trend of decrease in ion intensity at m / z of 895 (sialyl-core 1, Galf31-3GalNAc-), 1256 (disialylated core 1), 983 [core 2, GlcNAcf31-6(Galf31-3)-GalNAc-], and 1187 (di-galactosylated core 2).

[0126] Example 6 The effect of ManN on activating the UPR by increasing Bip and CHOP expression was evaluated. Asparagine-linked N-glycosylation is one of the most common modification reactions in eukaryotic cells, occurring on proteins that are cotranslationally transported across the ER or incorporated into the ER during biosynthesis. After N-linked oligosaccharides are transferred to nascent proteins by OSTs (oligosaccharyltransferases), ER-resident glucosidases and mannosidases generate a series of glycan trimming intermediates that are specifically recognized by ER-localized lectins to direct the nascent protein to protein folding, degradation, or export pathways. One consequence of inhibition of protein glycosylation is impaired protein folding, leading to ER stress. Physiological responses to the UPR are mediated by changes in gene expression, such as the regulation of the ER Hsp70 chaperone BiP (glucose-regulated protein 78, also known as immunoglobulin-binding protein) and another multifunctional transcription factor CHOP (CCAAT-enhancer-binding protein homologous protein). For example, UPR dysfunction in aging creates a permissive environment for protein aggregation, unresolved ER stress, and chronic inflammation.

[0127] To investigate the possible ManN-mediated ER stress, we studied the expression of Bip and CHOP in ManN- or mannose-treated cells by Western blot analysis. Our data showed that ManN, but not mannose or VEGF, could significantly turn on Bip expression in a concentration-dependent manner when growing cells were deprived of growth factor supply, with Bip accumulation evident at 24 h (Figures 5A and 5B) and 48 h (Figure 5A). CHOP induction appeared faster at about 6 h in a dose-dependent manner (Figure 5A). No synergy between ManN and VEGF in promoting Bip or CHOP expression was noted (Figure 5B).

[0128] We tested two well-known chemical chaperones, 4-PBA (4-phenylbutyric acid) and TUDCA (tauroursodeoxycholic acid), to alleviate ER stress in ManN-treated BCECs. Both were previously shown to attenuate the tunicamycin-induced eIF2a-ATF4-CHOP arm of the UPR and Bip expression. We found that 2 mM 4-PBA could prevent the induction of CHOP expression by ManN at 400 μM and 5 mM, and restore the expression of ATF-6 (activating transcription factor-6) by ManN at 400 μM, but not TUDCA at 500 μM (Figure 5C). Similarly, the restoration of ATF-6 expression by TUDCA was much weaker compared to 4-PBA. As a transmembrane ER glycoprotein, ATF-6 is cleaved to release a 50 kDa amino-terminal fragment, which translocates to the nucleus to activate the transcription of ER chaperones and activate 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 could effectively reverse the bell-shaped activity of ManN on BCEC proliferation in the absence or presence of VEGF. The additivity between ManN and VEGF was nearly abolished (Figure 5D).

[0129] Example 7 We evaluated the effect of ManN on non-endothelial cells. To extend the observations with ECs, we examined various non-EC types from different species. These included NIH3T3 fibroblasts and AML12 hepatocytes (mouse), ARPE-19 RPE cells (human), and freshly isolated bovine pituitary cells. We also tested several human cell types relevant to in vivo models, such as skin fibroblasts and keratinocytes. In addition, we screened four human or mouse cancer cell lines (A673, U87MG, Calu6, and 4T1) (Figure 6). To examine post-translational modifications of proteins in non-ECs, we monitored molecular weight changes using bFGFR1 or f31 integrin. Similar to BCECs, ManN can induce molecular weight changes in all these non-ECs, whereas mannose cannot (Figure 6 inset). However, unlike BCEC (Figure 1B), BREC (Figure 1C), hRMVEC, HUVEC, and hDMVEC, no proliferative effect by ManN was observed at μM-mM concentrations, 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 among different cell types, with AML12 being the most sensitive and human RPE cells and human keratinocytes being the least sensitive to 5 mM ManN levels (Figures 6E and 6H). Growth inhibition by 25 mM mannose in vitro has been reported in several tumor lines with low levels of PMI (phosphomannose isomerase). At 5 mM, ManN, but not mannose, showed significant toxicity on 4T1 cells, possibly due to higher PMI levels in 4T1 compared to all reported sensitive tumor lines (Figure 6D).

[0130] Example 8 Similar to ManN, inhibitors of protein N-glycosylation stimulate EC growth. To determine whether global changes in protein glycosylation promote cell proliferation, two well-characterized inhibitors, kifunensine (Kif) and castanospermine (Cas), were tested. BCEC proliferation was stimulated in a dose-dependent manner in the absence or presence of 5 ng / ml VEGF (Figures 7A and 7B). After treatment with Kif or Cas for 24 hours, a reduction in VEGFR2 molecular weight on SDS-PAGE was evident (Figure 7C).

[0131] At 40 μM, Kif significantly activated ERK and AKT in BCEC (Figure 7E), HUVEC, and hDMVEC. Activation of ERK by Cas was less evident in both BCEC and hDMVEC. However, both inhibitors were able to activate the JNK pathway in BCEC (Figure 7E). Blocking JNK activation with 5 μM SP600125 significantly reduced the effect of both glycosylation inhibitors on BCEC proliferation (Figure 7F). Figure 7C shows the dose-dependent induction of Bip expression when growing BCEC in the presence of Kif or Cas at concentrations that promote cell proliferation was switched to growth factor-free medium for 24 h.

[0132] Both Kif and Cas had significant activity in the BCEC "scratch" assay, with gaps being closed more rapidly by each molecule over a 48-h period compared to the control (Figure 7D). The inset panel of Figure 7D shows representative images from assays using Kif or Cas. Quantification analysis showed that there was a significant acceleration of gap closure in a dose-dependent manner compared to the control.

[0133] Example 9 The effect of ManN on endothelial cells in vitro and its relationship to angiogenesis in vivo was investigated through its effect in a mouse splint wound model. In this model, the repair process is entirely dependent on epithelialization, cell proliferation, and angiogenesis, which closely reflect the biological process of human wound healing. The effects of ManN and VEGF were tested alone or in combination. Topical application of 20 μg VEGF or 20 μg ManN per day was performed for the first 3 days after wounding. When VEGF and ManN were combined, a significant acceleration of wound closure was observed during the early phase of healing (Figure 8A). Compared to VEGF or ManN monotherapy, the combination had a significantly faster wound closure from day 2 onwards (Figure 8B). On day 4, the mean wound closure rates were 81.5%, 75.6%, 66.9%, and 29.8% in the PBS-, ManN-, VEGF-, and combination treatment groups, respectively. The number of small blood vessels around the wound area was quantified on day 4. A significant increase in CD31 positive blood vessels was found in the combination group compared to the PBS control, VEGF or ManN alone (FIGS. 8C and 8D).

[0134] Thus, ManN, in combination with VEGF, promotes angiogenesis in a skin injury model in which wound closure occurs rapidly without treatment.

[0135] The stability of ManN was evaluated in wound fluid contaminated by bacteria, a common feature of wounds. ManN was added to freshly collected wound fluid from a mouse model of skin infection with Staphylococcus aureus, a common cause of skin and soft tissue infections in humans. After incubation in such wound fluid for up to 24 h at 37 °C, no significant loss of free ManN was detected. Thus, ManN may be useful in the treatment of infected wounds, possibly in combination with antibacterial or other agents.

[0136] One of the known properties of VEGF is the rapid induction of vascular permeability after injection into the skin of guinea pigs. The effect of ManN in inducing vascular permeability was evaluated in the same assay. However, when tested at 1 ng to 5 μg, no permeability enhancing effect was elicited by ManN, whereas 25 ng of VEGF induced vascular permeability.

[0137] Example 10 The angiogenic effects of ManN and Kif in a mouse hindlimb ischemia model were evaluated. To evaluate the activity of ManN in a chronic ischemia model that may more specifically reflect its effect as an endothelial cell mitogen and angiogenic promoter, a mouse hindlimb ischemia model was considered. Depending on which vessel is occluded, several variants have been described. The variant chosen is the ligation and resection of the femoral artery, which results in more severe ischemia compared to simple femoral artery ligation. Occluding two vessels results in more severe ischemia, but has the disadvantage of inducing severe pain and distress in mice, as well as frequent ulceration and necrosis.

[0138] Oral administration of ManN was tested in this femoral artery ligation-resection model. Kif was previously administered intraperitoneally for in vivo studies, so this route was used. Laser Doppler perfusion imaging (LDPI) was used as a non-invasive method to observe the time and extent of blood flow recovery in the ischemic limb. Serial examination of blood flow was performed with LDPI, and the increase in the perfusion ratio of the ischemic (ligated, left side) vs. non-ischemic (sham, right side) hind limb after ligation was used to indicate blood flow recovery. Starting immediately after surgery, 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 HO-treated group showed approximately 25% blood flow recovery, a value in good agreement with published data on the same mouse strain and with the same type of lesion. However, the blood flow recovery rates in the ManN- and Kif-treated groups were approximately 40% and 47%, respectively, indicating an accelerated rate of blood flow recovery compared with mice treated with HO (Figs. 9A and 9B). The blood perfusion ratio continued to increase to approximately 50% of the sham-treated limb 3 weeks after ManN and Kif treatment, significantly higher than that of the control group (Figs. 9A and 9B).

[0139] Consistent with improved blood flow, the ischemic hindlimb of the ManN- and Kif-treated groups showed increased vascular density compared to the control group, as assessed by CD31 immunostaining of the surrounding muscle tissue 3 weeks after ligation. Compared to the HO-treated control group, vascular density was 2.3- and 1.8-fold higher in the ManN- and Kif-treated groups, respectively (Figures 9C and 9D).

[0140] After oral administration, there was a relatively rapid decline in ManN plasma levels. Plasma free ManN levels reached a peak level of approximately 100 nmol / ml plasma at 1 h. After 3 h, only about half of that amount was detectable. Muscle samples were taken from the ischemic leg 2 h after oral feeding of 20% ManN. Significant amounts of ManN reached the ischemic leg, with 0.17+1-0.18 nmol / mg protein for free ManN and 0.91+1-0.24 nmol / mg protein for ManN-6p. At least in BCECs, the effect of ManN on protein mass persisted for at least 8 h in the absence of exogenous ManN (Figure 4E), indicating that even a relatively short exposure may be sufficient to induce a pharmacological effect.

[0141] Example 11 The effects of ManN and Kif on the induction of retinal angiogenesis were evaluated. The findings in cultured eye-derived ECs were extended to a suitable in vivo model system. The mouse retina has been widely used over the past decades to study both physiological and pathological angiogenesis. To obtain a detailed description of the retinal vasculature, images from retinal flat mounts were processed for vascular area fraction (the ratio of the area covered by blood vessels to the total retinal area). Using this model, the effect of ManN on retinal angiogenesis was evaluated. Kif was also tested in this model because it is a water-soluble inhibitor and its mechanism of glycosylation inhibition is well established. Moreover, it shares with ManN the ability to activate ERK, AKT, and stress pathways in BCECs (Figure 7E).

[0142] 500 nanograms of ManN or Kif were injected intravitreally, and retinal vasculature was examined 7 days later. In this model, intravitreal administration of 200 ng of bFGF was used as a positive control. In the bFGF, ManN, and Kif treatment groups, the density of retinal blood vessels increased by approximately 35%, 30%, and 20%, respectively, compared to the PBS group (FIGS. 10A and 10B).

[0143] Example 12 The effect of ManN treatment on cerebral infarction (induced by MCAO) was tested. The experiment was performed in a mouse model of acute ischemia-reperfusion-induced brain injury, with a combination of pre-treatment and post-treatment with an N-glycosylation inhibitor. In this example, ManN was tested as an inhibitor of N-glycosylation. The mouse MCAO model is a well-studied and reproducible example of an acute ischemic event to one hemisphere of the brain. In contrast to other in vivo ischemia models disclosed herein (e.g., hindlimb ischemia model and blood flow assessment), the MCAO model has an acute phase of ischemia (2 hours) followed by the restoration of normal blood flow to the affected area. In this example, the effect of ManN treatment was evaluated in tissues (e.g., brain tissue) that are particularly susceptible to short periods of blood flow disruption, and assayed at a time point after normal blood flow had already been restored.

[0144] On day -1, mice were treated with 40 mg of ManN (20% ManN in 200 μL water) or 200 μL water for control animals by oral gavage. The timing of pretreatment, in which the test group mice were administered a dosage of ManN, was approximately 16 hours before surgery. Administration of this dosing regimen was continued once every 48 hours (e.g., on days 1, 3, etc.) until the extent of the infarcted area of ​​the animal's brain was evaluated. Approximately 16 hours after the first treatment (day 0), the animals underwent a 2-hour surgical occlusion of the right middle cerebral artery, thereby causing an acute ischemic event in a portion of the right brain. After the 2-hour occlusion, normal blood flow was restored. Figure 12A shows the timing of the ManN administration treatment regimen, as well as the timing of the procedure and subsequent assays. Figure 12B shows the results of the first experiment, in which four animals were treated with ManN paired with four control-treated animals for analysis. Coronal MRI results on day 4 show clearly demarcated and measurable infarct areas in ManN-treated and vehicle-treated animals. MRI was used to measure multiple CNS coronal slices from a single animal and to calculate the total infarct volume fraction per animal on day 4. ManN-treated animals had significantly smaller infarct areas as shown in the graph. Figure 12C shows the results of a second experiment in which four animals were treated with ManN paired with four control-treated animals for analysis. Coronal MRI results on day 4 show clearly demarcated and measurable infarct areas in ManN-treated and control-treated animals. MRI was used to measure multiple CNS coronal slices from a single animal and to calculate the total infarct volume fraction per animal on day 4. ManN-treated animals had significantly smaller infarct areas as shown in the graph. Figure 12D shows a combined analysis of the data from Figures 12B-12C, demonstrating a statistically significant effect of ManN treatment in reducing the overall infarct area assessed 4 days after the acute ischemic event. Statistical analysis was performed by two-tailed two-sample unequal variance t-test. These results show a measurable benefit of a treatment regimen that includes pre- and post-treatment with orally delivered ManN in reducing the total brain infarct area following an acute ischemic event. In additional animal studies, Figure 13A shows representative MRI images of the results in animals treated with ManN paired with control-treated animals for analysis.Coronal MRI results on days 2 and 4 show well-defined and measurable infarct areas in ManN-treated and vehicle-treated animals. Reduction of infarct area is significant in ManN-treated animals on both days 2 and 4 compared to vehicle (HO)-treated controls. All results on day 4 are graphed and analyzed in FIG. 13B. Multiple CNS coronal slices from a single animal were measured using MRI, and the total infarct volume factor on day 4 per animal was calculated. Quantification of MRI images was performed using ImageJ. Each point represents the calculated total CNS infarct volume per animal measured using MRI. Statistical analysis was performed using a two-tailed two-sample unequal variance t-test (p<0.001). This indicates that a multiple-dose treatment regimen involving administration of an effective amount of ManN to mice in the MCAO model significantly reduced CNS infarct volume on day 4 in ManN-treated animals compared to vehicle-treated animals. In FIG. 13C, TTC (2,3,5-triphenyltetrazolium chloride) staining was visualized in ManN-treated and vehicle-treated coronal slices of mouse CNS at day 4 from a representative animal. TTC staining is commonly applied for fast and reliable visualization of hypoxic brain tissue to clarify the size and area of ​​cerebral infarction. TTC-stained live cells turn healthy / normal tissue deep red. In contrast, damaged or dead tissue remains white indicating the absence or reduction of live cells, indicating the area of ​​infarction. The therapeutic effect is evident with reduced infarct area in ManN-treated animals at day 4, and the absence of TTN staining in widespread CNS areas in vehicle-treated animals, compared to the absence of white staining in widespread areas in ManN-treated animals.

[0145] To examine the vascular density in the infarct-affected area of ​​the cerebral cortex by examining the expression of CD31, animals were examined on day 6 according to the treatment protocol shown in FIG. 12A. CD31 (PECAM-1) is a marker highly expressed on endothelial cells. CD31 fluorescent immunohistochemistry confirmed and quantified the vascular density in the identified area of ​​the cerebral cortex affected by infarction in the MCAO model in ManN-treated and vehicle-treated animals. ManN-treated animals were gavaged with a therapeutically effective dose of ManN (40 mg) on ​​days -1, 1, 3, and 5. Vehicle-treated animals were given water on days -1, 1, 3, and 5. Brain tissue was harvested and fixed on day 6. The infarct area was identified and CD31 immunostaining was performed on coronal sections to label the vasculature. Representative CD31 stained sections of vehicle (HO)-treated and ManN-treated animals are shown in FIG. 14A. In Figure 14B, quantification of vascular density of representative sections was performed using Image J, and cumulative results are graphed according to CD31 staining density. Statistical analysis was performed using a two-tailed, two-sample, unequal variance t-test (p<0.001), indicating that a multi-dose treatment regimen involving administration of an effective dose of ManN to mice in the MCAO model significantly increased vascular density in the area of ​​the cerebral cortex affected by infarction in ManN-treated animals compared to vehicle-treated controls.

[0146] Animals were examined on day 4 according to the treatment protocol shown in FIG. 12A to examine histopathological changes within the infarct-affected area of ​​the right cerebral hemisphere by examining hematoxylin and eosin (H&E) staining of coronal sections. ManN-treated animals were gavaged with a therapeutically effective dose of ManN (40 mg) 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 using H&E. As seen in the top row of FIG. 15, the cerebral cortex and striatum regions show changes in brain morphology and cytoarchitecture in the brain coronal sections of vehicle-treated mice. The bottom row of FIG. 15 shows coronal sections of the CNS of ManN-treated mice, with the inset regions of the cerebral cortex and striatum shown at higher magnification. Vehicle-treated animals showed faint H&E staining of the infarcted area compared to H&E staining of normal brain tissue, with increased vacuolization and edema. Nuclei in the vehicle-treated infarcted area appeared smaller, shrunken, and stained more intensely than nuclei in comparable ManN-treated brain regions affected by infarction. These results indicate that pretreatment of ManN and continued posttreatment after an acute ischemic event ameliorates histopathological changes in the brain in a mouse MCAO model of stroke.

[0147] Materials and Methods Small Molecule Libraries The Mass Spectrometry Metabolite Library of Standards (MSMLS) (IROA TECHNOLOGIES, Bolton, MA; now Sigma) is a collection of 619 high-quality small molecules (>95% purity) spanning a broad range of primary metabolites including carboxylic acids, amino acids, biogenic amines, polyamines, nucleotides, coenzymes, vitamins, lipids, etc. Plates were spun at 300 g after reconstitution according to the manufacturer's instructions.

[0148] chemical compound D-mannosamine hydrochloride was obtained from Sigma (M4670) or Spectrum Chemical MFG Corp (M3220). 1-Amino-l-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), Streptomyces Tunicamycin (T7765) from Bacillus sp., and SP600125 (S5567) were obtained from Sigma. Hypure 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), 4-phenylbutyric acid (4-PBA) (P21005), castanospermine (Cas, C3784), kifunensine (K1140), DMSO (D2650) were from Sigma. DMSO (D2650) was used as the solvent for Cas.

[0149] antibody Antibodies used in this study were from Cell signaling Technology Inc (Danvers, MA) unless otherwise specified. 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). Phosphor-antibodies: 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, 9191), ACC (Ser79, 3661), eNOS (Ser1177, 9571), AMPKa (Thr172, 50081), c-Jun (Ser73, 9164), (31 integrin (4706&34971), av integrin (4711), JNK1 (3708), JNK2 (4672), JNK3 (2305). Anti-(3-actin was from Sigma.

[0150] cell Primary human umbilical vein endothelial cells (HUVECs, passages 4–10) were obtained from Lonza (C2519AS, lot no. 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 (BRECs, #BRMVEC-3) and bovine choroidal microvascular endothelial cells (BCECs, #BCME-4) (both from VEC Technologies, Renssellaer, NY) were maintained on fibronectin-coated plates (1 μg / cm2). Growth medium was low glucose DMEM supplemented with 10% bovine calf serum (BCS), 5 ng / ml bFGF, and 10 ng / ml human VEGF165. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. bFGF (233-FB) and VEGF165 (293-VE) were purchased from R&D systems. Human retinal microvascular endothelial cells (passage <15) were from Cell Systems Corporation (Kirkland, WA). Cells were grown on 0.1% gelatin-coated plates in medium 131, containing 5% fetal bovine serum, hydrocortisone (1 μg / ml), human fibroblast growth factor (3 ng / ml), heparin (10 μg / ml), human epidermal growth factor (1 ng / ml), and dibutyryl cyclic AMP (0.08 mM) (MVGS, S 005-25, Gibco Invitrogen). Human RPE cell line ARPE-19 was from ATCC. Cells were gently lifted with 0.025% trypsin and plated in RtEGM medium (Clonetics) containing 2% FBS, L-glutamine, human bFGF, GA-1000). Once the cells had attached to the plate, serum-free RtEGM medium was used to maintain the cultures for best results. ARPE-19 was obtained from ATCC (CRL-2302) and cultured according to the manufacturer's instructions. NIH3T3 cells were obtained from ATCC (CRL-1658). Human adult skin MVECs (CC-2543) were cultured in EGM-2MV (CC-4147, Lonza).Keratinocytes (ATCC, PCS-200-011) were cultured in Skin Cell Basal Medium (PCS-200-030) and Keratinocyte Growth Kit (PCS-200-040). Human primary skin fibroblasts (ATCC PCS-201-012) were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) and Growth Kit (ATCC, PCS-201-040). Growth stimuli 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 with 10% FBS (Omega Scientific, Tarzana, CA) and antibiotics. A673 (CRL-1598), A549 (CCL-185), U87MG (HTB-14) cells were 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 by the various suppliers.

[0151] Cell proliferation assay Proliferation assays were performed with BCECs and BRECs. Log-phase growing BCECs or BRECs (passages <10) were trypsinized, resuspended, and seeded at a density of 1200-1500 cells per well in 200 μl volume in 96-well plates (uncoated) in low-glucose DMEM supplemented with 10% calf serum, 2 mM glutamine, and antibiotics (growth medium). All reagents were added at the indicated final concentrations. After 3-6 days, cells were incubated with AlamarBlue for 4 h. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Experiments were repeated at least three times. To create hypoxic conditions, cells were placed in a hypoxic incubator containing a gas mixture consisting of 1% O2, 5% CO2, and 94% N2. Each 96-well plate included untreated and VEGF-treated (10 / ng / ml) wells to monitor plate-to-plate variations. 20% methanol or 0.05% DMSO served as negative controls. When Cas was tested in these cells, 0.05% DMSO served as negative controls. Human RMVEC and human adult DMVEC were split into gelatin-coated 96-well plates (2000 cells per well) of low glucose DMEM containing 10% FBS. 1200 cells / well were set up for proliferation assays in low glucose medium containing 0.5% FBS. Data were collected on days 4 or 5. HUVEC (p7-10) were grown on gelatin-coated plates until they reached 70-80% confluency.

[0152] On the day of the assay, cells were dissociated with 0.05% trypsin, which was neutralized with EBM containing 0.5% FBS. Cells were spun briefly and then resuspended in 0.5% FBS medium. Cells were counted and plated in 96-well plates, 1000 cells / well. Triplicate wells were used for each treatment. Data was collected on the third day, and then cells were fixed with 4% paraformaldehyde for 15 minutes before adding crystal violet. Cell coverage area was quantified after photography by Image J software.

[0153] Proliferation assays with fibroblasts were performed in low glucose DMEM containing 1% FBS with or without 10 ng / ml bFGF or 100 ng / ml human EGF, and assays were terminated on day 3. ARPE-19 cells were gently lifted with 0.025% trypsin and plated in RtEGM medium (Clonetics) containing 2% FBS, L-glutamine, human bFGF, and GA-1000. Once the cells had attached to the plate, serum-free RtEGM medium was used to maintain the cultures. For proliferation assays, 1500 human RPE cells were plated in 96-well plates in low glucose DMEM containing 1% FBS. A673, U87MG, Calu6, and AML12 cells were grown to confluence and then harvested and resuspended in the appropriate assay medium. For proliferation assays, cells were plated at a density of 1000-2000 cells / well in low glucose DMEM containing 5% FBS or as described elsewhere. Bovine pituitary cells (pituitary folliculo-stellate cells) were isolated as previously described. For proliferation assays with human epidermal keratinocytes, human DMVEC, and human dermal fibroblasts, 1000 cells / well were plated in low glucose DMEM containing 1% FBS with or without various growth factors. Assays were terminated on day 3 for bovine pituitary cells and day 4 for all other cell types. For 4T1, 1000 cells were plated in RPMI-1640 with 2% BME and 2% FBS on basement membrane extract (BME)-coated 96-wells and processed 4 hours later. After 4 days, tumor cell growth was measured by the MTS assay (Promega, Madison, WI), a colorimetric assay that measures the metabolic activity of viable cells. Recombinant human transferrin was obtained from EMD Millipore (Temecula, Calif.) Recombinant mouse apo-transferrin was obtained from Sigma.

[0154] SiRNA knockdown BCECs were plated in 6-well culture plates at a density of 1.5×105 cells / well and cultured overnight. 2 ml of antibiotic-free culture medium was used to replace the old medium. siRNAs including siNegative (Ambion, AM4611), siRNA against JNK1#2 (Invitrogen, NM_001192974.2_siRNA 266), JNK1#4 (Invitrogen, NM_001192974.2_siRNA_485), siRNA against JNK2#2 (Invitrogen, XM_005208371.4_siRNA_1240), and 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). Briefly, a mixture containing 25 pmol siRNA, 7.5 μL RNAiMAX reagent, and 125 μL Opti-MEM medium was used to transfect cells in each well, resulting in a final siRNA concentration of 12.5 nM. A mixture of RNAiMAX and Opti-MEM was used as a non-siRNA control. Cells were incubated with siRNA. After 8 hours, the siRNA-containing medium was replaced with fresh medium. 24 hours and / or 48 hours after transfection with siRNA, cells were used for proliferation assays and protein extraction.

[0155] PNGase F treatment Glycerol-free PNGase F was obtained from New England Biolabs (Ipswich, MA). Briefly, BCECs were lysed with NP-40 containing protease inhibitors (Thermo Scientific, Waltham, MA). Lysates were clarified at 5000g for 25 min at 4°C. Total protein content was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). 20 mg of protein was mixed with 10x denaturation buffer and H2O to a total volume of 10 ml. Glycoproteins were denatured at 100°C for 10 min followed by the addition of Glycobuffer and PNGase F. Reactions were carried out at 37°C for 2 h.

[0156] Western blot Cells were allowed to reach approximately 80% confluence in 12-well plates. Cells were pretreated with ManN, Kif, or Cas for various periods of time with or without subsequent addition of VEGF, with HO serving as a solvent control for ManN. At various time points, plates were removed from the incubator and kept on ice. Cell monolayers were first washed once with ice-cold PBS before lysing with 250 μI Pierce RIPA buffer (ThermoFisher Scientific, Rockford, IL) or lysed with 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 1% NP-40 containing protease / phosphatase inhibitor cocktail (100x) (Cell signaling, #5872). 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 (ThermoFisher scientific, #NP0007). Samples were subjected to SDS-PAGE (Bolt 4-12% Bis-Tris Plus, Invitrogen) using Bolt MES SDS running buffer or NuPAGE 3-8% Tris-Acetate gels using Tris-Acetate SDS running buffer (Novex). HUVECs (passages 6-8) were plated in EBM-2 basal medium (Lonza) containing 0.2% FBS. After overnight culture, cells were serum starved in EBM-2 medium for 4 hours and then treated with 50ng / ml VEGF165 or vehicle control for various lengths of time. Equal amounts of protein lysates were analyzed by SDS-PAGE and blotted with the indicated antibodies. Proteins were transferred using Tris-Glycine buffer containing 20% ​​methanol (proteomic grade) (Apex BioResearch Products). Membranes were first incubated with 5% milk in TBST, pH 7.6 (TEKnova, Hollister, CA) and subsequently blotted with primary and secondary antibodies.ECL anti-rabbit IgG, horseradish peroxidase-conjugated total antibodies from donkey or sheep anti-mouse were obtained from GE Healthcare (UK Limited). SuperSignal West Dura Extended Duration substrate was from ThermoFisher Scientific. In some cases, the same PVDF membranes were stripped by incubation in Restore Plus Western Blot Stripping Buffer (ThermoFisher Scientific) for 8 min to show total specific protein expression, followed by a second strip for (3-actin expression.

[0157] Migration assay HUVECs (passages 6-8) were cultured and serum-starved as described in "Western Blot". Ten thousand cells in 150 mL of EBM-2 medium were then added to the upper chamber of 8 gm pore size cell culture inserts (Falcon) coated with 0.1% gelatin. The lower compartment was filled with 600 mL of EBM-2 medium containing various drugs. The plates were 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) for 20 minutes at room temperature. The migrated cells on the bottom surface of the insert membrane were quantified by counting the entire area of ​​the insert at 40x magnification. Experiments were performed in triplicate and repeated three times. BCEC migration was set up similarly, except that the wells were coated with FN, the cells were suspended in 1% serum medium, and the migration time was 18-24 hours.

[0158] Scratch assay BCECs (passages 6–10) and HUVECs (passages 6–8) were used in this assay. Cells were grown to approximately 80% confluency in 6-well plates, washed twice with PBS, and then starved for 5 h in serum-free DMEM (low glucose, Hyclone) before creating a "scratch" using a 1 ml tip. Cell monolayers were briefly washed once with serum-free medium, followed by various treatments with medium containing 1% FBS. After 48 h, the assay was stopped by adding 2 ml of 4% paraformaldehyde. After 20 min, fixed cells were stained with 1 ml of crystal violet (Sigma). Plates were gently washed under running tap water and air-dried before being photographed. Images were acquired with a ZEISS Discovery V8 SteREO microscope equipped with a PixeLINK Megapixel FireWire camera. Quantification of wound closure was performed using AxioVision LE Re1.4.4 software. Six images were taken for each sample and six measurements (in pixels) were made for each image using AxioVision LE Re1.4.4 software.

[0159] N-Glycan, Monosaccharide, Sialic Acid, O-Glycan Analysis As soon as 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 min and washed once with cold PBS. Cells were homogenized and total protein was measured. All subsequent analyses were based on known protein amounts.

[0160] N-linked glycans were removed from glycoprotein samples using PNGase-F kit (New England BioLabs, P0705S). Briefly, 300 μg protein sample was reconstituted in 180 μl UltraPure water. 20 μl 10x denaturing buffer was added and boiled for 14 min using a 100°C water bath. The sample was cooled to room temperature and centrifuged at 2700g for 1 min. Then, 50 μl 10x NP-40 was added and the sample was kept at room temperature for 30 min with vortexing at 5 min intervals, followed by addition of 25 μL 10x reaction buffer and mixing thoroughly. Then, 5 μl PNGaseF (2500U) was added to the sample and mixed gently. The sample was incubated at 37°C for 16 h. Released N-glycans were purified using solid phase extraction method. Briefly, N-glycans were purified by sequentially passing the reaction mixture over a preconditioned Sep-Pak C18 1 cc cartridge (Waters) and a HyperSep PGC (polygraphitized carbon) cartridge (25 mg, 1 ml Thermo Scientific). The cartridge was washed with 4 ml of water, and only the PGC was washed with an additional 1 ml of water. N-glycans bound to the PGC were eluted using 30% acetonitrile containing 0.1% TFA in water. Finally, the purified N-glycans were lyophilized and labeled with 2-AB. Briefly, the samples were dissolved in a 10 μl solution of 0.44 M 2-AB (2-aminobenzamide) in 35% acetic acid in DMSO containing 1 M sodium cyanoborohydride. The samples were incubated at 65° C. for 2.5 h. The 2-AB labeled glycans were purified using a GlycoClean S cartridge (GLYKO) following their glycan clean-up protocol. Excess reagent was removed from the samples using Glycoclean S-cartridges (Prozyme) and the labeled glycans were dried using a SpeedVac and stored at −20° C. Profiles of 2-AB labeled glycans were obtained using a Dionex CarboPac PA1 (4×250 mm) anion exchange column with a guard column (4×50 mm) at a flow rate of 1 ml / min.Glycans were separated in 100 mM sodium hydroxide with a 0-250 mM sodium acetate gradient over 0-75 min. Data were collected using a Dionex ICS-3000 HPLC system equipped with an Ultimate 3000 fluorescence detector (Dionex) set at Aex 330 nm, Atm 420 nm with a sensitivity of 7. Data were processed using Chromeleon software (Thermo Scientific).

[0161] Monosaccharide composition analysis was performed using HPAEC-PAD (Thermo-Dionex ICS3000) to calculate the nmole amount of each monosaccharide present in 25 μg of protein. Samples were hydrolyzed using 2N trifluoroacetic acid (TFA) at 100 °C for 4 hours. Subsequently, a dry nitrogen flush was used to remove the acid. To ensure complete removal of the acid, samples were coevaporated twice with 100 μI of 50% isopropyl alcohol (IPA). Finally, samples were dissolved in Milli-Q water and injected into the HPAEC-PAD. Monosaccharide profile was performed using a Dionex CarboPae PA1 column (250 mm × 4 mm, with a 50 mm × 4 mm guard column). An isocratic solvent mixture of 19 mM sodium hydroxide and 0.95 mM sodium acetate was used at a flow rate of 1 ml per minute for 25 minutes. Data were acquired using the standard Quad waveform supplied by the manufacturer for carbohydrates. All neutral and amino sugars were 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.

[0162] Mild acid hydrolysis was used to release sialic acid. Briefly, samples were treated with 2M acetic acid at 80°C for 3 hours, followed by removal of excess acid using speed vacuum. Sialic acid was then tagged with DMB reagent and analyzed using a RP-UPLC-FL (Waters Acquity UPLC) system. Known amounts of standard Neu5Ac were used to quantify the amount of sialic acid in the samples.

[0163] For O-glycan analysis, homogenized cell samples were treated with 50 mM NaOH in the presence of 1 M NaBH4 at 45°C for 16 h. 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. The excess boric acid generated during neutralization was then removed by co-evaporation using acidified methanol and methanol, respectively. Finally, the O-glycans were purified by passing over 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 absolute methanol, mixed with SDHB (Super-DHB) MALDI matrix in a 1:1 v / v ratio, and spotted onto a maldi plate. Mass spectral data were acquired using a Bruker AutoFlex mass spectrometer in positive reflectron mode. Mass spectral data were analyzed and annotated using GlycoWork Bench software, and masses consistent with the proposed structures were annotated. Monoisotopic ion intensities were acquired for calculations.

[0164] To measure the cellular uptake of ManN and its subsequent conversion to ManN-6P, BCECs were grown in 60 mm dishes to a density of approximately 6 × 105 cells per dish. ManN was added to the cultures at a final concentration of 400 μM. The cells were then incubated for 2 h. The monolayers were washed three times with PBS at room temperature and lifted by a cell scraper in 10 ml of PBS on ice. The cell pellets were obtained by centrifugation at 400 g for 5 min and stored at -80 °C for further use. The cell pellets were suspended in 200 μl of Ultra-pure ice-cold water in the presence of 1 μl of protease inhibitors. The cells were sonicated for 1 min with 30 s pulses and vortexed to form a homogenous solution. 2.5 μl of the homogenate, in triplicate, was used for protein estimation using the BCA assay method. To quantify the total protein amount, a standard curve of BSA at concentrations from 0 to 800 μg / ml was performed. The cell homogenate was filtered through a pre-washed 3K filter and the filtrate was dried using a speed-vac. The dried sample was reconstituted in 100 μl of ultrapure water and a sample containing the equivalent of 200 μg of 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 samples. All standards except ManNH2-6P were obtained from Sigma-Aldrich. ManNH2-6P was obtained from Omicron Biochemicals, Inc. (South Bend, IN). The amount of monosaccharides present in the different cells is expressed as nmol / mg of total protein. All analyses were performed on a Thermo-Dionex ICS system using 100 mM NaOH and 250 mM NaOAc as the HPLC running buffer and a CarboPac-PA-1 column.

[0165] Biotinylation of surface proteins BCECs were plated on 10 cm cell culture dishes 3 days prior to cell surface protein isolation. Cells were washed three times with Dulbecco's PBS containing CaCl2 and MgCl2, followed by incubation with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA; 0.5 mg / ml in Dulbecco's) on ice for 30 min. Cells were washed twice with Dulbecco's and unreacted biotin was blocked with 20 mM glycine for 15 min. 100 μM oxidized glutathione (Sigma-Aldrich, St. Louis, MO) was added to the last wash to prevent reduction of disulfide bridges in biotin molecules during the cell lysis process. For cell lysis, 500 μL of lysis buffer (2% NP-40 in PBS, 1% Triton X-100, 10% glycerol, 100 μM oxidized glutathione, EDTA-free protease inhibitor tablets (Roche, Mannheim, Germany) were added to the cells. Lysed cell extracts were scraped off the plates and transferred to Eppendorf tubes, followed by incubation for 30 min on ice on a shaker. Cell extracts were incubated with 30 U of DNase (22°C 50 min, Roche, Mannheim, Germany) and centrifuged for 20 min (20,800 × g, 4°C) to pellet insoluble material. Protein concentrations of the supernatants were determined. Equal amounts of protein (approximately 2 mg) from each extract were used for cell surface protein isolation. Supernatants were transferred to biotin agarose beads (Pierce ImmunoPure Immobilized D-biotin, Thermo Scientific) and incubated for 20 min at 4°C for 3 h. The beads were pre-cleared using lysis buffer (Scientific, 20221) and the pre-cleared solution was used for cell surface protein isolation using streptavidin beads. The beads were washed four times with lysis buffer, four times with 300 mM NaCl in lysis buffer, and twice with 50 mM Tris-HCl, pH 7.8. Proteins were eluted twice with elution buffer (50 mM DTT in 50 mM Tris-HCl, pH 7.8) at 30°C, followed by pooling of the eluates. Three biological replicates and one non-biotinylated control were used in the study.

[0166] Gene expression analysis by real-time Q PCR RNA was purified using RNeasy mini kit (Qiagen). 50 ng of total RNA per reaction was used for real-time PCR (Taqman) analysis. Reactions were set up in MicroAmp Fast Optical 96-well reaction plates, sealed with MicroAmp optical adhesive film, and run on a ViiA7 real-time PCR system (Applied Biosystems) and absolute quantification using standard curves was used with Sequence Detection System (SDS) software. Expression levels of each gene were further quantified relative to the housekeeping gene RPL19 in the same sample. Taqman primer and probe mixes were obtained from Thermo Fisher Scientific. Bovine VEGF-A (Bt03213282), bovine RPL19 (Bt03229687), and bovine specific VEGFR2 (Bt03258877), GLUT1 (Bt03215313), and GLUT4 (Bt03215316).

[0167] α-Mannosidase, α- and -glucosidase activity assays a-Mannosidase activity was measured using the substrate p-nitrophenyl α-mannopyranoside (1 mM). Enzyme from Jack bean (M7257) (final concentration 0.077 U) was incubated at 37°C in a final volume of 50 μl of 50 mM potassium phosphate buffer, pH 7.5. α-Glucosidase was assayed with the substrate p-nitrophenyl a-glucoside (7 mM). Enzyme from Saccharomyces cerevisiae type 1 (Sigma, G5003) (final concentration 0.1 U) was incubated at 37°C in a final volume of 50 μl of PBS, pH 7.5. β-Glucosidase was assayed with the substrate 4-nitrophenyl β-D-glucopyranoside (Roche). Enzyme from almond (Sigma, G0395) (final concentration 0.002 U) 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). Enzyme activity was measured at 405 nm. α-Glucosidase from Saccharomyces cerevisiae type I (G5003) was assayed using p-nitrophenyl α-D-glucopyranoside (Sigma, N1377) as substrate.

[0168] Measurement of ManN in wound fluid from S. aureus -infected mice To test the stability of ManN in wound fluid collected from mice with skin infections, as described below, 1.5 μl of 5% ManN solution was added to each 200 μl of wound fluid, which was first diluted 1:1 (v / v) in PBS. At each time point, samples were removed from the 37° C. incubator and stored at −80° C. Plasma proteins were precipitated by adding ice-cold acetonitrile in a ratio of 1:3 (v / v) plasma:acetonitrile. Samples were kept on ice for 1 h and then centrifuged at 12000 g for 10 min at 7° C. to form a pellet. The supernatant was transferred to another tube, dried on a Speed ​​Vac, then reconstituted in UltraPure distilled water and filtered through a pre-washed Nanosep 3K Omega filter (Pall Corporation). The filtrate was dried on a Speed ​​Vac. The dried samples were dissolved in 100 μl of water and 2 μl of plasma or wound fluid sample was subjected to HPLC analysis. Neutral and amino sugars were separated on a Dionex CarboPae PA1 column 4mm x 250mm with a 4mm x 50mm guard column. An isocratic gradient of 19mM sodium hydroxide with 0.95mM sodium acetate was used at a flow rate of 1ml / min for 20 minutes. Data was collected using a Dionex ICS-3000 HPLC system equipped with a pulsed amperometric detector using a standard Quad waveform. ManN was identified and quantified by comparison to monosaccharide standards using Thermo Scientific Chromeleon software. No ManN samples served as negative controls.

[0169] Skin Wound Healing Model All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego and were performed in an ethical manner and in accordance with the Animal Care Program (ACP) guidelines.

[0170] The model has been described previously. Briefly, C57BL / 6 female mice (8–10 weeks old) were obtained from Jackson labs (Sacramento, CA). A fresh full-thickness punch wound (4 mm diameter) was created on the back of the animal in a class II biological safety cabinet using a punch (Acu Punch, Acuderm inc. Ft. Lauderdale, FL), splinted with a sterile neoprene ring (6 mm outer diameter, 4 mm inner diameter), and secured with 5–6 sutures (4–0 nylon) under the influence of isoflurane. Sterile technique was followed for all surgical procedures. Buprenorphine was administered subcutaneously before awakening from anesthesia for expected pain. Mice were monitored until fully awakened and housed individually to minimize injury / biting / fighting to the surgical site. Recombinant human VEGF was a gift from Roche-Genentech (Telbermin, recombinant human VEGF165). Treatments were prepared in PBS, sterile filtered, and 25 μl of the solution was applied directly to the wound bed daily for the first 4-5 days under the influence of isoflurane, followed by daily observation. Wound closure was monitored by routine imaging, and wound area was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).

[0171] On day 4 after wounding, the wounds were excised, including a 2 mm margin of surrounding tissue, and placed in 10% formalin for up to 24 hours. The wounds were then bisected centrally and 5 μm paraffin sections were processed for hematoxylin and eosin (H&E) and Masson's trichrome staining. Epithelial gaps were measured histomorphometrically using AxioVision LE Re1.4.4 software. Skin tissues were fixed in 10% formalin for 24 hours. Paraffin embedding and sectioning were performed by the Moores Cancer Center Histology Core, UCSD. The 5 μm paraffin sections were deparaffinized and rehydrated, followed by heat-induced antigen retrieval in 10 mM citrate buffer (pH 6.0). Immunostaining was performed as previously described. Anti-CD31 (SZ31, rat IgG2a) (Dianova, Warburgstrasse 45, 20354 Hamburg, Germany) was used at 2 μg / ml. CD31 positively stained small vessels were counted microscopically on 10 fields (20x magnification) taken around the wound.

[0172] Vascular permeability assay Vascular permeability was evaluated using a modified Miles assay. Hairless male guinea pigs (control: 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 of 1% Evans Blue dye. 15 min later, an intradermal injection (0.05 ml / site) of different doses of ManN was administered in the area of ​​the trunk behind the shoulder. All reagents were diluted in PBS for intradermal administration. 25 ng of VEGF165 per site was used as a positive control. Thirty min after the intradermal injection, the animals were euthanized by ip injection of pentobarbital (200 mg / kg). Skin tissue was cut from connective tissue and photographed.

[0173] Mouse skin infection model A mouse skin infection model was established. Briefly, mid-log phase Staphylococcus aureus subcultured from an overnight culture in Todd Hewitt broth was used in this study. 6-8 week old C57BL / 6 mice were obtained from Charles River Laboratories. Mice were infected after shaving and depilation with Nair cream. 5 × 107 CFU of S. aureus were injected intradermally into the left groin of the mice. Three days later, the abscess was surgically removed and homogenized on ice. Fluid was collected and spun at 14000 rpm. The clarified supernatant was diluted 1:1 with PBS for further use. Animals were housed in clean cages and experimental procedures were subsequently performed under pathogen-free conditions. The presence of bacteria in the wound fluid was confirmed using Todd Hewitt broth (THB) plates.

[0174] Hindlimb ischemia model and evaluation of blood flow C57BL / 6 male mice (6-8 weeks old) underwent unilateral hindlimb surgery under anesthesia with ketamine / xylazine cocktail. Briefly, the left femoral artery was isolated from the vein and nerve, ligated proximally, and excised. The right hindlimb served as a control. Blood flow was measured using a laser Doppler perfusion imager (PeriScan PSI, Perimed). Ischemic and nonischemic limb perfusion was measured before and after surgery and at 1, 2, and 3 weeks after surgery. After surgery, mice were randomly assigned to different groups (8 mice per group). 200 μl of 20% ManN was administered orally every other day starting on the third day after surgery. 200 μl of 1 mg / nil Kif was administered by ip injection every other day. HO was used as a vehicle control. Final blood flow values ​​were expressed as the ratio of ischemic to nonischemic hindlimb perfusion from the same animal. Quantification of vascular area was performed as described.

[0175] retinal neovascularization Assessment of retinal neovascularization after intravitreal administration was performed. Briefly, 6-8 week old C57BL / 6 male mice were randomly assigned to different groups and anesthetized with ketamine / xylazine cocktail. The indicated amounts of ManN, Kif, or bFGF (R&D systems, AF-233-NA) in 1 μl of PBS and PBS vehicle control were injected intravitreally with a 33-gauge Hamilton syringe. Seven days after injection, animals were euthanized. Eyes were then enucleated and fixed in 4% paraformaldehyde (PFA) for 30 min. Retinas were isolated and stained with anti-CD31 immunofluorescence (IF) to demonstrate vasculature. Assessment was performed by an investigator blinded to treatment. For CD31 IF, rat anti-mouse antibody (BD Biosciences, CAT# 550274) was diluted 1:100 and incubated overnight at 4 °C. After 4 h of incubation with Alexa Fluor-488-labeled anti-rat antibody (Life Technologies, A11006), whole mounts were imaged with the 488 nm channel using an A1R Confocal STORM super-resolution system (Nikon). Quantification of choroidal and retinal vascular density was performed by ImageJ. Each experiment was repeated three times with similar results, and each treatment group consisted of five individual samples.

[0176] Middle cerebral artery occlusion stroke model An evaluation of the extent of cerebral infarct size modulated by ManN treatment was performed. A mouse model of acute stroke was selected to investigate the effect of pretreatment with ManN and the continuation of obvious posttreatment of causative ischemia. This experiment was performed twice. Adult C57BL / 6J mice (Jackson Laboratory, Bar Harbor, ME) weighing 20-25 g were randomly assigned to each group. Hexosamine D-mannosamine (ManN) treatment was started the day before surgery (day -1). The drug (ManN) was administered by oral gavage. 40 mg was administered to each mouse (20% ManN in 200 μL of water). Control group mice were given water each time as a vehicle control. This treatment regimen was repeated every 48 hours (e.g., day 1, day 3, etc.) until the extent of the infarct area of ​​the animals was examined. Surgery was performed as described above. Briefly, on day 0, mice were anesthetized with ketamine / xylazine cocktail. Mice were then placed in a supine position and prepared in a sterile manner. A midline cervical incision was made, and the soft tissues over the trachea were gently retracted. The left common carotid artery (CCA) was carefully exposed and dissected away. After double ligation of the proximal end of the CCA, the external carotid artery (ECA) was ligated to prevent bleeding, and the internal carotid artery (ICA) was temporarily occluded with a microarterial clamp. A small incision was made 2 mm distal to the ECA-CCA bifurcation, and a blunt-tipped 6-0 silicone-coated monofilament nylon suture (6022910PK10Re, Doccol Corporation, MA, USA) was gently inserted through the CCA. After removing the microarterial clamp on the ICA, the filament was advanced into the ICA until it blocked the origin of the middle cerebral artery (approximately 9–10 mm). After 2 h of occlusion, the suture was removed to restore blood supply to the MCA territory. Body temperature was maintained at 36.5-37.5 °C using a heating pad on the operating table throughout the procedure, from the start of surgery until the animals recovered from anesthesia. For sham-operated animals, vascular stripping was performed without the insertion of sutures. One day after surgery, neurological scores were assessed based on the behavior of the mice. MRI imaging of the brain was performed 24 h after reperfusion and every other day until the determined endpoint was reached. T2-weighted (T2w) imaging was performed for cerebral infarction. Ten slices with a field of view of 2 × 2 cm at a thickness of 1 mm were positioned on the brain.Alternatively, harvest mouse brains 24 h after reperfusion for TTC staining. Obtain brain slices at 1 mm intervals in the coronal plane. Place fresh brain slices in 1% TTC solution to determine the infarct area.

[0177] statistics and fertility Statistical parameters including n values ​​are shown in the figure legends. Sample sizes were determined to ensure sufficient power as recommended by the Biostatistics and Bioinformatics Department, Moores Cancer Center. We used two-tailed, two-sample, unequal variance t-tests. For several in vitro data sets, statistical significance was further confirmed using Wilcoxon rank sum tests between treatment groups of interest, as the method does not require the usual assumptions about the variables. Statistical inference was based on the p-values ​​for each comparison using the R function "wilcox.test". We used linear mixed effects (LME) models to investigate the wound area (percentage) between the three treatment groups (ManN, VEGF, VEGF+ManN) and the PBS group. Two LME models were fitted. In the first LME model, the control group was considered as the reference group. We included the day effect (considering days as a categorical variable rather than a continuous variable) and its interaction with treatment as fixed effects, and we also included subject id as a random effect to capture correlations between measurements on different days for the same subject. At baseline, there are no differences between the different groups. In the second LME model, we re-leveled the VEGF+ManN group as the reference group to investigate the comparison between single treatment groups and combined treatment. For each LME model, we investigated the different treatment effects and their corresponding p-values ​​on days 3, 5, and 8, respectively, with respect to the reference group. Data were considered significant if p<0.05. Significant p-values ​​are depicted in the figures as follows: ***p<0.001, **p<0.01, *p<0.05. For each experiment, representative experimental results from 2-5 independent studies are shown.

[0178] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to one skilled in the art from a reading and understanding of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all of the techniques and devices described above can be used in various combinations. All publications, 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 publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

[0179] Aspects Embodiment 1: A pharmaceutical composition for treating stroke, comprising an effective amount of an N-glycosylation inhibitor administered to a subject in need thereof.

[0180] Embodiment 2: The pharmaceutical composition of embodiment 1, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof.

[0181] Embodiment 3: The pharmaceutical composition of embodiment 1, wherein the N-glycosylation inhibitor comprises ManN.

[0182] Embodiment 4: The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the subject has experienced an ischemic stroke.

[0183] Embodiment 5: The pharmaceutical composition of embodiment 4, wherein the ischemic stroke comprises thrombotic stroke or embolic stroke.

[0184] Embodiment 6: The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the subject has experienced a hemorrhagic stroke.

[0185] Embodiment 7: The pharmaceutical composition of embodiment 6, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

[0186] Embodiment 8: The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the subject has experienced a brain stem stroke.

[0187] Embodiment 9: The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the subject is experiencing a transient ischemic attack.

[0188] Embodiment 10: The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the subject is experiencing a cryptogenic stroke.

[0189] Embodiment 11: A pharmaceutical composition according to any one of embodiments 1 to 10, wherein the effective amount of the N-glycosylation inhibitor comprises a single dose treatment regimen.

[0190] Embodiment 12: The pharmaceutical composition according to embodiment 11, wherein the single-dose treatment regimen comprises pre-treatment, concomitant treatment, or post-treatment associated with an ischemic event affecting the brain.

[0191] Embodiment 13: A pharmaceutical composition according to any one of embodiments 1 to 10, wherein the effective amount of the N-glycosylation inhibitor comprises a multiple dose treatment regimen.

[0192] Embodiment 14: The pharmaceutical composition according to embodiment 13, wherein the multiple dose treatment regimen comprises pre-treatment, concomitant treatment, and / or post-treatment related to an ischemic event affecting the brain.

[0193] Embodiment 15: A pharmaceutical composition according to any one of embodiments 1 to 14, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

[0194] Embodiment 16: The pharmaceutical composition according to any one of embodiments 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises from about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0195] Embodiment 17: The pharmaceutical composition according to any one of embodiments 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises from about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0196] Embodiment 18: The pharmaceutical composition according to any one of embodiments 1 to 15, wherein the effective amount of the N-glycosylation inhibitor comprises from about 5 g to about 100 g of the N-glycosylation inhibitor.

[0197] Embodiment 19: The pharmaceutical composition according to any one of embodiments 1 to 18, wherein the pharmaceutical composition further comprises an effective amount of a pro-angiogenic factor.

[0198] Embodiment 20: The pharmaceutical composition according to embodiment 19, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

[0199] Embodiment 21: The pharmaceutical composition according to embodiment 20, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placenta growth factor (PLGF), or a combination thereof.

[0200] Embodiment 22: A pharmaceutical composition according to embodiment 20 or 21, wherein the VEGF is recombinant VEGF.

[0201] Embodiment 23: A pharmaceutical composition according to any one of embodiments 20 to 22, wherein VEGF is administered locally near the site of the subject's brain affected by the ischemic event.

[0202] Embodiment 24: A method of treating stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor.

[0203] Embodiment 25: The method according to embodiment 24, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof.

[0204] Embodiment 26: The method of embodiment 25, wherein the N-glycosylation inhibitor comprises ManN.

[0205] Embodiment 27: The method according to any one of embodiments 24 to 26, wherein the subject has experienced an ischemic stroke.

[0206] Embodiment 28: The method of embodiment 27, wherein the ischemic stroke is thrombotic stroke or embolic stroke.

[0207] Embodiment 29: The method according to any one of embodiments 24 to 26, wherein the subject has experienced a hemorrhagic stroke.

[0208] Aspect 30: The method of aspect 29, wherein the hemorrhagic stroke comprises subarachnoid hemorrhage or intracerebral hemorrhage.

[0209] Embodiment 31: The method of any one of embodiments 24 to 26, wherein the subject has experienced a brain stem stroke.

[0210] Embodiment 32: The method according to any one of embodiments 24 to 26, wherein the subject has experienced a transient ischemic attack.

[0211] Embodiment 33: The method of any one of embodiments 24 to 26, wherein the subject has experienced a cryptogenic stroke.

[0212] Embodiment 34: The method according to any one of embodiments 24 to 33, wherein the effective amount of the N-glycosylation inhibitor comprises a single dose treatment regimen.

[0213] Embodiment 35: The method of embodiment 34, wherein the single administration treatment regimen comprises pre-treatment, concomitant treatment, or post-treatment associated with an ischemic event affecting the brain.

[0214] Embodiment 36: The method according to any one of embodiments 24 to 35, wherein the effective amount of the N-glycosylation inhibitor comprises a multiple dose treatment regimen.

[0215] Embodiment 37: The method of embodiment 36, wherein the multiple dose treatment regimen comprises pre-treatment, concomitant treatment, and / or post-treatment associated with an ischemic event affecting the brain.

[0216] Embodiment 38: The method according to any one of embodiments 24 to 37, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

[0217] Embodiment 39: The method according to any one of embodiments 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises from about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0218] Embodiment 40: The method according to any one of embodiments 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises from about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0219] Embodiment 41: The method according to any one of embodiments 24 to 38, wherein the effective amount of the N-glycosylation inhibitor comprises from about 5 g to about 100 g of the N-glycosylation inhibitor.

[0220] Embodiment 42: The method according to any one of embodiments 34 to 41, wherein the single dose treatment regimen comprises administering a formulation of the N-glycosylation inhibitor at a dosage of from about 20 mg to about 900 mg / kg of the subject's body weight.

[0221] Embodiment 43: The method according to any one of embodiments 34 to 41, wherein the single dose treatment regimen comprises administering a formulation of the N-glycosylation inhibitor at a dosage of about 200 mg to about 700 mg / kg of the subject's body weight.

[0222] Embodiment 44: The method according to any one of embodiments 36 to 41, wherein the multiple dose treatment regimen comprises administering a formulation of the N-glycosylation inhibitor at a dosage of from about 10 mg to about 900 mg / kg of the subject's body weight.

[0223] Embodiment 45: The method according to any one of embodiments 36 to 41, wherein the multiple dose treatment regimen comprises administering a formulation of the N-glycosylation inhibitor at a dosage of from about 200 mg to about 700 mg / kg of the subject's body weight.

[0224] Embodiment 46: The method of any one of embodiments 24 to 45, wherein administering to the subject further comprises administering an effective amount of a pro-angiogenic factor.

[0225] Embodiment 47: The method of embodiment 46, wherein the pro-angiogenic factor comprises vascular endothelial growth factor (VEGF) or a derivative thereof.

[0226] Embodiment 48: The method of embodiment 47, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, placenta growth factor (PLGF), or a combination thereof.

[0227] Embodiment 49: A method according to embodiment 47 or 48, wherein the VEGF is recombinant VEGF.

[0228] Embodiment 50: A method according to any one of embodiments 47 to 49, wherein the VEGF is administered locally near the site of the subject's brain affected by the ischemic event.

[0229] Embodiment 51: A method according to any one of embodiments 24 to 50, wherein N-linked glycosylation of proteins is inhibited in endothelial cells of the subject.

[0230] Embodiment 52: The method according to any one of embodiments 24 to 51, wherein the administration is effective to stimulate endothelial cell proliferation and angiogenesis in blood vessels near the brain.

[0231] Aspect 53: The method of any one of aspects 24 to 52, wherein the administration is effective to stimulate endothelial cell proliferation and angiogenesis in the brain.

[0232] Embodiment 54: The method according to any one of embodiments 24 to 53, wherein the administration is effective to activate JNK signaling and enhance the unfolded protein response caused by endoplasmic reticulum stress.

[0233] Embodiment 55: A method for preventing stroke, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor.

[0234] Embodiment 56: The method of embodiment 55, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof.

[0235] Embodiment 57: The method according to embodiment 56, wherein the N-glycosylation inhibitor comprises ManN.

[0236] Embodiment 58: A method according to any one of embodiments 55 to 57, wherein the effective amount of the N-glycosylation inhibitor comprises a single dose treatment regimen.

[0237] Embodiment 59: A method according to any one of embodiments 55 to 57, wherein the effective amount of an N-glycosylation inhibitor comprises a multiple dose treatment regimen.

[0238] Embodiment 60: The method according to any one of embodiments 55 to 59, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

[0239] Embodiment 61: The method according to any one of embodiments 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises from about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0240] Embodiment 62: A method according to any one of embodiments 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises from about 200 mg to about 200 g of the N-glycosylation inhibitor.

[0241] Embodiment 63: A method according to any one of embodiments 55 to 60, wherein the effective amount of the N-glycosylation inhibitor comprises from about 5 g to about 100 g of the N-glycosylation inhibitor.

[0242] Embodiment 64: A method according to any one of embodiments 55 to 63, wherein the risk of recurrent stroke is reduced in the subject.

[0243] Embodiment 65: The method of embodiment 64, wherein the subject has previously experienced an ischemic stroke, a hemorrhagic stroke, a brain stem stroke, a transient ischemic attack, or a cryptogenic stroke.

[0244] Embodiment 66: The method of embodiment 64 or 65, wherein the risk of stroke recurrence comprises risk of ischemic stroke, hemorrhagic stroke, brain stem stroke, transient ischemic attack, or cryptogenic stroke.

[0245] Embodiment 67: A method according to any one of embodiments 55 to 66, wherein the measurement of C-reactive protein (CRP) in the blood in the subject is reduced compared to a previous measurement of the blood CRP level in the subject.

[0246] Embodiment 68: The method of embodiment 67, wherein the measured CRP level in the blood of the subject is reduced to less than about 10 mg / L.

[0247] Embodiment 69: A method of alleviating an ongoing ischemic event affecting the brain, comprising administering to a subject in need thereof an effective amount of an N-glycosylation inhibitor.

[0248] Embodiment 70: The method according to embodiment 69, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifunensine (Kif), castanospermine (Cas), or a combination thereof.

[0249] Embodiment 71: The method according to embodiment 70, wherein the N-glycosylation inhibitor comprises ManN.

[0250] Embodiment 72: The method of any one of embodiments 69 to 71, wherein the subject is experiencing an ischemic stroke, a hemorrhagic stroke, a brain stem stroke, a transient ischemic attack, or a cryptogenic stroke.

[0251] Embodiment 73 A method according to any one of embodiments 69 to 72, wherein the effective amount of the N-glycosylation inhibitor is administered orally, intravenously, intrathecally, or intraperitoneally.

[0252] Embodiment 74: A method according to any one of embodiments 69 to 73, wherein the effective amount of the N-glycosylation inhibitor comprises from about 10 mg to about 300 g of the N-glycosylation inhibitor.

[0253] Embodiment 75: The method according to any one of embodiments 69 to 73, wherein the effective amount of the N-glycosylation inhibitor comprises from about 5 g to about 100 g of the N-glycosylation inhibitor.

[0254] Embodiment 76: The method according to any one of embodiments 69 to 75, wherein the neurological assessment of acute stroke in the subject improves compared to a previous assessment.

[0255] Embodiment 77: A method according to any one of embodiments 69 to 76, wherein blood flow to the subject's brain is improved.

[0256] Aspect 78: The method of aspect 77, wherein blood flow to the brain is assessed by transcranial Doppler ultrasound.

[0257] Aspect 79: A method of treating stroke in a subject in need thereof, comprising:

[0258] a) identifying a subject who has experienced a stroke event;

[0259] b) administering to the subject a therapeutically effective amount of one or more N-glycosylation inhibitors;

[0260] c) determining whether the subject has suffered an ischemic stroke or a hemorrhagic stroke; and

[0261] d) administering a therapeutically effective amount of an anticoagulant if the subject is determined to have suffered an ischemic stroke, or withholding administration of a therapeutically effective amount of an anticoagulant if the subject is determined to have suffered a hemorrhagic stroke;

[0262] e) administering at least one N-glycosylation inhibitor over a treatment period, wherein the N-glycosylation inhibitors of steps b) and e) are the same or different.

[0263] Aspect 80: The method of aspect 79, wherein step c) includes performing a CT scan or an MRI scan of the subject.

[0264] Embodiment 81: A method according to embodiment 79 or 80, wherein in step e) the N-glycosylation inhibitor is administered 1 to 7 times per week.

[0265] Aspect 82: A method according to any one of aspects 79 to 81, wherein the treatment period of step e) ranges from about 1 day to about 12 weeks.

[0266] Embodiment 83: The method according to any one of embodiments 79 to 82, wherein infarct size is reduced in the subject compared to anticoagulant therapy alone, TPA therapy alone, or a combination of anticoagulant therapy alone and TPA therapy alone.

[0267] Embodiment 84: The method of any one of embodiments 79 to 83, wherein blood-brain barrier disruption is reduced in the subject compared to anticoagulant therapy alone, TPA therapy alone, or a combination of anticoagulant therapy alone and TPA therapy alone.

[0268] Embodiment 85: The method of any one of embodiments 79 to 84, wherein hemorrhagic damage due to ischemic stroke is reduced in the subject compared to anticoagulant therapy alone, TPA therapy alone, or a combination of anticoagulant therapy alone and TPA therapy alone.

[0269] Embodiment 86: The method according to any one of embodiments 79 to 85, wherein mortality rate is reduced in the subject compared to anticoagulant therapy alone, TPA therapy alone, or a combination of anticoagulant therapy alone and TPA therapy alone.

[0270] Embodiment 87: A method of treating degenerative (pathological) myopia in a subject in need thereof, comprising intravitreally administering to the subject an effective amount of one or more N-glycosylation inhibitors, wherein following administration, one or more symptoms of degenerative (pathological) myopia are ameliorated.

[0271] Embodiment 88: The method according to embodiment 88, wherein the one or more N-glycosylation inhibitors comprise ManN, Kif, Cas, or any combination thereof.

[0272] Embodiment 89: The method according to embodiment 87 or 88, wherein the one or more N-glycosylation inhibitors comprise ManN.

[0273] Embodiment 90: A method according to any one of embodiments 87 to 89, wherein retinal neovascularization is induced in the eye of the subject by administration of an effective amount of one or more N-glycosylation inhibitors.

[0274] Embodiment 91: A method according to any one of embodiments 87 to 90, wherein choroidal neovascularization is induced in the eye of the subject by administration of an effective amount of one or more N-glycosylation inhibitors.

[0275] Aspect 92: A method according to aspect 90 or 91, wherein the induction of retinal neovascularization or the induction of choroidal neovascularization restores vascular perfusion in the subject's eye.

[0276] Embodiment 93: A method according to any one of embodiments 87 to 92, wherein myopic choroidal neovascularization (CNV) is reduced in the eye of the subject.

[0277] Embodiment 94: The method according to any one of embodiments 87 to 93, wherein the presence or progression of chorioretinal atrophy (CRA) adjacent to an area of ​​myopic CNV in the subject's eye is reduced following administration.

[0278] Embodiment 95: The method according to any one of embodiments 87 to 94, wherein the progression of further visual impairment in the subject is reduced or halted following administration.

[0279] Aspect 96: The method of any one of aspects 87 to 95, wherein the subject exhibits delayed disease progression of one or more symptoms selected from the group consisting of diffuse chorioretinal atrophy, patchy chorioretinal atrophy, lacquer cracks, myopic CNV, CNV-associated macular atrophy, progressive vision loss, foveal separation, metamorphopsia, blurred vision, scotoma, difficulty reading, floaters or photopsia, reduced central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and reduced contrast sensitivity.

[0280] Aspect 97: The method of any one of aspects 87 to 96, wherein the subject exhibits a halt 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-associated macular atrophy, progressive vision loss, foveal separation, metamorphopsia, blurred vision, scotoma, difficulty reading, floaters or photopsia, reduced central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and reduced contrast sensitivity.

[0281] Aspect 98: The method of 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-associated macular atrophy, progressive vision loss, foveal separation, metamorphopsia, blurred vision, scotoma, difficulty reading, floaters or photopsia, reduced central vision, retinal thinning, difficulty recognizing faces, gray spots in the visual field, and reduced contrast sensitivity.

[0282] Embodiment 99: The method according to any one of embodiments 87 to 98, wherein disease progression and efficacy of treatment are monitored using Spectral Domain Optical Coherence Tomography.

[0283] Embodiment 100: The method of embodiment 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 an N-glycosylation inhibitor as part of a treatment regimen, the therapeutic dose of an N-glycosylation inhibitor to be administered to a subject, or cessation of administration of an N-glycosylation inhibitor to a subject after one or more symptoms of degenerative (pathological) myopia have improved.

Claims

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

2. The pharmaceutical composition according to claim 1, wherein the N-glycosylation inhibitor comprises hexosamine D-mannosamine (ManN), kifenensin (Kif), or castanospermine (Cas), or a combination thereof.

3. The pharmaceutical composition according to claim 2, further comprising administering an effective amount of angiogenic factor.

4. The pharmaceutical composition according to claim 1, wherein the N-glycosylation inhibitor comprises ManN.

5. The pharmaceutical composition according to claim 4, wherein the effective amount of ManN is approximately 10 mg to approximately 300 g per administration.

6. The pharmaceutical composition according to claim 4, wherein the effective amount of ManN is approximately 200 mg to approximately 100 g per administration.

7. The pharmaceutical composition according to claim 4, wherein the effective amount of ManN is approximately 5 g to approximately 40 g per administration.

8. The pharmaceutical composition according to claim 4, wherein N-linked glycosylation of a protein is inhibited in the target endothelial cells.

9. The pharmaceutical composition according to claim 4, wherein the administration is effective in stimulating endothelial cell proliferation and angiogenesis in the brain.

10. The pharmaceutical composition according to claim 4, wherein the subject shows improvement in one or more symptoms of stroke after administration.

11. The pharmaceutical composition according to claim 4, wherein the subject shows a significant increase in vascular density in or near the area of ​​brain injury due to stroke after administration.

12. The pharmaceutical composition according to claim 1, wherein the subject has experienced an ischemic stroke.

13. The pharmaceutical composition according to claim 1, wherein the subject has experienced a hemorrhagic stroke.

14. The pharmaceutical composition according to claim 13, wherein the hemorrhagic stroke includes subarachnoid hemorrhage or intracerebral hemorrhage.

15. The pharmaceutical composition according to claim 1, wherein the subject has experienced a brainstem stroke.

16. The pharmaceutical composition according to claim 1, wherein the subject has experienced a transient ischemic attack.

17. The pharmaceutical composition according to claim 1, wherein the subject has experienced a cryptogenic stroke.

18. The pharmaceutical composition according to claim 1, further comprising administering the effective amount of the N-glycosylation inhibitor orally, intravenously, intrathecally, or intraperitoneally.

19. The pharmaceutical composition according to any one of claims 1 to 18, further comprising delivering the effective amount of the N-glycosylation inhibitor to the subject by a single-dose treatment regimen or a multi-dose treatment regimen.

20. The pharmaceutical composition according to claim 19, wherein the single-dose treatment regimen or the multi-dose treatment regimen comprises administering the preparation of the N-glycosylation inhibitor at a dosage of approximately 20 mg to approximately 2000 mg / kg of body weight of the target.

21. The pharmaceutical composition according to claim 19, wherein the single-dose treatment regimen includes administering the preparation of the N-glycosylation inhibitor at a dosage of approximately 100 mg to approximately 300 mg / kg of the target body weight.

22. The pharmaceutical composition according to claim 19, wherein the single-dose treatment regimen or the multi-dose treatment regimen includes pre-treatment, concurrent treatment, or post-treatment related to an ischemic event affecting the brain of the subject.

23. The aforementioned pre-treatment, simultaneous treatment, or post-treatment may last 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, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, or 40 hours. The pharmaceutical composition according to claim 22, comprising administering at a frequency of once every 42 hours, 44 hours, 46 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 18 days, 20 days, 21 days, 24 days, 28 days, or 30 days.

24. The pharmaceutical composition according to claim 22, wherein the single-dose treatment regimen or the multi-dose treatment regimen comprises the prior treatment, and the prior treatment is initiated at least one hour before the stroke.

25. The pharmaceutical composition according to claim 22, wherein the single-dose treatment regimen or the multi-dose treatment regimen comprises the concurrent treatment, and the concurrent treatment is initiated within approximately 48 hours of the stroke.

26. The pharmaceutical composition according to claim 22, wherein the single-dose treatment regimen or the multi-dose treatment regimen comprises the post-treatment, and the post-treatment is initiated within approximately 120 hours after the stroke.

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