Dendrimer compositions for targeted delivery of therapeutic agents to neurons - Patents.com

Abstract

Glucose dendrimers synthesized using hypercore and glucose monosaccharide-based branching units have significantly enhanced accumulation in brain and retina neurons when administered in vivo, compared to dendrimers that do not contain glucose monosaccharide-based branching units, such as PAMAM. Compositions of glucose dendrimers conjugated with one or more therapeutic, prophylactic or diagnostic agents for preventing, treating or diagnosing a disease or disorder in a subject in need thereof, and methods of using the same have been developed. The compositions are particularly suitable for treating and / or ameliorating diseases or disorders associated with diseased neurons in the eye or brain. Methods of treating a human subject having or at risk of having a neurological disease or disorder are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 327,610, filed April 5, 2022, the contents of which are incorporated herein in their entirety.

[0002] FIELD OF THEINVENTION The present invention is generally in the field of drug delivery, and in particular, dendrimer compositions and methods for selectively delivering drugs to neurons. [Background technology]

[0003] 2. Background of the Invention Preserving neurons in the context of brain injury is a major challenge and a major goal in the treatment of many neurological disorders. However, targeting specifically damaged neurons, whether by local administration to the brain or from systemic administration, has been a major challenge, especially considering their relatively low plasma membrane invagination rate compared to immune cells (Krol, S., Journal of Controlled Release. 164(2), 145-55(2012)).

[0004] For example, brain injury can result in neuronal hyperexcitability, which can contribute to several neurological conditions such as epilepsy, chronic pain, and Parkinson's disease (Anastacio, et al, Translational Psychiatry 12:1-14 (2022); G. Carola et al., Parkinson's Disease 7:1-14 (2021)). Therapeutic targeting of selected hyperactive neurons can not only rescue neurons from excitotoxic death but also limit disease propagation. However, selective targeting of neurons remains challenging, mainly due to the blood-brain barrier (BBB) ​​and the spectrum of neuronal properties and functions exhibited in different brain regions affected by diverse neurological conditions. Systemically administered drugs need to cross the blood-brain barrier, diffuse freely through brain tissue, and be selectively taken up by target cells. This is a major challenge and many ligand, antibody and viral vector based approaches have been explored with mixed results (S. Krol, J Control Release 164: 145-155 (2012);ES Smith, et al., Advanced drug delivery reviews 148: 181-203 (2019);J. Garcia-Chica et al., NanoMedicine (Lond) 15: 1617-1636 (2020);AP Spencer et al., Pharmaceutics:12 (2020)). Even if a therapeutic drug achieves crossing the BBB, selectively targeting neurons remains a significant challenge and must be carefully considered as it may result in unwanted side effects.Neurons function in complex and diverse ways in different brain regions, and because receptor overexpression varies regionally, targeting neuronal subtypes in different brain regions using ligand-based strategies may be impractical for widespread application (J. Garcia-Chica et al., NanoMedicine (Lond) 15: 1617-1636 (2020);F. Zhang, Y. et al., J. Control Release 240: 212-226 (2016)). Among the myriad differences, one common characteristic in most brain injuries is the high metabolic activity and high demand for glucose in neurons, at least during the acute phase of the injury or neurological disease. Glucose is the main metabolic source for the brain (B. Siesjoe, Journal of neural transmission, 17-22 (1978)), and is transported across the BBB and made available to neurons and glia via specific glucose transporters (L. Pellerin, Proc. Nat.Acad. Sci. 91:10625-10629 (1994);LK Bak et al., J. Neurochem.109: 87-93 (2009);Diaz-Garcia et al., Cell metabolism 26: 361-374. e364 (2017);Tredern et al., Cell Reports 36: 109620 (2021)). In the presence of excitotoxic insults, such as those found in epilepsy, glucose transport to affected neurons is increased due to increased metabolic demand.Glut3 and SGLT transporters expressed in neurons can transport glucose from the interstitium and generate ATP via glycolysis, the pentose phosphate pathway and oxidative metabolism, depending on the degree of neuronal stimulation (CM Diaz-Garcia et al., Cell metabolism 26: 361-374. e364 (2017);I. Lundgaard et al., Nat Commun 6, 6807 (2015);Pellerin et al., Glia, 1251-1262 (2007);Herrero-Mendez et al., Nature cell biology 11:747-752 (2009);J. Jurcovicova, Endocr Regul 48:35-48 (2014)). Currently, it is a challenge to deliver therapeutic agents, including small molecule drugs and high molecular weight biologics, to the nucleus of neurons.To treat neurological disorders, such as Alzheimer's, Parkinson's, cerebral palsy, autism, multiple sclerosis, spinal muscular atrophy, traumatic brain injury, glaucoma, and other retinal disorders, new systems are needed for targeted delivery to damaged neurons and selective delivery of drugs to the site of lesion. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Krol, S., Journal of Controlled Release. 164(2), 145-55(2012) [Non-Patent Document 2] Anastacio, et al, Translational Psychiatry 12:1-14 (2022) [Non-Patent Document 3] G. Carola et al., Parkinson's Disease 7:1-14 (2021) [Non-Patent Document 4] S. Krol, J Control Release 164: 145-155 (2012)

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[0006] It is therefore an object of the present invention to provide compositions that selectively deliver therapeutic, prophylactic, or diagnostic agents to target cells within the site of a lesion in the CNS, including the eye, brain, or neurons, as well as methods of making and using the same.

[0007] It is also an object of the present invention to provide compositions for treating or preventing one or more symptoms of neurological and retinal disorders by directly targeting diseased cells. [Means for solving the problem]

[0008] Summary of the Invention Dendrimer compositions have been developed that are termed "glucose dendrimers that can selectively accumulate inside neurons, particularly in the nuclei of injured and / or hyperactive neurons." These dendrimers can accumulate at high levels inside activated microglia. In contrast to hydroxyl dendrimers, which accumulate primarily in microglia, these dendrimers primarily travel to neurons.

[0009] A glucose dendrimer comprises (a) a central core; (b) one or more branching units, which are monosaccharide glucose-based branching units, optionally having a linker conjugated thereto; and optionally (c) one or more therapeutic, prophylactic and / or diagnostic agents. Typically, the one or more branching units are conjugated to the central core, and the surface groups of the dendrimer are monosaccharide glucose molecules. In some embodiments, the central core is dipentaerythritol or a hexapropargylated derivative thereof. In some embodiments, the branching units are conjugated to the central core via a linker, e.g., a hydrocarbon or oligoethylene glycol chain. In a preferred embodiment, the branching units are β-D-glucopyranoside tetraethylene glycol azide having the following structure: [ka] or a peracetylated derivative thereof.

[0010] In some embodiments, the glucose dendrimer is a first, second, third, fourth, fifth, or sixth generation dendrimer. In one embodiment, the dendrimer has the following structure: [ka] It is a first generation dendrimer having the following structure.

[0011] In a preferred embodiment, the dendrimer has the following structure: [ka] It is a second generation dendrimer having the following structure.

[0012] In some embodiments, one or more therapeutic, prophylactic, and / or diagnostic agents are encapsulated, associated, and / or conjugated to the dendrimer at a concentration of between about 0.01% and about 30% by weight, preferably between about 1% and about 20% by weight, more preferably between about 5% and about 20% by weight. In some embodiments, the dendrimer is conjugated to a small molecule, an antibody or an antigen-binding fragment thereof, a nucleic acid, or a polypeptide. In some embodiments, the therapeutic agent conjugated to the dendrimer is an anti-inflammatory agent, an antioxidant, or an immunomodulatory agent. In other embodiments, the dendrimer is conjugated to one or more diagnostic agents, such as fluorescent dyes, near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes.

[0013] In some embodiments, the dendrimer and the therapeutic, prophylactic, or diagnostic agent are conjugated via one or more linkers or coupling agents, such as one or more hydrocarbon or oligoethylene glycol chains. Exemplary bonds are disulfide bonds, ester bonds, ether bonds, thioester bonds, and amide bonds.

[0014] Pharmaceutical preparations that include glucose dendrimers typically include a dendrimer composition and one or more pharma- ceutically acceptable carriers or excipients. In some embodiments, the preparation is formulated for systemic administration. In some embodiments, the preparation is formulated for enteral or parenteral administration, for example, intramuscular, intraperitoneal, intravenous, or subcutaneous injection administration.

[0015] Also provided is a method for treating or preventing one or more diseases, conditions, and / or damage of the eye, brain, and / or nervous system (CNS) by administering a pharmaceutical formulation of glucose dendrimer to a subject in need thereof. Typically, the one or more diseases, conditions, and / or damage of the eye, brain, and / or nervous system are diseases, conditions, and damage associated with neurons and / or activated microglia. In some embodiments, the one or more diseases, conditions, and / or damage of the eye are eye diseases associated with retinal ganglion cells, such as glaucoma, diabetic retinopathy, acute retinal ischemia, traumatic optic nerve injury, optic atrophy, and Leber's hereditary optic neuropathy, and the one or more therapeutic agents encapsulated, associated, and / or conjugated to the dendrimer are ROCK inhibitors, alpha-2 adrenergic receptor agonists, or caspase inhibitors. In some embodiments, the one or more diseases, conditions, and / or injuries of the brain and / or nervous system are neurological and / or neurodegenerative diseases, such as traumatic brain injury, demyelinating diseases, epilepsy, neuralgia, Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebral palsy, autism, multiple sclerosis, spinal muscular atrophy, neuronal ceroid lipofuscinosis, and neuronopathic Goucher disease. In these cases, exemplary therapeutic agents encapsulated, associated, and / or conjugated to the dendrimer include calpain inhibitors, GPR52 antagonists, NMDA antagonists, mTOR inhibitors, LLRK2 inhibitors, nuclear factor erythroid 2-related factor 2 activators, and SMN-2 promoters. In other embodiments, the one or more diseases, conditions, and / or injuries of the brain and / or nervous system are neurological diseases associated with motor neurons, such as amyotrophic lateral sclerosis, primary lateral sclerosis, progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease. In one embodiment, the neurological disease is spinal muscular atrophy, and optionally the therapeutic agent encapsulated, associated, and / or conjugated to the dendrimer is an HDAC inhibitor or an antisense oligonucleotide, such as nusinersen.The dendrimer formulations may be administered orally, intravenously, intraperitoneally, or intravitreally. In a preferred embodiment, the amount of a therapeutic, prophylactic or diagnostic agent effective to treat or prevent one or more symptoms is less than the amount of the same therapeutic, prophylactic or diagnostic agent administered in the absence of a glucose dendrimer or administered as a formulation in combination with a dendrimer in the absence of surface glucose molecules.

[0016] Also provided is a method for labeling one or more neurons and / or activated microglia associated with one or more diseases, conditions, and / or injuries of the eye, brain, and / or nervous system (CNS). The method includes administering to a subject an effective amount of a pharmaceutical formulation of a glucose dendrimer to label one or more cells associated with one or more diseases, conditions, and / or injuries of the eye, brain, and / or nervous system (CNS). In some embodiments, the label is used to diagnose or identify one or more diseases, conditions, and / or injuries of the eye, brain, and / or nervous system (CNS) in a subject. In other embodiments, the label is used to monitor or guide a treatment and / or surgery. The dendrimer formulation is administered orally, intravenously, subchondrially, or intravitreally.

[0017] Also provided is a method for delivering one or more therapeutic, preventive or diagnostic agents to one or more neurons in a subject in need of delivery. The method comprises administering to the subject an effective amount of a pharmaceutical formulation of glucose dendrimer. In some embodiments, the dendrimer delivers to one or more neurons, including cerebral cortical neurons, motor neurons, dopaminergic neurons, hypothalamic neurons, thalamic neurons, brainstem neurons, raphe neurons, Purkinje neurons, retinal ganglion cells, and other neurons in the central nervous system. In a preferred embodiment, the amount of dendrimer administered causes one or more therapeutic, preventive or diagnostic agents to accumulate in one or more neurons at least 5 times, 10 times, 20 times, 30 times, 40 times, or 50 times more than the amount of the same therapeutic, preventive or diagnostic agent administered in the absence of dendrimer or administered as a formulation combined with dendrimer in the absence of surface glucose molecules. [Brief description of the drawings]

[0018] [Figure 1A] 1A-1B are schematic diagrams showing molecular structures in a step-by-step synthetic route for producing an exemplary glucose dendrimer (FIG. 1A) and for conjugating an exemplary drug, Cy5, to the dendrimer (FIG. 1B). [Figure 1B] 1A-1B are schematic diagrams showing molecular structures in a step-by-step synthetic route for producing an exemplary glucose dendrimer (FIG. 1A) and for conjugating an exemplary drug, Cy5, to the dendrimer (FIG. 1B). [Figure 2A-B] 2A-2E are schematic diagrams showing an exemplary synthetic route for glucose dendrimer-drug conjugates (FIG. 2A) and step-by-step synthetic routes for conjugating exemplary therapeutic, prophylactic or diagnostic agents loperamide (FIG. 2B), rapamycin (FIG. 2C), and valproic acid (FIGS. 2D and 2E) to dendrimers using copper-catalyzed alkyne-azide click (CuAAC) chemistry. [Figure 2C-D] 2A-2E are schematic diagrams showing an exemplary synthetic route for glucose dendrimer-drug conjugates (FIG. 2A) and step-by-step synthetic routes for conjugating exemplary therapeutic, prophylactic or diagnostic agents loperamide (FIG. 2B), rapamycin (FIG. 2C), and valproic acid (FIGS. 2D and 2E) to dendrimers using copper-catalyzed alkyne-azide click (CuAAC) chemistry. [Figure 2E] 2A-2E are schematic diagrams showing an exemplary synthetic route for glucose dendrimer-drug conjugates (FIG. 2A) and step-by-step synthetic routes for conjugating exemplary therapeutic, prophylactic or diagnostic agents loperamide (FIG. 2B), rapamycin (FIG. 2C), and valproic acid (FIGS. 2D and 2E) to dendrimers using copper-catalyzed alkyne-azide click (CuAAC) chemistry. [Figure 3A] 3A-3B are schematic diagrams showing an exemplary synthetic route for glucose dendrimer-drug conjugates using a combination of click and esterification / amidation reactions (FIG. 3A), and a stepwise synthetic route for conjugating an exemplary active agent, N-acetylcysteine, to a dendrimer (FIG. 3B). [Figure 3B] 3A-3B are schematic diagrams showing an exemplary synthetic route for glucose dendrimer-drug conjugates using a combination of click and esterification / amidation reactions (FIG. 3A), and a stepwise synthetic route for conjugating an exemplary active agent, N-acetylcysteine, to a dendrimer (FIG. 3B). [Figure 4] FIG. 4 is a scheme showing an exemplary synthetic route for glucose dendrimer-drug conjugates using copper-free biorthogonal click chemistry. [Diagram 5] Figure 5 is a bar graph showing the mean dendrimer fluorescence intensity (Cy5 channel) from contralateral CA1 neurons in mice intracranially injected with PAMAM-OH-Cy5 or GD2-Cy5 (n=32 neurons from 2 mice). *p<0.001. [Figure 6A-B] Figure 6A is a scheme showing that Thy1-YFP mouse brains were removed, transferred to culture medium, and incubated with the indicated agents before formalin fixation and imaging analysis. Figure 6B is a bar graph showing the mean dendrimer fluorescence intensity in Mg2+-free artificial cerebrospinal fluid (ACSF) in control conditions (n=187 neurons) or in samples pretreated with cytochalasin B (n=104 neurons) or glutor (n=128 neurons) or phlorizin (n=80 neurons), and in medium with high Mg2+ and N-methyl-D-glucamine (NMDG) (n=69 neurons). *p<0.001. [Figure 7] FIG. 7 is a scheme showing an exemplary synthetic route for making glucose dendrimers (GD) and fluorescently labeled glucose dendrimers (GD-Cy5). [Figure 8A-B] Figures 8A and 8B demonstrate that GD2 targets CA1 neurons in a mouse model of temporal lobe epilepsy. Figure 8A is a schematic illustrating the experimental timeline. Cy5-conjugated PAMAM-OH or GD2 was administered intracranially to the right hemisphere. After overnight recovery, pilocarpine was injected to induce seizures, and 30 minutes after active behavioral seizures (Racine scale 3 or higher) were observed, mice were sacrificed, perfused, and brains were harvested for immunohistochemistry. Figure 8B is a bar graph showing the mean dendrimer fluorescence intensity (Cy5 channel) from contralateral CA1 neurons, showing that uptake of GD2 is approximately 100-fold higher (n=56 neurons from 2 mice) than PAMAM-OH (n=32 neurons from 2 mice). *p<0.001. [Figure 9A]Figures 9A and 9B demonstrate that neuronal activity and GLUT transporters mediate GD2 uptake. Figure 9A shows a scheme of an experiment in which 300 μm cortical brain slices were pretreated for 30 min with control ACSF (without Mg2+, which increases neuronal firing) or ACSF containing Mg2+ / NMDG (which inhibits neuronal activity) or control ACSF containing either cytochalasin B (5 μM) or glutol (10 μM). After pretreatment, brain slices were incubated with GD2-Cy5 (10 μg / ml) for 30 min, followed by fixation in 10% formalin and confocal imaging. The mean fluorescence intensity for GD-Cy5 was assessed from YFP-expressing cortical neurons. FIG. 9B is a bar graph showing that GD2-Cy5 uptake was significantly reduced when neuronal activity was inhibited (n=69 neurons) and when pretreated with cytochalasin B (n=104 neurons) or glutol (n=128 neurons) compared to control incubation conditions (n=187 neurons). *p<0.001 [Figure 9B] Figures 9A and 9B demonstrate that neuronal activity and GLUT transporters mediate GD2 uptake. Figure 9A shows a scheme of an experiment in which 300 μm cortical brain slices were pretreated for 30 min with control ACSF (without Mg2+, which increases neuronal firing) or ACSF containing Mg2+ / NMDG (which inhibits neuronal activity) or control ACSF containing either cytochalasin B (5 μM) or glutol (10 μM). After pretreatment, brain slices were incubated with GD2-Cy5 (10 μg / ml) for 30 min, followed by fixation in 10% formalin and confocal imaging. The mean fluorescence intensity for GD-Cy5 was assessed from YFP-expressing cortical neurons. FIG. 9B is a bar graph showing that GD2-Cy5 uptake was significantly reduced when neuronal activity was inhibited (n=69 neurons) and when pretreated with cytochalasin B (n=104 neurons) or glutol (n=128 neurons) compared to control incubation conditions (n=187 neurons). *p<0.001 [Figure 10A]Figures 10A and 10B show the synthetic route to clickable VPA-azide (Figure 10A); glucose-dendrimer-VPA conjugate (GD2-VPA) (Figure 10B). At pH 7.4, the GD2-VPA conjugate is stable, with no release of VPA observed for up to 24 h. Under intracellular conditions, the GD2-VPA conjugate shows rapid release of VPA, with approximately 15% released in 1-2 h and 25% released in 24 h. [Figure 10B] Figures 10A and 10B show the synthetic route to clickable VPA-azide (Figure 10A); glucose-dendrimer-VPA conjugate (GD2-VPA) (Figure 10B). At pH 7.4, the GD2-VPA conjugate is stable, with no release of VPA observed for up to 24 h. Under intracellular conditions, the GD2-VPA conjugate shows rapid release of VPA, with approximately 15% released in 1-2 h and 25% released in 24 h. [Figure 11A-B] Figures 11A and 11B show that intranasal GD2-VPA reduces the severity of seizures induced by pilocarpine. Figure 11A is a schematic timeline of the experiment. Figure 11B is a bar graph showing that the locomotion and activity of mice treated with GD2-VPA is superior to saline-treated animals 1 hour after administration of pilocarpine. [Figure 12A-C] 12A-12I show uptake of GD2-Cy5 dendrimers by selected neurons (Syngap mouse seizure model). FIG. 12A is a timeline of the experiment. FIG. 12B and 12C are bar graphs of seizure duration scores and latency to high grade seizures on day 1. FIG. 12D-12F are bar graphs showing seizure duration scores for low grade seizures (FIG. 12D), medium grade seizures (FIG. 12E), and high grade seizures (FIG. 12F). FIG. 12G-12I are bar graphs showing seizure duration and latency to high grade seizures on day 2 for low grade seizures (FIG. 12G), medium grade seizures (FIG. 12H), and high grade seizures (FIG. 12I). [Fig. 12D-F]12A-12I show uptake of GD2-Cy5 dendrimers by selected neurons (Syngap mouse seizure model). FIG. 12A is a timeline of the experiment. FIG. 12B and 12C are bar graphs of seizure duration scores and latency to high grade seizures on day 1. FIG. 12D-12F are bar graphs showing seizure duration scores for low grade seizures (FIG. 12D), medium grade seizures (FIG. 12E), and high grade seizures (FIG. 12F). FIG. 12G-12I are bar graphs showing seizure duration and latency to high grade seizures on day 2 for low grade seizures (FIG. 12G), medium grade seizures (FIG. 12H), and high grade seizures (FIG. 12I). [Fig. 12G-I] 12A-12I show uptake of GD2-Cy5 dendrimers by selected neurons (Syngap mouse seizure model). FIG. 12A is a timeline of the experiment. FIG. 12B and 12C are bar graphs of seizure duration scores and latency to high grade seizures on day 1. FIG. 12D-12F are bar graphs showing seizure duration scores for low grade seizures (FIG. 12D), medium grade seizures (FIG. 12E), and high grade seizures (FIG. 12F). FIG. 12G-12I are bar graphs showing seizure duration and latency to high grade seizures on day 2 for low grade seizures (FIG. 12G), medium grade seizures (FIG. 12H), and high grade seizures (FIG. 12I). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention I. Definition The terms "active agent" or "biologically active agent" are used interchangeably to refer to chemical or biological compounds that induce a desired pharmacological and / or physiological effect, which may be prophylactic, therapeutic, or diagnostic. These may be nucleic acids, nucleic acid analogs, small molecules with a molecular weight of less than 2 kD, more typically less than 1 kD, peptidomimetics, proteins or peptides, carbohydrates or sugars, lipids, or combinations thereof. The term also encompasses pharma- ceutically acceptable and pharmacologically active derivatives of the agent, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs. The term "therapeutic agent" refers to an agent that may be administered to treat one or more symptoms of a disease or disorder. The term "diagnostic agent" generally refers to an agent that may be administered to localize, identify, and define the localization of a pathological process. Diagnostic agents may label target cells, allowing for subsequent detection or imaging of these labeled target cells. In some embodiments, diagnostic agents may selectively target neurons, particularly neurons within the site of a lesion in the eye, brain, or CNS, via the dendrimer. The term "prophylactic agent" generally refers to an agent that can be administered to prevent a disease or to prevent a particular condition.

[0020] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and / or onto a dendrimer, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on factors such as the disease or condition being treated, the particular targeted construct being administered, the size of the subject, or the severity of the disease or condition. Those of ordinary skill in the art may empirically determine the effective amount of a particular compound without undue experimentation. In some embodiments, the term "effective amount" refers to an amount of a therapeutic or prophylactic agent to reduce or reduce the symptoms of one or more ocular or neurological diseases.

[0021] The term "inhibit" or "reduce" in the context of inhibition means to decrease or lower the activity and amount. This can be a complete inhibition or reduction or a partial inhibition or reduction in activity or amount. The inhibition or reduction can be compared to a control or standard level. The inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a dendrimer composition comprising one or more inhibitors may inhibit or reduce the activity and / or amount of diseased neurons by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% over the activity and / or amount of the same cells in a comparable tissue of a subject not given or treated with the dendrimer composition. In some embodiments, the inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ levels. For example, inhibition and reduction in the rate of neuronal loss, rate of brain weight loss, or rate of hippocampal volume loss compared to untreated control subjects.

[0022] The term "treating" or "preventing" refers to alleviating, reducing or otherwise halting a disease, disorder or condition from occurring or progressing in an animal that may be susceptible to the disease, disorder and / or condition but has not yet been diagnosed as having it; inhibiting a disease, disorder or condition, e.g., preventing its progression; and alleviating a disease, disorder or condition, e.g., causing regression of a disease, disorder and / or condition. Treating a disease or condition includes improving at least one symptom of a particular disease or condition even if the underlying pathophysiology is not affected, e.g., treating pain in a subject by administering an analgesic agent includes improving at least one symptom of a particular disease or condition, even though such an agent does not treat the cause of the pain. Desirable effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and ameliorating or improving prognosis. For example, an individual has been successfully "treated" if one or more symptoms associated with Alzheimer's disease are alleviated or eliminated, including, but not limited to, a slowing of the rate of neuronal loss, a reduction in symptoms attributable to the disease, an increase in the quality of life of those afflicted with the disease, a reduction in the doses of other pharmaceutical agents needed to treat the disease, a slowing of disease progression, and / or an increase in the survival time of the individual.

[0023] The phrase "pharmacologically acceptable" or "biocompatible" refers to compositions, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. The phrase "pharmacologically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material involved in carrying or transporting any subject composition from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other components of the subject composition and not harmful to the patient.

[0024] The term "biodegradable" generally refers to a material that is broken down or removed under physiological conditions into smaller units or species that can be metabolized, excreted, or excreted in vivo. Degradation time is a function of composition and morphology.

[0025] The term "dendrimer" includes, but is not limited to, molecular configurations with an inner core, inner layers, or "generations" of repeat units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation.

[0026] The term "functionalization" means modifying a compound or molecule in a manner that results in the attachment of a functional group or moiety. For example, a molecule can be functionalized by introducing a molecule that makes it a strong nucleophile or a strong electrophile.

[0027] The term "targeting moiety" refers to a moiety that localizes at or away from a specific location. The moiety may be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The entity may be, for example, a therapeutic compound, for example, a small molecule, or a diagnostic entity, for example, a detectable label. The location may be a tissue, a specific cell type, or a subcellular compartment. In one embodiment, the targeting moiety localizes the agent. In a preferred embodiment, the dendrimer composition can selectively target neurons, particularly damaged / hyperactive neurons, in the absence of additional targeting moieties.

[0028] The term "prolonged residence time" refers to an increase in the time required for an agent to clear from a patient's body or from an organ or tissue of the patient. In certain embodiments, "prolonged residence time" refers to an agent clearing with a half-life that is 10%, 20%, 50% or 75% longer than a standard of comparison, such as a comparable agent that is not conjugated to a delivery vehicle, such as a dendrimer. In certain embodiments, "prolonged residence time" refers to an agent clearing with a half-life that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 or 10000 times longer than a standard of comparison, such as a comparable agent without a dendrimer that specifically targets a particular cell type.

[0029] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or containing an agent in and / or on a composition that allows for release, e.g., sustained release, of such agent in a desired application. Drugs or other materials can be incorporated into dendrimers by binding to one or more surface functional groups of such dendrimers (by covalent, ionic, or other binding interactions), by physical mixing, by enveloping the agent within the dendritic structure, and / or by encapsulating the agent within the dendritic structure.

[0030] II. Composition Dendrimers have been developed that are suitable for delivering one or more agents to neurons, preferably within the site of a lesion in the eye, brain, or CNS. These dendrimers are particularly suitable for delivering one or more agents for preventing, treating, or diagnosing one or more ocular diseases, one or more neurological and neurodegenerative diseases, especially dementia, and other disorders associated with neuroinflammation.

[0031] A. Dendrimer Dendrimers are three-dimensional hyperbranched, monodisperse, spherical, multivalent macromolecules containing surface end groups (Tomalia, DA, et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). Due to their unique structural and physical features, dendrimers have shown unprecedented potential as nanocarriers for various biomedical applications including targeted drug / gene delivery, imaging and diagnostics (Sharma, A., et al., RSC Advances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of Materials Chemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today, 6, 427 (2001); and Kannan, RM, et al., Journal of Internal Medicine, 276, 579 (2014)).

[0032] Dendrimers are useful for a variety of biomedical applications including drug / gene delivery, targeting, imaging and diagnostics (Soliman, GM et al., Chem. Commun. 2011, 47, 9572; and Tomalia, DA et al., Biochem. Soc. Trans. 2007, 35, 61). Among several different types of dendrimers, polyamidoamine (PAMAM) dendrimers have been extensively investigated for drug delivery applications due to their commercial availability, water solubility and biocompatibility (Tomalia, DA et al., Polym J 1985, 17, 117). The small size and the presence of multiple, easily tunable surface groups make these nanoparticles excellent carriers for transporting drugs to the CNS. Previous studies have shown that non-cytotoxic hydroxyl-terminated fourth generation PAMAM dendrimers (approximately 4 nm in size, without any targeting ligand) can cross the impaired BBB and target activated microglia at the site of injury in the brain several-fold higher than in healthy controls (Lesniak, WG et al., Mol Pharm 2013, 10). These dendrimers are non-toxic even at intravenous doses of over 500 mg / kg and are cleared intact via the kidney. These findings have been demonstrated in various small and large animal models (Kannan, S et al., Sci. Transl. Med. 2012, 4, 130ra46; Kambhampati, SP et al., Invest Ophthalmol Vis Sci 2015, 56; Nance, E et al., J. Control. Release 2015, 214, 112; Mishra, MK et al., ACS Nano 2014, 8, 2134; and Nanomedicine 2010, 5, 1317). The selective uptake and localization of these neutral dendrimers in activated microglia may be due to their ability to cross the impaired BBB and rapidly diffuse in the brain parenchyma and subsequently be taken up by glial cells whose phagocytosis is constantly activated.

[0033] Dendrimer surface groups can significantly affect their biodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)). More specifically, hydroxyl-terminated fourth generation PAMAM dendrimers (approximately 4 nm in size), without any targeting ligand, have been shown to cross the impaired BBB significantly more (>20-fold) upon systemic administration in a rabbit model of cerebral palsy (CP) compared to healthy controls, and to selectively target activated microglia and astrocytes (Lesniak, WG, et al., Mol Pharm, 10 (2013); Kannan, S., et al., Science Translational Medicine, 4, 130ra46 (2012); Iezzi, R., et al., Biomaterials, 33, 979 (2012); Mishra, MK, et al., ACS Nano, 8, 2134 (2014); Kambhampati, SP, et al., European Journal of Pharmaceutics and Biopharmaceutics, 95, Part B, 239 (2014). (2015); Zhang, F., et al., Journal of Controlled Release, 249, 173 (2017); Guo, Y., et al., PLOS ONE, 11, e0154437 (2016); and Inapagolla, R., et al., International Journal of Pharmaceutics, 399, 140 (2010)).

[0034] The term "dendrimer" includes, but is not limited to, molecular configurations having an inner core ("GO") and layers (or "generations") of repeating units attached to and extending from the inner core, each layer having one or more branch points with the outer surface of the terminal groups attached to the outermost generation. In some embodiments, dendrimers have a regular dendrimer or "starburst" molecular structure. The dendrimers may have carboxylic acid, amine, or hydroxyl termini and may be of any generation, including, but not limited to, a first generation ("G1") dendrimer ("D1"), second generation ("G2") dendrimer ("D2"), third generation ("G3") dendrimer ("D3"), fourth generation ("G4") dendrimer ("D4"), fifth generation ("G5") dendrimer ("D5"), sixth generation ("G6") dendrimer ("D6"), seventh generation ("G7") dendrimer ("D7"), eighth generation ("G8") dendrimer ("D8"), ninth generation ("G9") dendrimer ("D9"), or tenth generation ("G10") dendrimer ("D10").

[0035] Generally, the diameter of the dendrimer is between about 1 nm and about 50 nm, more preferably between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. In some embodiments, the diameter is between about 1 nm and about 2 nm, inclusive; between about 2 nm and about 3 nm, inclusive; between about 3 nm and about 5 nm, inclusive; or between about 4 nm and about 5 nm, inclusive. In preferred embodiments, the dendrimer has a diameter effective to penetrate brain tissue and be retained in target cells for an extended period of time.

[0036] In some embodiments, the molecular weight of the dendrimer is between about 500 and about 100,000 daltons (inclusive), preferably between about 500 and about 50,000 daltons (inclusive), and most preferably between about 1,000 and about 20,000 daltons (inclusive).

[0037] In some embodiments, the dendrimer has a hypercore (e.g., dipentaerythritol) and one or more monosaccharide branching units. In some embodiments, the monosaccharide branching units are conjugated to the core or pre-monomer layer via a linker, e.g., a polyethylene glycol chain. In a preferred embodiment, the hypercore is dipentaerythritol and the monosaccharide branching units are glucose-based branching units, e.g., those shown in structures I-III.

[0038] In some embodiments, the dendrimers specifically target certain tissue regions and / or cell types after administration to the body. In preferred embodiments, the dendrimers specifically target certain tissue regions and / or cell types without a targeting moiety. In one embodiment, the glucose dendrimers selectively target or enrich in neurons, particularly the nuclei of neurons. In preferred embodiments, the glucose dendrimers selectively target or enrich in damaged, diseased, and / or hyperactive neurons.

[0039] 1. Central Core The multifunctional core moiety allows for the stepwise addition of branching units (ie, generations) around the core.

[0040] Exemplary chemical structures suitable as the core moiety include dipentaerythritol, pentaerythritol, 2-(aminomethyl)-2-(hydroxymethyl)propane-1,3-diol, 2-ethyl-2-(hydroxymethyl)propane-1,3-diol, 3,3',3'',3'''-silanetetrayltetrakis(propane-1-thiol), 3,3-divinylpenta-1,4-diene, 3,3',3''-nitrilotripropionic acid, 3,3',3''-nitrilotris(N -(2-aminoethyl)propanamide), 3,3',3'',3'''-(ethane-1,2-diylbis(azanetriyl))tetrapropanamide, 3-(carboxymethyl)-3-hydroxypentanedioic acid, 2,2'-((2,2-bis((2-hydroxyethoxy)methyl)propane-1,3-diyl)bis(oxy))bis(ethan-1-ol), tetrakis(3-(trichlorosilyl)propyl)silane, 1-thioglycerol, 2,2,4,4,6,6-hexafluoropropane, 1,3,5,2l5,4l5,6l5-Triazatriphosphinine, 3-(hydroxymethyl)-5,5-dimethylhexane-2,4-diol, 4,4',4''-(ethane-1,1,1-triyl)triphenol, 2,4,6-trichloro-1,3,5-triazine, 5-(hydroxymethyl)benzene-1,2,3-triol, 5-(hydroxymethyl)benzene-1,3-diol, 1,3,5-tris(dimethyl(vinyl)silyl)benzene, carbosiloxane The core moiety may be a chitosan. The chitosan core may be a chitosan, a cyclodextrin ...

[0041] In a preferred embodiment, the core moiety is dipentaerythritol.

[0042] 2. Branching Unit Exemplary chemical structures suitable as branching units include monosaccharides. In some embodiments, the monosaccharide branching units are conjugated to the core or the monomer prelayer via a linker, such as a polyethylene glycol chain. In a preferred embodiment, the monosaccharide branching units are glucose-based branching units. Exemplary glucose-based branching units are shown in Structures I-III.

[0043] These are spacer molecules and therefore alkyl (CH 2 ) n - It can also be a hydrocarbon-like unit.

[0044] The branching units are PEG or alkyl chain linkers between different dendrimer generations, for example, glucose layers are linked via PEG linkers and triazole rings.

[0045] In a preferred embodiment, the branching units are the same for each generation of dendrimers produced from the core. Thus, in one embodiment, the branching units are glucose-based branching units for producing first generation dendrimers as shown in structures IV-VI, and for producing second generation dendrimers as shown in Figures 1A and 1B.

[0046] In some embodiments, the branching unit is a hyper-monomer, i.e., AB n Exemplary hyper-monomers include AB 4 , A.B. 5 , A.B. 6 , A.B. 7 , A.B. 8 The number of available end groups is dramatically increased by the hyper-monomer strategy. 4The hypermonomer is a peracetylated β-D-glucopyranoside tetraethylene glycol azide as shown in structure II.

[0047] 3.Surface groups The surface group or terminal functional group is preferably the hydroxyl group of the terminal glucose of the branching unit.In some embodiments, the desired surface group can also be added by modifying one of the conjugation methods for the core and the branching unit.Exemplary surface groups include hydroxyl end groups, amine end groups, carboxylic acid end groups, and thiol end groups.

[0048] In some embodiments, dendrimers can specifically target specific tissue regions and / or cell types, preferably cells and tissues of the central nervous system (CNS) and eye. In some embodiments, dendrimers specifically target neurons of the CNS and eye. Unmodified PAMAN dendrimers with hydroxyl end groups are not as enriched in neurons of the brain and / or retinal ganglion cells (RGC) in the eye as these glucose dendrimers are. Glucose dendrimers with terminal glucose monosaccharides and high density of hydroxyl functional groups effectively target neurons in a generation-dependent manner. Examples demonstrate efficacy, and second generation (G2), as well as G3 and G4 should be effective. G5 and above are more difficult to use.

[0049] In a preferred embodiment, the dendrimer comprises a number of terminal glucose and / or hydroxyl groups effective for targeting one or more neurons of the CNS or eye. The hydroxyl groups on the surface of the dendrimer are part of the glucose molecule. There are no extra hydroxyls other than the glucose molecules on the surface. The number of sugar molecules on the surface is determined by the number of generations. It is expected that all generations will target neurons.

[0050] In some embodiments, the dendrimers are made from glucose and oligoethylene glycol building blocks. Exemplary glucose dendrimers are shown in the Examples, such as the first generation dendrimers shown in Structures IV-VI, and the second generation dendrimers shown in Figures 1A and 1B. Some exemplary glucose dendrimers include a first generation glucose dendrimer with 24 hydroxyl (-OH) end groups, a second generation glucose dendrimer with 96 hydroxyl (-OH) end groups, a third generation glucose dendrimer with 396 hydroxyl (-OH) end groups, and a fourth generation glucose dendrimer with 1584 hydroxyl (-OH) end groups. In a preferred embodiment, the glucose dendrimer is a second generation glucose-based dendrimer with 24 glucose molecules on the exterior surface and 6 embedded glucose molecules held by PEG segments in the backbone.

[0051] B. Conjugation Chemistry and Spacers Dendrimer conjugates can be formed from therapeutic, preventive and / or diagnostic agents or compounds conjugated or bound to glucose dendrimers. Optionally, the therapeutic, preventive or diagnostic agents are conjugated to the dendrimer via one or more spacers / linkers through different bonds, such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazide and amide bonds. One or more spacers / linkers between the dendrimer and the therapeutic, preventive or diagnostic agent can be designed to provide a releasable or non-releasable form of the dendrimer-active complex in vivo. In some embodiments, the binding occurs through a suitable spacer that provides an ester bond between the agent and the dendrimer. In some embodiments, one or more spacers / linkers between the dendrimer and the agent are added to achieve the desired effective release kinetics in vivo. These can be cleavable (ester, SS) or non-cleavable (amide, ether). The conjugation chemistry can be click chemistry, acid-amine coupling, Stecklich esterification, and the like.

[0052] 1. Coupling Agent In some embodiments, the therapeutic, preventive or diagnostic agent is attached to the dendrimer via a linking moiety designed to be cleaved in vivo. The linking moiety can be designed to be cleaved hydrolytically, enzymatically, or a combination thereof to provide sustained release of the agent in vivo. Both the composition of the linking moiety and its point of attachment to the agent are selected such that cleavage of the linking moiety releases either the therapeutic, preventive or diagnostic agent or a prodrug thereof. The composition of the linking moiety can also be selected taking into account the desired release rate of the agent.

[0053] In some embodiments, the linkage occurs through one or more of a disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide bond. In preferred embodiments, the linkage occurs through a suitable spacer that provides an ester or amide bond between the drug and the dendrimer depending on the desired release kinetics of the drug.

[0054] The binding moiety generally comprises one or more organic functional groups. Examples of suitable organic functional groups include secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O) 2 -NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonates (-OC(O)-O-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinols (-CHOH-, -CROH-), disulfide groups, hydrazones, hydrazides, ethers (-O-), and esters (-COO-, -CH 2 O 2 C-, CHRO 2 C-), where R is an alkyl group, an aryl group, or a heterocyclic group. In general, the identity of the organic functional group or groups in the linking moiety is selected having regard to the desired release rate of the drug. Additionally, the organic functional group or groups may be selected to facilitate covalent attachment of the drug to the dendrimer.

[0055] 2. Spacer In certain embodiments, the attachment moiety comprises one or more of the above organic functional groups in combination with a spacer group. The term "spacer" includes compositions used to attach therapeutic, prophylactic and / or diagnostic agents to dendrimers. A spacer can be either a single chemical entity or two or more chemical entities linked together to bridge the polymer and the therapeutic or imaging agent. Spacers can include sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and any small chemical, peptide or polymer with a carbonate terminus.

[0056] In some embodiments, the spacer group is comprised of an assembly of atoms, including oligomeric and polymeric chains; however, the total number of atoms in the spacer group is preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, and most preferably between 3 and 50 atoms. Examples of suitable spacer groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Varying the spacer group provides additional control over the release of the agent in vivo. In embodiments where the linking moiety includes a spacer group, one or more organic functional groups are typically used to attach the spacer group to both the therapeutic, prophylactic or diagnostic agent and the dendrimer. In some embodiments, the spacer is selected from the class of compounds terminating in sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and carbonate groups. In some embodiments, the spacer includes a thiopyridine-terminated compound, such as dithiodipyridine, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or sulfo-LC-SPDP. In some embodiments, the spacer includes a peptide, which is linear or cyclic and has a sulfhydryl group, such as glutathione, homocysteine, cysteine ​​and its derivatives, arg-gly-asp-cys (RGDC), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-D-Tyr-Cys), cyclo(Arg-Ala-Asp-d-Tyr-Cys). In some embodiments, the spacer is a mercapto acid derivative, such as 3 mercaptopropionic acid, mercaptoacetic acid, 4 mercaptobutyric acid, thiolan-2-one, 6 mercaptohexanoic acid, 5 mercaptovaleric acid or other mercapto derivatives, such as 2 mercaptoethanol and 2 mercaptoethylamine.In some embodiments, the spacer is thiosalicylic acid or a derivative thereof, (4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridylthio]propionyl hydrazide. In other embodiments, the spacer has a maleimide terminus, and the spacer includes a polymer or small molecule chemical, such as bis-maleimidodiethylene glycol and bis-maleimidotriethylene glycol, bis-maleimidoethane, bismaleimidohexane. In some embodiments, the spacer includes a vinyl sulfone, such as 1,6-hexane-bis-vinyl sulfone. In some embodiments, the spacer is a thioglycoside, such as thioglucose. In some embodiments, the spacer is a reduced protein, such as bovine serum albumin and human serum albumin, any thiol-terminated compound capable of forming a disulfide bond. In some embodiments, the spacer includes maleimide, succinimidyl, and thiol-terminated polyethylene glycol.

[0057] C. Therapeutic, Prophylactic and Diagnostic Agents The glucose dendrimers are complexed, covalently conjugated, or intramolecularly dispersed or encapsulated therein one or more therapeutic, prophylactic and / or diagnostic agents.

[0058] A wide range of drugs may be included in the particles to be delivered. The drug may be a protein or peptide, a sugar or carbohydrate, a nucleic acid or oligonucleotide, a lipid, a small molecule, or a combination thereof. The nucleic acid may be an oligonucleotide encoding a protein, such as a DNA expression cassette or an mRNA. Representative oligonucleotides include siRNA, microRNA, DNA, RNA, and aptamers. In some embodiments, the therapeutic, preventive, or diagnostic agent is a therapeutic antibody. One or more types of therapeutic, preventive, or diagnostic agents may be encapsulated, complexed, or conjugated to the dendrimer. For example, the dendrimer is conjugated to one or more NAC molecules via disulfide bridges and to one or more antibodies via amide bonds.

[0059] Exemplary therapeutic agents include anti-inflammatory agents, anti-proliferative agents, chemotherapeutic agents, vasodilators, neuroactive agents, and anti-infective agents. In some embodiments, the dendrimer is conjugated to a targeting moiety, an imaging agent, and / or a therapeutic agent.

[0060] 1. Therapeutic Agents One or more therapeutic agents may be complexed, covalently attached, or intramolecularly dispersed or encapsulated with the dendrimer. In some embodiments, two or more different therapeutic agents may be associated with the dendrimer via covalent and / or non-covalent interactions.

[0061] Dendrimer conjugates can preferentially cross the blood-brain barrier (BBB) ​​when administered by intravenous injection. Preferably, the drug is bound or conjugated to a dendrimer, which can preferentially release the drug at the target site, i.e., the site of disease and / or injury. For example, some drugs can be released intracellularly under reducing conditions found in vivo. Dendrimer conjugates bound to drugs can be used to perform several functions, including targeting, localization at disease sites, drug release, and imaging purposes. Dendrimer complexes can be tagged with or without targeting moieties.

[0062] In some embodiments, the one or more therapeutic agents target an underlying cause of the disease or condition, and the one or more therapeutic agents alleviate one or more symptoms of the disease or condition.

[0063] Preferred therapeutic or prophylactic agents include agents that reduce neuroinflammation (e.g., N-acetylcysteine, pioglitazone, vitamin E) and RNA oligonucleotides that interfere with gene transcription or translation. In particularly preferred embodiments, the agent is N-acetylcysteine, 4-phenylbutyrate, bezafibrate, thyroid hormone (T3), sobetirome, pioglitazone, resveratrol, VBP15, vitamin E, erucic acid, coenzyme Q10, clemastine, galactosylceramidase (GALC), aspartoacylase (ASPA), or arylsulfatase A (ARSA). Other suitable agents include anti-inflammatory agents, neuroactive agents, and imaging agents. Dendrimers can be conjugated to more than one agent and more than one type of agent.

[0064] Anti-inflammatory agent In some embodiments, the composition comprises one or more anti-inflammatory agents that reduce inflammation, including steroidal and non-steroidal drugs.

[0065] Preferred anti-inflammatory drugs are antioxidant drugs containing N-acetylcysteine.Preferred NSAIDs include mefenamic acid, aspirin, diflunisal, salsalate, ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, meclofenamic acid, flufenamic acid, tolfenamic acid, elecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, sulfonanilide, nimesulide, niflumic acid, and licofelone.

[0066] Representative small molecules include steroids, such as methylprednisone, dexamethasone, nonsteroidal anti-inflammatory agents including COX-2 inhibitors, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressants, anti-inflammatory and anti-angiogenic agents, anti-excitotoxic agents, such as valproic acid, D-aminophosphonovalerate, D-aminophosphonoheptanoate, glutamate formation / release inhibitors, such as baclofen, NMDA receptor antagonists, salicylic acid anti-inflammatory agents, ranibizumab, anti-VEGF agents including aflibercept, and rapamycin.Other anti-inflammatory agents include nonsteroidal drugs, such as indomethacin, aspirin, acetaminophen, diclofenac sodium, and ibuprofen.Corticosteroids can be fluocinolone acetonide and methylprednisolone.

[0067] Exemplary immunomodulatory agents include cyclosporine, tacrolimus and rapamycin. In some embodiments, the anti-inflammatory agent is a biological drug that blocks the action of one or more immune cell types, such as T cells, or blocks proteins in the immune system, such as tumor necrosis factor-alpha (TNF-alpha), interleukin 17-A, interleukin 12 and 23.

[0068] In some embodiments, the anti-inflammatory drug is a synthetic or natural anti-inflammatory protein.An antibody specific to selected immune components may be added to the immunosuppressive therapy.In some embodiments, the anti-inflammatory drug is an anti-T cell antibody (e.g., anti-thymocyte globulin or anti-lymphocyte globulin), an anti-IL-2Rα receptor antibody (e.g., basiliximab or daclizumab), or an anti-CD20 antibody (e.g., rituximab).

[0069] In preferred embodiments, the one or more anti-inflammatory agents are released from the dendrimer conjugate in an amount effective to inhibit inflammation for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, preferably at least 1 week, 2 weeks, or 3 weeks, and more preferably at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months after administration to a mammalian subject.

[0070] b. Neuroactive agents A number of drugs have been developed to interrupt, affect, or temporarily halt the glutamate excitotoxicity cascade toward neuronal damage. One strategy is to decrease glutamate release "upstream." This category of drugs includes riluzole, lamotrigine, and rifariidine, which are sodium channel blockers. The commonly used nimodipine is a voltage-dependent channel (L-type) blocker. Some agents affect the bound glutamate receptor site. Some of these drugs include felbamate, ifenprodil, magnesium, memantine, and nitroglycerin. These "downstream" drugs attempt to affect intracellular events, such as the formation of free radicals, the formation of nitric oxide, protein degradation, endonuclease activity, and ICE-like protease formation (a key component in the process leading to programmed cell death or apoptosis).

[0071] The drugs for treating neurodegenerative diseases are well known in the art and can be changed based on the symptoms and diseases to be treated.For example, the conventional treatment for Parkinson's disease can include levodopa (usually combined with dopa decarboxylase inhibitor or COMT inhibitor), dopamine agonist, or MAO-B inhibitor.

[0072] Treatments for Huntington's disease can include dopamine blockers to help reduce abnormal behavior and movement, or drugs such as amantadine and tetrabenazine to control movement. Other drugs that help reduce chorea include neuroleptics and benzodiazepines. Compounds such as amantadine or remacemide have shown good results. Hypokinesia and rigidity, especially in juvenile cases, can be treated with antiparkinsonian drugs, and myoclonic hyperkinesia can be treated with valproic acid. Psychiatric symptoms can be treated with medications similar to those used in the general population. Selective serotonin reuptake inhibitors and mirtazapine are recommended for depression, while atypical antipsychotics are recommended for psychosis and behavioral disorders.

[0073] The anti-excitotoxic drug riluzole (RILUTEK®) (2-amino-6-(trifluoromethoxy)benzothiazole) has resulted in improved survival in subjects with ALS. Other drug therapies and interventions may reduce symptoms caused by ALS. Some treatments improve quality of life and some appear to extend lifespan. Common ALS-related therapies are reviewed in Gordon, Aging and Disease, 4(5):295-310 (2013), see, e.g., Table 1 therein. Numerous other drugs have been tested in one or more clinical trials, with efficacy ranging from ineffective to promising. Exemplary drugs are reviewed in Carlesi, et al., Archives Italiennes de Biologie, 149:151-167 (2011). For example, treatments include agents that reduce excitotoxicity, such as talampanel (8-methyl-7H-1,3-dioxolo(2,3)benzodiazepine), cephalosporins, such as ceftriaxone, or memantine; agents that reduce oxidative stress, such as coenzyme Q10, manganese porphyrin, KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride, RPPX], or edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186); agents that reduce apoptosis, such as These may include histone deacetylase (HDAC) inhibitors, including valproic acid, TCH346 (dibenzo(b,f)oxepin-10-ylmethyl-methylprop-2-ynylamine), minocycline, or tauroursodeoxycholic acid (TUDCA); agents that reduce neuroinflammation, such as thalidomide and cerlastol; neurotropic agents, such as insulin-like growth factor 1 (IGF-1) or vascular endothelial growth factor (VEGF); heat shock protein inducers, such as arimoclomol; or autophagy inducers, such as rapamycin or lithium.

[0074] Treatments for Alzheimer's disease can include, for example, acetylcholinesterase inhibitors, such as tacrine, rivastigmine, galantamine, or donepezil; NMDA receptor antagonists, such as memantine; or antipsychotics.

[0075] Treatments for Lewy body dementia can include, for example, acetylcholinesterase inhibitors such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine; dopaminergic therapies such as levodopa or selegiline; antipsychotics such as olanzapine or clozapine; REM disorder therapies such as clonazepam, melatonin, or quetiapine; antidepressant and anti-anxiety therapies such as selective serotonin reuptake inhibitors (such as citalopram, escitalopram, sertraline, paroxetine) or serotonin and noradrenaline reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, for example, Macijauskiene, et al., Medicina (Kaunas), 48(1):1-8 (2012)).

[0076] Exemplary neuroprotective agents include, for example, glutamate antagonists, antioxidants, and NMDA receptor stimulants. Other neuroprotective agents and treatments include caspase inhibitors, trophic factors, anti-protein aggregation agents, therapeutic hypothermia, and erythropoietin.

[0077] Other common therapeutic, prophylactic or diagnostic agents for treating neurological dysfunction include amantadine and anticholinergics to treat motor symptoms, clozapine to treat psychosis, cholinesterase inhibitors to treat dementia, and modafinil to treat daytime sleepiness.

[0078] C. Anti-infective agents Antibiotics include beta-lactams such as penicillin and ampicillin, cephalosporins such as cefuroxime, cefaclor, cephalexin, cephydroxil, cepfodoxime and proxetil, tetracycline antibiotics such as doxycycline and minocycline, macrolide antibiotics such as azithromycin, erythromycin, rapamycin and clarithromycin, fluoroquinolones such as ciprofloxacin, enrofloxacin, ofloxacin, gatifloxacin, levofloxacin and norfloxacin, tobramycin, colistin, or aztreonam, and antibiotics known to have anti-inflammatory activity such as erythromycin, azithromycin, or clarithromycin.

[0079] 2. Diagnostic agents In some cases, the agent that is delivered to target cell or tissue via glucose dendrimer is a diagnostic agent.The examples of diagnostic agents that can be delivered to brain by glucose dendrimer conjugate include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, X-ray imaging agents, and contrast agents.The glucose dendrimer conjugate can include agents that are useful for determining the location of administered composition.The agents that are useful for this purpose include fluorescent tags, radionuclides, and contrast agents.

[0080] Exemplary diagnostic agents include dyes, fluorescent dyes, near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. Representative dyes include carbocyanine, indocarbocyanine, oxacarbocyanine, thueicarbocyanine and merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor647, HiLyte Fluor680, HiLyte Fluor750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0081] Exemplary SPECT or PET imaging agents include chelating agents such as di-ethylenetri-aminepentaacetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), di-aminedithiols, activated mercaptoacetyl-glycyl-glycyl-glycine (MAG3), and hydrazidonicotinamide (HYNIC).

[0082] Exemplary isotopes include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Gd3+, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, F-18, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-166.

[0083] In a preferred embodiment, the dendrimer composition comprises one or more radioisotopes suitable for positron emission tomography (PET) imaging. Exemplary positron-emitting radioisotopes include carbon-11 ( 11 C), copper-64( 64 Cu), Nitrogen-13( 13 N), oxygen-15( 15 O), Gallium-68( 68 Ga), and Fluorine-18( 18 F), for example, 2-deoxy-2- 18 F-fluoro-β-D-glucose ( 18 F-FDG).

[0084] In a further embodiment, a single glucose dendrimer conjugate composition may simultaneously treat and / or diagnose a disease or condition at one or more locations in the body.

[0085] D. Dendrimer Conjugates The surface groups allow for the attachment of small molecules, imaging agents, and small biological agents, such as siRNA, regardless of the charge or water solubility of the payload. The glucose dendrimers may contain one or more therapeutic or prophylactic agents that are complexed, covalently conjugated, or intramolecularly dispersed or encapsulated with the dendrimer.

[0086] In some embodiments, one or more agents are covalently attached to one or more terminal groups of the glucose dendrimer. In some embodiments, the glucose dendrimer conjugate comprises one or more therapeutic, preventive or diagnostic agents conjugated or complexed to the glucose dendrimer via one or more linking moieties. In further embodiments, the linking moiety incorporates or is conjugated to one or more spacer moieties. The linking and / or spacer moieties can be cleaved, for example, by exposure to the intracellular compartment of the target cell in vivo. The therapeutic, preventive or diagnostic agents and / or targeting moieties can be either covalently attached or intramolecularly dispersed or encapsulated. The glucose dendrimers are preferably second, third, fourth, fifth, sixth and up to tenth generation, which have hydroxyl surface groups on the terminal glucose monosaccharides. In a preferred embodiment, the glucose dendrimer is linked to the agent via a spacer that terminates in a disulfide, ester or amide bond.

[0087] Optimal drug loading necessarily depends on many factors, including the choice of drug, the structure and size of the dendrimer, and the tissue to be treated.In some embodiments, one or more therapeutic, preventive, or diagnostic agents are encapsulated, associated, and / or conjugated to the dendrimer at a concentration of about 0.01% to about 45% by weight (inclusive); preferably about 0.1% to about 30% by weight (inclusive); about 0.1% to about 20% by weight (inclusive); about 0.1% to about 10% by weight (inclusive); about 1% to about 10% by weight (inclusive); about 1% to about 5% by weight (inclusive); about 3% to about 20% by weight (inclusive); and about 3% to about 10% by weight (inclusive).However, the optimal drug loading for any given drug, dendrimer, and target site can be identified by routine methods, such as those described.

[0088] In some embodiments, drug / linker conjugation occurs through about 1%, 2%, 3%, 4%, or 5% of the total available surface functional groups of the dendrimer prior to conjugation, preferably hydroxyl groups. In other embodiments, drug / linker conjugation occurs through less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75% of the total available surface functional groups of the dendrimer prior to conjugation and / or modification with a therapeutic, prophylactic or diagnostic agent. In a preferred embodiment, the dendrimer complex retains an effective amount of surface functional groups for targeting the target cells while being conjugated to an effective amount of an agent for treating, preventing and / or imaging a disease or disorder.

[0089] Typically, dendrimer conjugates have a hydrodynamic volume in the nanometer range.For example, in some embodiments, the diameter of glucose dendrimer conjugates with one or more therapeutic, preventive or diagnostic agents complexed or conjugated to the dendrimer is about 2 nm to about 100 nm, or more than 100 nm, or up to 500 nm, depending on the generation of the dendrimer, chemical composition and the amount of therapeutic, preventive or diagnostic agents loaded.Preferably, glucose dendrimer conjugates with one or more therapeutic, preventive or diagnostic agents complexed or conjugated to the dendrimer have a diameter that is effective to penetrate brain tissue and be retained in target cells for a long time.

[0090] The presence of therapeutic, preventive or diagnostic agent(s) may affect the zeta potential or surface charge of the dendrimer conjugate. In one embodiment, the zeta potential of the dendrimer conjugated or complexed with a therapeutic, preventive or diagnostic agent is between -100mV and 100mV, between -50mV and 50mV, between -25mV and 25mV, between -20mV and 20mV, between -10mV and 10mV, between -10mV and 5mV, between -5mV and 5mV, or between -2mV and 2mV. The above ranges include all values ​​between -100mV and 100mV. In a preferred embodiment, the surface charge is neutral or near neutral, i.e., about -10mV to about 10mV inclusive.

[0091] III. Methods for Making Glucose Dendrimers Glucose dendrimers can be prepared via various chemical reaction steps. Dendrimers are usually synthesized according to methods that allow control of the dendrimer structure at every stage. Dendrimer structures are mainly synthesized by one of two different approaches: divergent or convergent.

[0092] In some embodiments, dendrimers are prepared using a divergent methodology, in which dendrimers are assembled from a multifunctional core and extended outward by a series of reactions. The strategy involves coupling monomer molecules bearing reactive and protecting groups to the multifunctional core moiety, resulting in the stepwise addition of generations around the core, followed by removal of the protecting groups. For example, dendrimers can be prepared using the AB 4 It is first synthesized by coupling a peracetylated β-D glucose-PEG4-azide monomer to a hexapropargylated core.

[0093] In some embodiments, the dendrimer core, one or more branching units, one or more linkers / spacers, and / or one or more surface groups are modified to allow conjugation to further functional groups (branching units, linkers / spacers, surface groups, etc.), monomers, and / or drugs via click chemistry using one or more of copper-assisted azide-alkyne cycloaddition (CuAAC), Diels-Alder reactions, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules. 2015 May 20;20(5):9263-94). "Click chemistry" involves the coupling of two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or equivalent) on the surface of a first moiety and an azide moiety (e.g., present in a triazine composition or its equivalent) on a second moiety, or any active end group such as, for example, a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.

[0094] In some embodiments, dendrimer synthesis relies on one or more reactions, such as thiol-ene click reaction, thiol-yne click reaction, CuAAC, Diels-Alder click reaction, azide-alkyne click reaction, Michael addition, epoxy ring opening, esterification, silane chemistry, and combinations thereof.

[0095] In some embodiments, the method involves protecting and deprotecting functional groups (e.g., hydroxyl groups) on the central core, the branching units, and / or the therapeutic, preventive or diagnostic agent one or more times to facilitate the addition of the branching units to generate the desired dendrimer molecule, or the addition of the therapeutic, preventive or diagnostic agent to generate the desired dendrimer conjugate. In the case of hydroxyl groups, they may be protected by forming ethers, esters, or acetals. Other exemplary protecting groups include Boc and Fmoc.

[0096] 1. Synthesis of Hypercore In a preferred embodiment, the hypercore is prepared from dipentaerythritol, for example, by propargylation of dipentaerythritol to achieve a hexapropargylated core. [ka]

[0097] 2. Synthesis of Hypermonomer In some embodiments, the branching unit is a hypermonomer, i.e., AB n Exemplary hypermonomers include AB 3 , A.B. 4 , A.B. 5 , A.B. 6 , A.B. 7 , A.B. 8 The building blocks include: the hypermonomer strategy dramatically increases the number of available end groups; an exemplary hypermonomer is the AB 1211, which contains one azide functionality and four allyl groups, prepared from the reaction of dipentaerythritol, which has five allyl groups, with monotosylated triethylene glycol azide. 4 It is an orthogonal hypermonomer (Scheme 2).

[0098] In some embodiments, the branching unit is a linear or branched polyethylene glycerol as shown in Formula II. Other monomers include disaccharides and oligosaccharides, as well as saccharides such as fructose, lactose, and sucrose.

[0099] A.B. 4 Building Block Synthesis Hypermonomer AB 4 Some exemplary syntheses of the hypermonomer AB are described below. 4 is based on a glucose molecule. In a preferred embodiment, the hypermonomer AB 4is conjugated to a polyethylene glycerol, e.g., tetraethylene glycol (PEG4). In one embodiment, the hypermonomer AB 4 is peracetylated β-D-glucopyranoside tetraethylene glycol azide.

[0100] In some embodiments, the synthesis of glucose-OAc-TEG-OT involves the following steps: A solution of peracetylated β-D-glucopyranoside (10 g, 25.6 mmol) was dissolved in 50 mL of anhydrous dichloromethane (DCM), followed by the addition of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.2 g, 17.9 mmol) and the reaction mixture was cooled to 0° C. Boron trifluoride diethyl etherate (2.5 eq.) was added and the reaction was allowed to warm to room temperature. The reaction was monitored with the aid of TLC and quenched after 5 h by the addition of saturated sodium bicarbonate solution at 0° C. After stirring for 10 min, DCM (300 mL) was added and the organic layer was washed three times with saturated sodium bicarbonate solution until the effervescence was quenched. The reaction mixture was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified by CombiFlash chromatography using a mixture of ethyl acetate / hexane (70:30) as the eluent. The desired compound was obtained in 60% yield. The structure of Glucose-OAc-TEG-OT is shown below: [ka]

[0101] In some embodiments, glucose-OAc-TEG-N 3 The synthesis of Glucose-OAc-TEG-OT involves the following steps: A solution of Glucose-OAc-TEG-OT (6 g, 8.8 mmol) is dissolved in 40 mL of anhydrous DMF, followed by the addition of sodium azide (2 eq) and heating the reaction mixture to 50 °C overnight. Upon completion, the reaction mixture is filtered and the DMF is evaporated. Once dry, the crude reaction mixture is passed through a Combi-Flash using ethyl acetate:hexane (70:30) as the eluent. Glucose-OAc-TEG-N 3The structure of is shown below: [ka]

[0102] In some embodiments, Glucose-OH-TEG-N 3 The synthesis of peracetylated β-D-glucopyranoside involves the following steps: Tetraethylene glycol azide is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH to approximately 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and the pH is adjusted to approximately 6-7 with Amberlist IR-120+. The reaction mixture is isolated by filtration and the solvent is removed by rotary evaporation. Glucose-OH-TEG-N 3 The structure is shown below. [ka]

[0103] 3. Synthesis of glucose dendrimers In some embodiments, the glucose dendrimer is an AB 4 It is synthesized by coupling a peracetylated β-D glucose-PEG4-azide monomer to a hexapropargylated core. In a preferred embodiment, the hexapropargylated core (1) is converted to an AB 4 β-D-glucose-PEG4-azide building block (2) is coupled to give the first generation dendrimer.

[0104] In some embodiments, the first generation dendrimer D1-Glu6-OAc24 (compound 3a in FIG. 1A) is prepared as follows: the hexapropargylated compound (0.5 g, 1 mmol) and the azide derivative (1.2 eq. per (4.1 g, 7.4 mmol) acetylene) are suspended in a 1:1 mixture of DMF and water in a 20 mL microwave vial equipped with a magnetic stir bar. CuSO dissolved in a minimum amount of water. 4 5H 2O (5 mol% / acetylene, 75 mg) and sodium ascorbate (5 mol% / acetylene, 60 mg) are added. The reaction is irradiated in a microwave at 50° C. for 6 h. The reaction mixture is dialyzed against DMF followed by water containing EDTA. EDTA is further removed by extensive water dialysis. The product is lyophilized to give D1-Glu6-OAc24. The structure of D1-Glu6-OAc24 is shown below. [ka]

[0105] In some embodiments, the first generation dendrimer D1-Glu 6 -OH 24 (Compound 3b in Figure 1A) is prepared as follows: Peracetylated first generation glucose dendrimer (1 g, 0.26 mmol) is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH to approximately 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and the pH adjusted to approximately 6-7 with AMBERLIST® IR-120+. The reaction mixture is isolated by filtration and the solvent is removed by rotary evaporation, followed by water dialysis. The structure of D1-Glu6-OH24 is shown below. [ka]

[0106] In some embodiments, the first generation dendrimer D1-Glu6-OH24 is propargylated to give D1-acetylene 24 (compound 4 in FIG. 1A) as follows: D1-GLu6-OH24 (2 g, 0.721 mmol) was dissolved in anhydrous dimethylformamide (DMF, 50 mL) by sonication. Sodium hydride [60% dispersion in mineral oil] (951 mg, 39.65 mmol) was added to the solution slowly in portions at 0° C. with stirring. The solution was stirred at 0° C. for another 15 min. This was followed by the addition of propargyl bromide (3.85 mL, 34.608 mmol, 80% w / w solution in toluene) at 0° C. and stirring was continued for another 6 h at room temperature. The reaction mixture was quenched with ice and water, filtered, and dialyzed against DMF, followed by water to give D1-acetylene 24. The structure of D1-acetylene 24 is shown below. [ka]

[0107] In some embodiments, the first generation dendrimer D1-acetylene 24 is AB 4 Further reaction with β-D-glucose-PEG4-azide gives a second generation dendrimer with 24 glucose molecules containing 96 surface hydroxyl groups.

[0108] An exemplary second generation dendrimer D2-Glu24-OAc96 (compound 5a in FIG. 1A) is prepared as follows: D1-acetylene dendrimer 24 (0.5 g, 0.13 mmol) and glucose-OAc-TEG-azide (2.2 g, 4 mmol) are suspended in a 1:1 mixture of DMF and water in a 20 mL microwave vial equipped with a magnetic stir bar. To this is added CuSO dissolved in a minimum amount of water. 4 5H 2O (5 mol% / acetylene, 5 mg) and sodium ascorbate (5 mol% / acetylene, 10 mg) are added. The reaction is irradiated in a microwave at 50° C. for 8 h. Upon completion, the reaction mixture is dialyzed against DMF followed by water containing EDTA. EDTA is further removed by extensive water dialysis. The product is lyophilized to give D2-Glu24-OAc96.

[0109] In some embodiments, the second generation dendrimer D2-Glu24-OH96 (compound 5b in FIG. 1A) is prepared as follows: Peracetylated second generation glucose dendrimer D2-Glu24-OH96 is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH to approximately 8.5-9.0. The reaction is stirred overnight at room temperature, then diluted with methanol and the pH adjusted to approximately 6-7 with AMBERLIST® IR-120+. The reaction mixture is filtered to remove the resin, and the filtrate is evaporated by rotary evaporation followed by dialysis against water to give the product as an off-white solid.

[0110] In some embodiments, the second generation dendrimer D2-Glu24-OH96 is propargylated at one or more terminal hydroxyl groups suitable for further conjugation to one or more therapeutic, prophylactic or diagnostic agents. In some embodiments, one or more terminal hydroxyl groups of the second generation dendrimer D2-Glu24-OH96 are propargylated as follows: D2-Glu24-OH96 (5b) (200 mg, 0.016 mmol) is dissolved in anhydrous dimethylformamide (DMF, 10 mL) by sonication. Sodium hydride [60% dispersion in mineral oil] (22 mg, 0.934 mmol) is added slowly in portions to this stirred solution at 0° C. The solution is further stirred at 0° C. for 15 minutes. Propargyl bromide (18.0 μL, 80% w / w solution in toluene) is then added at 0° C. and stirring is continued for another 6 hours at room temperature. The solvent is evaporated using a V10 evaporator system and the crude product is purified by passing through a PD10 SEPHADEX® G25M column. The aqueous solution is lyophilized to give the product as an off-white solid.

[0111] In some embodiments, one or more fluorescent dyes, such as infrared fluorescent Cy5 dyes, are conjugated to the second generation dendrimer D2-Glu24-OH96. In one embodiment, Cy5-D2-Glu24-OH96 (compound 7 in FIG. 1B) is prepared as follows: Compound 6 (200 mg, 0.016 mmol) and Cy5 azide (20.7 mg, 0.02 mmol) are suspended in a 1:1 mixture of DMF and water in a 25 mL round bottom flask equipped with a magnetic stir bar. To this is added CuSO dissolved in a minimum amount of water. 4 5H 2 O (5 mol% / acetylene, 0.3 mg) and sodium ascorbate (10 mol% / acetylene, 0.5 mg) are added. The reaction is stirred at room temperature for 24 hours. Upon completion, the DMF is evaporated using a V10 and purification is performed using a PD10 Sephadex G25M column. The aqueous solution is lyophilized to give the product as a blue solid.

[0112] In some embodiments, the total hydroxyl groups for further conjugation to active agents, including therapeutic and / or diagnostic agents, are about 1-30, 2-20, or 5-10 of the 96 total available hydroxyl groups for an exemplary second generation dendrimer having 24 glucose molecules containing 96 surface hydroxyl groups.

[0113] 4. Synthesis of Glucose Dendrimer Conjugates Methods for conjugating drugs to dendrimers are known in the art and are described, for example, in U.S. Published Application Nos. 2011 / 0034422, 2012 / 0003155, and 2013 / 0136697.

[0114] Reactions and strategies that are useful for covalently attaching drugs to dendrimers are known in the art.See, for example, March, "Advanced Organic Chemistry," 5th Edition, 2001, Wiley-Interscience Publication, New York and Hermanson, "Bioconjugate Techniques," 1996, Elsevier Academic Press, USA.The appropriate method for covalently attaching a given drug can be selected by considering the structure of the drug and dendrimer in relation to the desired binding moiety, as well as the compatibility of functional groups, protecting group strategy, and the presence of labile bonds.

[0115] In a preferred embodiment, glucose dendrimer-drug conjugate is prepared using click chemistry.Click chemistry makes synthesis easy and robust, and therefore provides good control over synthesis and ligand loading (Sharma, R. et al., Journal of Controlled Release 2020;Sharma, R. et al., Bioconjugate Chemistry 2017, 28 (11), 2874-2886;Sharma, A. et al., Theranostics 2018, 8 (20), 5529). Techniques for obtaining high purity dendrimer-drug conjugates using CuAAC chemistry have been published (Sharma, A. et al., Science Advances 2020, 6 (4), eaay8514; Sharma, R. et al., Biomacromolecules 2021, 22 (8), 3574-3589; Sharma, A. et al., Biomacromolecules 2020, 21 (12), 5148-5161; and Sharma, A. et al., Journal of Controlled Release 2018, 283, 175-189).

[0116] Exemplary glucose dendrimer conjugates are shown in Figures 2A-2C, 3A-3B, and 4. The structural confirmation and purity of the conjugates are shown in Figures 2A-2C, 3A-3B, and 4, respectively. 1 It can be determined by 1 H NMR and HPLC.

[0117] a. Synthesis using copper-catalyzed alkyne-azide click (CuAAC) In some embodiments, glucose dendrimer-drug conjugates are prepared using copper-catalyzed alkyne-azide click (CuAAC). In some embodiments, the synthesis of glucose dendrimer-drug conjugates starts with partial modification of OH groups to provide propargyl groups (FIG. 2A). Meanwhile, drugs are modified using releasable or non-releasable chemical bonds to provide azide end groups through polyethylene glycol linkers. An exemplary synthesis of glucose dendrimer-loperamide conjugates is shown in FIG. 2B: loperamide hydrochloride is reacted with azide-PEG4-acid at room temperature in the presence of DCC and DMAP in DCM to obtain loperamide-PEG-azide. Meanwhile, partial modification of the OH groups of glucose dendrimer is performed to provide approximately 10 propargyl groups, which are reacted with loperamide-PEG-azide to obtain glucose dendrimer-loperamide conjugates.

[0118] An exemplary synthesis of rapamycin-azide is achieved using the protocol of Sharma, A. et al., Biomacromolecules 2020, 21, (12), 5148-5161. Meanwhile, partial modification of the OH groups of glucose dendrimer is carried out to provide about four propargyl groups, which are reacted with rapamycin-azide to obtain glucose dendrimer-r conjugate (Figure 2C). Structural confirmation and purity of the conjugate are achieved by 1H NMR and HPLC, respectively.

[0119] b. Synthesis using a combination of click and esterification / amidation reactions In some embodiments, glucose dendrimer-drug conjugates are prepared using a combination of click and esterification / amidation reactions. Synthesis is accomplished by partially modifying the hydroxyl (-OH) groups of glucose dendrimers to provide propargyl groups, which are reacted with linkers containing azide and amine termini to provide surface amine groups (Figure 3A). The drug is modified using a carboxylic acid or -NHS ester-terminated linker (hydrocarbon or PEG chains containing disulfide, ester, or amide bonds). The drug and dendrimer are then reacted using an amidation reaction using a coupling agent, e.g., EDC and DMAP.

[0120] An exemplary synthesis of glucose dendrimer-NAC conjugates is achieved by partially modifying the OH groups of glucose dendrimers to provide propargyl groups, which are reacted with a linker containing azide and amine termini to provide surface amine groups (Figure 3B). SPDP-NAC is obtained by a previously published procedure. Reaction of NAC-SPDP and dendrimer at pH 7.4 gives glucose dendrimer-NAC conjugates.

[0121] c. Synthesis using copper-free biorthogonal click chemistry In some embodiments, glucose dendrimer-drug conjugates are prepared using copper-free biorthogonal click chemistry. In preferred embodiments, copper-free click reactions, such as TCO-triazine (Figure 4), strain-promoted azide-alkyne, Staudinger ligation, DBCO-azide click reactions, are used to conjugate drugs, siRNA, peptides, mRNA, oligonucleotides, antibodies, and other biologics, where the presence of copper and reducing agents may reduce the efficacy of therapeutic, prophylactic, or diagnostic agents.

[0122] IV. Pharmaceutical Preparations Pharmaceutical compositions comprising a glucose dendrimer and one or more therapeutic, prophylactic or diagnostic agents may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may optionally contain excipients and auxiliaries to facilitate processing of the active compounds into preparations and which may be pharma- ceutical used for oral, intranasal, subcutaneous, intraperitoneal or intramuscular administration.

[0123] The appropriate formulation depends on the selected route of administration. In a preferred embodiment, the composition is formulated for parenteral delivery. In some embodiments, the composition is formulated for intravenous injection. Typically, the composition is formulated in a sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition can be stored in a lyophilized state in a single-use vial for rehydration immediately before use. Other means for rehydration and administration are known to those skilled in the art.

[0124] Representative excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof. Suitable pharma- ceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing undesired biological side effects or undesirable interactions.

[0125] Generally, pharmaceutically acceptable salts can be prepared by reacting the free acid or free base form of the drug with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of drugs derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reacting the drug with a suitable organic ligand (e.g., quaternary ammonium salts). A list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of ophthalmic drugs that are sometimes administered in the form of pharmaceutically acceptable salts include timolol maleate, brimonidine tartrate, and diclofenac sodium.

[0126] The glucose dendrimer composition is preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The phrase "unit dosage form" refers to a physically discrete unit of the conjugate appropriate for the patient to be treated. However, it is understood that the total amount of a single administration of the composition will be determined by the attending physician within the scope of sound medical judgment. Therapeutically effective doses can be estimated initially either in cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. Animal models are also used to achieve a desired concentration range and route of administration. Such information should therefore be useful in determining effective doses and routes for administration in humans. The therapeutic efficacy and toxicity of the conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of the therapeutic effect to the toxicity is the therapeutic index, and the ratio, LD 50 / ED 50Pharmaceutical compositions that exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies can be used in formulating various dosages for human use.

[0127] In certain embodiments, the glucose dendrimer composition is administered locally, for example, by direct injection at the site to be treated. In some embodiments, the composition is injected, applied topically, or administered directly to the vasculature on vascular tissue at or adjacent to the site of injury, surgery, or transplantation. For example, in embodiments, the composition is applied topically to vascular tissue exposed during surgery. Typically, local administration results in an increase in the local concentration of the composition, which exceeds that which can be achieved by systemic administration.

[0128] Pharmaceutical compositions of glucose dendrimers formulated for parenteral (intramuscular, intraperitoneal, intravenous or subcutaneous injection) and enteral routes of administration are described.

[0129] A. Parenteral Administration The glucose dendrimer composition may be administered parenterally. The phrases "parenteral administration" and "administered parenterally" are art-recognized terms and include modes of administration other than enteral and topical administration. The dendrimer may be administered orally, nasally, subcutaneously, intraperitoneally, or intramuscularly. For liquid formulations, the pharma- ceutically acceptable carrier may be, for example, an aqueous or non-aqueous solution, a suspension, an emulsion, or an oil. Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media. The dendrimer may also be administered in an emulsion, for example, water in oil. Examples of oil are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0130] Preparations suitable for parenteral administration may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the preparation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0131] Injectable pharmaceutical carriers for injectable compositions are well known to those of skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0132] B. Enteral Administration The glucose dendrimer composition may be administered enterally. The carrier or diluent may be a solid carrier, such as a capsule or tablet, or a diluent for a solid formulation, a liquid carrier or a diluent for a liquid formulation, or a mixture thereof.

[0133] For liquid preparations, pharma- ceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions containing saline and buffered media.

[0134] Examples of oil are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0135] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Formulations include, for example, aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Vehicles may include, for example, fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose, and related sugar solutions are preferred liquid carriers. They may also be formulated with proteins, fats, saccharides, and other components of infant formula.

[0136] In a preferred embodiment, the composition is formulated for oral administration.Oral formulations can be in the form of chewing gum, gel strips, tablets, capsules or lozenges.Encapsulating materials for preparing enteric coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and methacrylic acid ester copolymers.Solid oral formulations, such as capsules or tablets, are preferred.Elixirs and syrups are also well-known oral formulations.

[0137] V. Usage Methods of using glucose dendrimer compositions are described. In preferred embodiments, the glucose dendrimer conjugates cross the blood-brain barrier (BBB) ​​and selectively target or enrich in neurons, preferably in the neuronal nuclei of damaged / hyperactive neurons. In further embodiments, the glucose dendrimer conjugates also accumulate in activated microglia and astrocytes.

[0138] A. Treatment The preparation can be administered to treat infection, inflammation or cancer-related disorders, particularly certain disorders that have systemic inflammation that extends to the nervous system, particularly the CNS and eye.More specifically, the envisioned preparation can be particularly effective during the early stages of brain injury and ischemic injury, where it is important to prevent early neuronal death.

[0139] Typically, an effective amount of a dendrimer complex comprising a combination of a dendrimer and one or more therapeutically, prophylactically and / or diagnostically active agents is administered to an individual in need thereof. The dendrimer may also contain targeting agents, although as demonstrated in the examples, these are not required for delivery to neurons in the spinal cord and brain and / or to retinal ganglion cells (RGCs) of the eye.

[0140] In some embodiments, the dendrimer conjugate can release a therapeutic, preventive or diagnostic agent intracellularly under conditions found in vivo. The amount of dendrimer conjugate administered to a subject is selected to deliver an amount effective to reduce, prevent or otherwise alleviate one or more clinical or molecular symptoms of the disease or disorder being treated, compared to a control, e.g., a subject treated with a therapeutic, preventive or diagnostic agent without a dendrimer. In some embodiments, the method includes a step of selecting a subject who may benefit from treatment with a glucose dendrimer composition.

[0141] B. Condition to be Treated The composition is suitable for treating one or more diseases, conditions, and injuries in the eye, brain, and nervous system, particularly those associated with the pathological activation of neurons, microglia, and / or astrocytes.The composition can also be used for treating eye diseases, and other tissues in which nerves play a role in disease or disorder.The composition and method are also suitable for prophylactic use.

[0142] The dendrimer complex composition selectively targets neurons that play a key role in the pathogenesis of many disorders and conditions, including neurodevelopmental diseases, neurodegenerative diseases, and brain cancer.Therefore, the dendrimer complex is administered in a dosage unit amount effective to treat or alleviate conditions associated with the pathological condition of neurons.Generally, by targeting these cells, the dendrimer specifically delivers drugs to treat diseased neurons.

[0143] In a preferred embodiment, the dendrimers are administered in an amount effective to treat disease neuron-mediated pathology in a subject in need thereof without any associated toxicity.

[0144] In some embodiments, the subject to be treated is a human. In some embodiments, the subject to be treated is a child or infant. All methods may include a step of identifying and selecting a subject in need of treatment or who would benefit from administration with the described composition.

[0145] 1. Eye diseases and injuries The compositions and methods are suitable for treating eye-related discomfort, pain, dryness, excessive tearing, injuries, infections, and burns.

[0146] Examples of eye disorders that may be treated include amebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, oncorcerca keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophic diseases, Fuchs' endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, cicatrical scarring and fibrosis, punctate epithelial keratopathy, linear keratitis, corneal erosion, thinning, ulcers and perforations, Sjogren's syndrome, Stevens-Johnson syndrome, autoimmune dry eye disease, environmental dry eye disease, and ocular diseases. The present invention relates to a method for treating ocular diseases, including ocular diseases, ocular neovascularization diseases, prevention and treatment of corneal transplant rejection, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), panuveitis, vitreous or retinal inflammatory diseases, prevention and treatment of endophthalmitis, macular edema, macular degeneration, age-related macular degeneration, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, autoimmune diseases of the retina, primary and metastatic intraocular melanoma, other intraocular metastatic tumors, glaucoma, open-angle glaucoma, angle-closure glaucoma, pigmentary glaucoma, and combinations thereof. Other disorders include corneal damage, burns, or detachment, cataracts, and age-related degeneration of the eye or associated vision loss.

[0147] In a preferred embodiment, the eye disorder to be treated is age-related macular degeneration (AMD).Age-related macular degeneration (AMD) is a neurodegenerative, neuroinflammatory disease of the macula, which causes central vision loss.The pathogenesis of age-related macular degeneration involves chronic neuroinflammation of the choroid (blood layer under the retina), retinal pigment epithelium (RPE), cell layer under the neurosensory retina, Bruch's membrane, and the neurosensory retina itself.

[0148] 2. Neurological and neurodegenerative diseases The glucose dendrimer compositions and formulations thereof can be used to diagnose and / or treat one or more neurological and neurodegenerative diseases. The compositions and methods are particularly suitable for treating one or more neurological or neurodegenerative diseases associated with defective or diseased neurons. In some embodiments, the disease or disorder is selected from, but is not limited to, some psychiatric disorders (e.g., depression, schizophrenia (SZ), alcohol use disorder, and morphine antinociceptive tolerance), neurological and neurodegenerative disorders (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS)). In one embodiment, the dendrimer complex is used to treat Alzheimer's disease (AD) or dementia.

[0149] Neurodegenerative diseases are chronic progressive disorders of the nervous system that affect neurological and behavioral functions, with biochemical changes that result in obvious histopathological and clinical syndromes (Hardy H, et al., Science. 1998;282:1075-9). Abnormal proteins that are resistant to cellular degradation mechanisms accumulate within cells. The pattern of neuronal loss is selective in the sense that one group is affected while the other remains intact. There is often no obvious precipitating event for the disease. Diseases classically described as neurodegenerative are Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0150] The composition and method can also be used to deliver drugs for the treatment of neurological or neurodegenerative disease or disorder or central nervous system disorder.In a preferred embodiment, the composition and method are effective in treating and / or reducing the neuroinflammation associated with neurological or neurodegenerative disease or disorder or central nervous system disorder.The method typically comprises administering to a subject an effective amount of the composition for increasing cognition or reducing cognitive decline, increasing cognitive function or reducing cognitive decline, enhancing memory or reducing memory decline, enhancing learning ability or capacity or reducing learning ability or capacity decline, or a combination thereof.

[0151] Neurodegeneration refers to the progressive loss of neuronal structure or function, including neuronal death. For example, the compositions and methods can be used to treat diseases or disorders such as Parkinson's disease (PD) and PD-related disorders, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and other dementias, prion diseases such as Creutzfeldt-Jakob disease, corticobasal degeneration, frontotemporal dementia, HIV-associated cognitive impairment, mild cognitive impairment, motor neuron disease (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Lewy body disease, Alpers' disease, neuronal ceroid lipofuscinosis, Batten disease, cerebro-oculo-facial-skeletal syndrome, corticobasal degeneration, Gerstmann-Sträussler-Schaefer syndrome, and others. It can be used to treat subjects with Inker's disease, Rickets, Leigh's disease, unilateral muscular atrophy, multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis (MS), Duchenne muscular dystrophy (DMD), neurodegeneration with brain iron accumulation, opsoclonus-myoclonus, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, vascular dementia, progressive multifocal leukoencephalopathy, dementia with Lewy bodies (DLB), lacunar syndrome, hydrocephalus, Wernicke-Korsakoff syndrome, post-encephalitic dementia, cognitive impairment and dementia associated with cancer and chemotherapy, and depression-induced dementia and pseudodementia.

[0152] In a preferred embodiment, the disease or disorder is spinal muscular atrophy.In such a case, HDAC inhibitor, antisense oligonucleotide (ASO) drug nusinersen or gene therapy drug ZOLGENSMA® can be conjugated to glucose dendrimer to deliver to neuron or neuron nucleus to treat spinal muscular atrophy.

[0153] In other embodiments, the disease or disorder is injection-limited amyloidosis, cerebral amyloid angiopathy, myopathy, neuropathy, brain trauma, frontotemporal dementia, Pick's disease, multiple sclerosis, prion disorder, type 2 diabetes, fatal familial insomnia, cardiac arrhythmia, isolated atrial amyloidosis, atherosclerosis, rheumatoid arthritis, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, systemic AL amyloidosis, neuropathic Gaucher disease, or Down's syndrome. In a preferred embodiment, the disease or disorder is Alzheimer's disease or dementia.

[0154] Criteria for evaluating improvement in a particular neurological factor include methods for evaluating cognitive skills, motor skills, memory capacity, etc., as well as methods for evaluating physical changes in selected areas of the central nervous system, such as magnetic resonance imaging (MRI) and computed tomography scanning (CT) or other imaging methods. Such evaluation methods are well known in the fields of medicine, neurology, psychology, etc., and can be appropriately selected to diagnose the state of a particular neurological disorder. A selected assessment or evaluation test, or multiple evaluation tests, is performed to evaluate changes in Alzheimer's disease or associated neurological changes, and then administration of the dendrimer composition is initiated. After this initial assessment, a treatment regimen for administering the dendrimer composition is initiated and continued for various time intervals. At selected time intervals following the initial assessment of the neurological deficit disorder, the same assessment or evaluation test(s) are used again to reassess the changes or improvements in the selected neurological criteria.

[0155] Alzheimer's disease and dementia The dendrimer composition is suitable for reducing or preventing one or more pathological processes associated with the onset and progression of neurological diseases, such as Alzheimer's disease and dementia.Therefore, a method is provided for treating, reducing, and preventing pathological processes associated with Alzheimer's disease, comprising administering the dendrimer composition in an amount and dosage regimen effective to reduce brain and / or serum exosomes, brain and / or serum ceramide levels, serum anti-ceramide IgG, glial activation, total Aβ42 and plaque load, tau phosphorylation / proliferation, and improved cognition in learning tasks, such as fear-conditioned learning tasks, in individuals suffering from Alzheimer's disease or dementia.A method is provided for reducing, preventing, or reversing learning and / or memory defects in individuals suffering from Alzheimer's disease or dementia.

[0156] In some embodiments, the dendrimer composition is administered in an amount and dosage regimen effective to induce neuroenhancement in a subject in need of administration. Neuroenhancement resulting from administration of the dendrimer composition includes stimulating or inducing neuronal mitosis resulting in the generation of new neurons, i.e., exhibiting a neurogenic effect, preventing or delaying neuronal loss, including reducing the rate of neuronal loss, i.e., exhibiting a neuroprotective effect, or one or more of these modes of action. The term "neuroprotective effect" includes preventing, delaying, and / or stopping the deterioration, damage, or death of an individual's neurons, neurites, and neural networks. Administration of the composition results in improvement or enhancement of neurological function in individuals with neurological disease, neurological injury, or age-related neuronal loss or damage.

[0157] Neuronal deterioration may be the result of any condition that impairs neuronal function, which may result in neuronal loss. Neuronal function may be impaired by, for example, changes in the biochemical, physiological, or anatomical structure of neurons, including their neurites. Neuronal deterioration may include membrane, dendritic, or synaptic changes, which are detrimental to normal neuronal function. The cause of neuronal deterioration, impairment, and / or death may be unknown. Alternatively, it may be the result of neurological changes associated with aging, injury, and / or disease that occur in the nervous system of an individual.

[0158] In Alzheimer's patients, neuronal loss is most prominent in the hippocampus, frontal cortex, parietal cortex, anterior temporal cortex, amygdala, and olfactory system. The most prominently affected zones of the hippocampus include the CA1 region, subiculum, and entorhinal cortex. Because the hippocampus is well known to play an important role in memory, memory loss is considered the earliest and most representative cognitive change.

[0159] Neuronal loss due to disease, age-related decline or physical injury leads to neurological disease and disorders.The composition can counteract the harmful effects of neuronal loss by promoting the development of new neurons, new neurites and / or neural connections, and provides neuroprotection of existing neurons, neurites and / or neural connections, or one or more of these processes.Therefore, the neurostrengthening properties of the composition provide an effective strategy for generally reversing the neuronal loss associated with degenerative disease, aging and physical injury or trauma.

[0160] Administration of the glucose dendrimer composition to an individual who is or has been affected by neuronal loss as a result of Alzheimer's disease reduces any one or more of the symptoms of Alzheimer's disease or related cognitive disorders, including dementia. Clinical symptoms of AD or dementia that can be treated, reduced or prevented include clinical symptoms of mild AD, moderate AD, and / or severe AD or dementia.

[0161] In mild Alzheimer's disease, a person may appear healthy, but will have increasing difficulty understanding the world around them. The realization that something is wrong often comes gradually to the individual and his / her family. Exemplary symptoms of mild Alzheimer's disease / mild dementia include memory loss; impaired judgment resulting in incorrect decisions; loss of initiative and independence; slower completion of normal daily tasks; repetitive questioning; difficulty handling money and paying bills; wandering and getting lost; losing things or leaving them in strange places; mood and personality changes, and increased anxiety and / or aggression.

[0162] Symptoms of moderate Alzheimer's disease / moderate dementia include: forgetfulness; increased memory loss and confusion; inability to learn new things; difficulty with language and problems with reading, writing and working with numbers; or difficulty organizing thoughts and reasoning; a shorter attention span; problems dealing with new situations; difficulty doing multi-step tasks, such as getting dressed; problems recognizing family and friends; hallucinations, delusions, and delusional disorders; impulsive behavior, such as undressing at inappropriate times or places or using vulgar language; inappropriate outbursts of anger; restlessness, agitation, anxiety, tearfulness, wandering (especially in the late afternoon or evening); repetitive speech or movements and occasional muscle twitching.

[0163] Symptoms of severe Alzheimer's disease / severe dementia include inability to communicate; weight loss; seizures; skin infections; difficulty swallowing; moaning, groaning, or growling; increased sleeping; and loss of bowel and bladder control.

[0164] Physiological symptoms of Alzheimer's disease / dementia include loss of brain mass, such as loss of hippocampal volume.Thus, in some embodiments, the method of administering the dendrimer composition increases the brain mass of the subject and / or reduces or prevents the rate of loss of brain mass compared to untreated control subjects; increases the hippocampal volume of the subject and reduces or prevents the rate of loss of hippocampal volume.

[0165] The dendrimer composition is administered to provide an effective amount of one or more therapeutic agents when administered to an individual. As used in this context, an "effective amount" of one or more therapeutic agents is an amount effective to improve or alleviate one or more symptoms associated with Alzheimer's disease or dementia, including neurological deficits or cognitive decline or impairment. Such therapeutic effects are generally observed within about 12 to about 24 weeks of initiating administration of a composition comprising an effective amount of one or more neuroenhancing agents, although therapeutic effects may be observed in less than 12 weeks or more than 24 weeks.

[0166] The individual is preferably an adult human, more preferably a human over the age of 30 who has lost some neurological function as a result of Alzheimer's disease or dementia. Generally, neuronal loss refers to any neuronal loss at the cellular level, including loss of neurites, neural mechanisms, or neural networks.

[0167] In other embodiments, the method includes selecting subjects who may benefit from treatment with the dendrimer composition. For example, the ceramide level in the patient's CSF is first determined and compared to that of a healthy control. In some embodiments, the dendrimer composition is administered to a patient who has a high ceramide concentration in CSF or serum compared to that of a healthy control. In other embodiments, the dendrimer composition is administered to a patient who has a high amount of brain and / or serum exosomes compared to that of a healthy control. In other embodiments, the dendrimer composition is administered to a patient who has a high level of serum anti-ceramide IgG compared to that of a healthy control.

[0168] In some embodiments, the subject has a nervous system disorder or is in need of neuroprotection. Exemplary conditions and / or subjects include, but are not limited to, subjects who have suffered or have suffered from stroke, traumatic brain injury, spinal cord injury, post-traumatic stress syndrome, or combinations thereof, or subjects who may suffer or have suffered from stroke, traumatic brain injury, spinal cord injury, post-traumatic stress syndrome, or combinations thereof.

[0169] In some embodiments, the compositions and methods are administered to a subject in need thereof in an amount effective to reduce or prevent one or more molecular or clinical symptoms of a neurodegenerative disease, or one or more mechanisms leading to neurodegeneration.

[0170] The drugs for treating neurodegenerative diseases are well known in the art and can be changed based on the symptoms and diseases to be treated.For example, the conventional treatment for Parkinson's disease can include levodopa (usually combined with dopa decarboxylase inhibitor or COMT inhibitor), dopamine agonist, or MAO-B inhibitor.

[0171] Treatments for Huntington's disease can include dopamine blockers to help reduce abnormal behavior and movement, or drugs such as amantadine and tetrabenazine to control movement. Other drugs that help reduce chorea include neuroleptics and benzodiazepines. Compounds such as amantadine or remacemide have shown preliminary good results. Hypokinesia and rigidity, especially in juvenile cases, can be treated with antiparkinsonian drugs, and myoclonic hyperkinesia can be treated with valproic acid. Psychiatric symptoms can be treated with medications similar to those used in the general population. Selective serotonin reuptake inhibitors and mirtazapine are recommended for depression, while atypical antipsychotics are recommended for psychosis and behavioral disorders.

[0172] The anti-excitotoxin drug riluzole (RILUTEK®) (2-amino-6-(trifluoromethoxy)benzothiazole) has resulted in improved survival in subjects with ALS. Other medications and interventions, mostly used off-label, may reduce symptoms resulting from ALS. Some treatments improve quality of life, and only a few treatments appear to be effective in extending lifespan. Common ALS-related therapies are reviewed in Gordon, Aging and Disease, 4(5):295-310 (2013), see, e.g., Table 1 therein. Numerous other drugs have been tested in one or more clinical trials, with efficacy ranging from ineffective to promising. Exemplary drugs are reviewed in Carlesi, et al., Archives Italiennes de Biologie, 149:151-167 (2011). For example, treatments include agents that reduce excitotoxicity, such as talampanel (8-methyl-7H-1,3-dioxolo(2,3)benzodiazepine), cephalosporins, such as ceftriaxone, or memantine; agents that reduce oxidative stress, such as coenzyme Q10, manganese porphyrin, KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride, RPPX], or edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186); agents that reduce apoptosis, such as These may include histone deacetylase (HDAC) inhibitors, including valproic acid, TCH346 (dibenzo(b,f)oxepin-10-ylmethyl-methylprop-2-ynylamine), minocycline, or tauroursodeoxycholic acid (TUDCA); agents that reduce neuroinflammation, such as thalidomide and cerlastol; neurotropic agents, such as insulin-like growth factor 1 (IGF-1) or vascular endothelial growth factor (VEGF); heat shock protein inducers, such as arimoclomol; or autophagy inducers, such as rapamycin or lithium.

[0173] Treatments for Lewy body dementia can include, for example, acetylcholinesterase inhibitors such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine; dopaminergic therapies such as levodopa or selegiline; antipsychotics such as olanzapine or clozapine; REM disorder therapies such as clonazepam, melatonin, or quetiapine; antidepressant and anti-anxiety therapies such as selective serotonin reuptake inhibitors (such as citalopram, escitalopram, sertraline, paroxetine) or serotonin and noradrenaline reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, for example, Macijauskiene, et al., Medicina (Kaunas), 48(1):1-8 (2012)).

[0174] Other common therapeutic, prophylactic or diagnostic agents for treating neurological dysfunction include amantadine and anticholinergics to treat motor symptoms, clozapine to treat psychosis, cholinesterase inhibitors to treat dementia, and modafinil to treat daytime sleepiness.

[0175] 3. Neurodevelopmental disorders Neurodevelopmental disorders generally suggest that the brain does not form normally from the beginning. Basic neurodevelopmental processes may be dysregulated or may be disrupted by injury, which may take a variety of forms. Autism and attention-deficit hyperactivity disorder are classically described as neurodevelopmental disorders.

[0176] Cerebral palsy (CP) is one of the most common childhood neurological / neurodevelopmental disorders, currently estimated to affect approximately 2 to 3 per 1,000 live births (Kirby, RS et al., Research in Developmental Disabilities, 32, 462 (2011)). CP is recognized in early infancy and the condition persists throughout life. The most common causes of CP include prematurity, hypoxia-ischemia and placental insufficiency, neonatal asphyxia and maternal-fetal inflammation (Dammann, O. Acta Paediatrica 2007, 96, 6; Yoon, BH et al., American Journal of Obstetrics and Gynecology 2000, 182, 675; and O'Shea, TM et al., Journal of child neurology 2012, 27, 22). Although the etiology of CP is heterogeneous and the disease mechanisms are highly complex, neuroinflammation is a common pathophysiological mechanism regardless of etiology. Targeting neuroinflammation and delivering drugs directly to the site of injury may be beneficial.

[0177] The compositions and methods can also be used to deliver therapeutic, prophylactic or diagnostic agents for treating neurodevelopmental disorders, such as cerebral palsy. In a preferred embodiment, the compositions and methods are effective in treating and / or reducing neuroinflammation associated with neurodevelopmental disorders, such as cerebral palsy.

[0178] In some embodiments, the dendrimer complex is effective for treating, imaging, and / or preventing brain inflammation in neurodevelopmental disorders, including Rett syndrome. In a preferred embodiment, the dendrimer complex is used to deliver anti-inflammatory agents (D-NAC) and anti-excitotoxic agents and D-anti-glutamic agents. Preferred candidates are MK801, memantine, 1-MT.

[0179] In some embodiments, the dendrimer complexes are effective in treating, imaging, and / or preventing brain inflammation in autism spectrum disorders. The term "spectrum" refers to the wide range of symptoms, skills, and levels of impairment or disability that children with ASD may have. Some children are mildly impaired by their symptoms, while others are severely impaired. Although characteristics of Asperger syndrome are included within the broad category of ASD, Asperger syndrome is no longer included in the latest edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5).

[0180] At this time, the only medications approved by the FDA to treat any aspect of ASD are the antipsychotics risperidone (Risperdal) and aliplipazole (Abilify). Some medications that may be prescribed off-label for children with ASD include:

[0181] Antipsychotic drugs are more commonly used to treat serious mental illnesses, such as schizophrenia. These medications may help reduce aggression and other serious behavioral disorders in children, including those with ASD. They may also help reduce repetitive behaviors, hyperactivity, and attention disorders.

[0182] Antidepressant medications, such as fluoxetine or sertraline, are usually prescribed to treat depression and anxiety, but are sometimes prescribed to reduce repetitive behaviors. Some antidepressants may also help control aggression and anxiety in children with ASD.

[0183] Stimulant medicines, such as methylphenidate (RITALIN®), are safe and effective in treating people with attention deficit hyperactivity disorder (ADHD).Methylphenidate has also been shown to effectively treat hyperactivity in children with ASD.However, many children with ASD do not respond to treatment, and those who do respond show more side effects than children with ADHD but not ASD.

[0184] 4. Brain tumors The compositions and methods should be useful for treating subjects with benign or malignant tumors by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth.In a preferred embodiment, the tumor treated is neuronal tumor and mixed neuronal-glial tumor.Neuronal tumor and mixed neuronal-glial tumor are rare tumor types that occur in the brain or spinal cord.In most cases, tumor is not cancerous (benign), but tumor can press on nearby brain tissue and cause disorders, such as seizures.

[0185] Thus, the glucose dendrimer conjugates may be administered in combination with one or more additional therapeutically active agents known to be capable of treating brain tumors or one or more symptoms associated therewith.

[0186] For example, the dendrimers may be administered to the brain via intravenous administration or during surgery to remove all or part of a tumor. The dendrimers may be used to deliver chemotherapy agents, agents to enhance adjuvant therapy, such as those in subjects who have been treated with radiation therapy, and the hydroxyl-terminated dendrimers are covalently linked to at least one radiosensitizer in an amount effective to suppress or inhibit the activity of DDX3 in proliferative disorders of the brain.

[0187] In addition to chemotherapy, surgical intervention and radiation therapy are also used in the treatment of cancer of the nervous system, as understood by those skilled in the art.Radiation therapy refers to administering ionizing radiation to the subject close to the location of cancer in the subject.In some embodiments, the radiosensitizer is administered in two or more doses, and then ionizing radiation is administered to the subject close to the location of cancer in the subject.In further embodiments, the administration of the radiosensitizer followed by the administration of ionizing radiation can be repeated for two or more cycles.

[0188] Typically, the dose of ionizing radiation will vary with the size and location of the tumor, but the dose will range from 0.1 Gy to about 30 Gy, preferably from 5 Gy to about 25 Gy.

[0189] In some embodiments, the ionizing radiation is in the form of stereotactic ablative radiation therapy (SABR) or stereotactic body radiation therapy (SBRT).

[0190] C. Dosage and Effective Amount Dosage and dosage regimen depend on the severity and location of the injury or damage, and / or the method of administration, and the therapeutic or prophylactic agent delivered. This can be determined by those skilled in the art. The therapeutically effective amount of the glucose dendrimer composition used in treating brain proliferative disease or disorder is typically sufficient to reduce or alleviate one or more symptoms of brain cancer and / or brain proliferative disorder. Typically, dosage will be in the range of micrograms / kg body weight up to about 100 mg / kg body weight.

[0191] Preferably, the therapeutic, prophylactic or diagnostic agent does not target or otherwise modulate the activity or amount of healthy cells that are not present in or associated with diseased / damaged tissue, or targets or otherwise modulates the activity or amount of healthy cells at a reduced level compared to cells associated with disease or disorder, such as cancer and / or proliferative disorder.In this way, the by-products and other side effects associated with the composition are reduced.Thus, in a preferred embodiment, the glucose dendrimer composition is administered in an amount that results in improved or enhanced function in individuals with disease or disorder, such as cancer and / or proliferative disorder.

[0192] The actual effective amount of the glucose dendrimer composition may vary according to factors including the specific agent administered, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the subject being treated. Generally, for intravenous injection or infusion, the dosage is lower than for oral administration.

[0193] Dosages may vary and may be administered daily for one or several days in one or multiple dose administrations. Guidelines can be found in the literature regarding appropriate dosages for a given class of pharmaceuticals. Optimal dosing schedules can be calculated from measurements of drug accumulation in the subject's or patient's body. Those skilled in the art can easily determine optimal dosages, dosing methodologies, and repetition rates. Optimal dosages may vary depending on the relative potency of individual pharmaceutical compositions and may be determined based on ECs that have been found to be effective in in vitro and in vivo animal models. 50 can be generally inferred based on

[0194] Dosage forms of pharmaceutical compositions comprising dendrimer compositions are also provided. "Dosage form" refers to the physical form of a dose of therapeutic compound, e.g., a capsule or vial, intended to be administered to a patient. The term "dosage unit" refers to the amount of therapeutic compound administered to a patient in a single dose.

[0195] In general, the timing and frequency of administration is adjusted to balance the effectiveness of a given treatment or diagnostic schedule and the side effects of a given delivery system.Exemplary dosing frequencies include continuous infusion, single and multiple administrations, such as hourly, daily, weekly, monthly, or yearly administrations.

[0196] In some embodiments, dosages are administered daily, biweekly, weekly, biweekly or less frequently in an amount that results in a therapeutically effective increase in blood levels of the therapeutic agent. If administration is by other than an oral route, the composition may be delivered over more than one hour, for example, 3-10 hours, to produce a therapeutically effective dose within 24 hours. Alternatively, the composition may be formulated for controlled release, and the composition is administered as a single dose that is repeated on a weekly or less frequent regimen.

[0197] It is understood by those skilled in the art that the dosing regimen can be any duration sufficient to treat the disorder in the subject.In some embodiments, the regimen comprises one or more cycles of a course of treatment followed by a rest period (e.g., drug-free).The rest period can be 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0198] In a preferred embodiment, the method for treating or preventing one or more symptoms of injury, disorder, or disease in the brain / CNS of a subject in need of such treatment or prevention comprises administering to the subject a formulation comprising a glucose dendrimer complexed to, covalently conjugated to, or intramolecularly dispersed or encapsulated within one or more therapeutic or prophylactic agents in an amount effective to treat or prevent one or more symptoms of injury, disorder, or disease in the brain / CNS of the subject. Those skilled in the art will appreciate that the dosing regimen relates to an amount and duration sufficient to treat injury, disorder, or disease in the brain / CNS to reduce one or more symptoms, such as swelling, pain, or seizures. The duration and amount of treatment administered is usually determined by a physician. Typically, the glucose dendrimer conjugates containing one or more therapeutic, prophylactic, or diagnostic agents are administered systemically and transported across the blood-brain barrier (BBB) ​​to enter the brain and are selectively taken up by damaged and / or diseased neurons. Typically, glucose dendrimer conjugates accumulate in neuronal nuclei and deliver therapeutic, prophylactic or diagnostic agents to these cells. The accumulation of glucose dendrimer conjugates in neurons is up to 100-fold higher than that of dendrimer conjugates that do not contain glucose-based monosaccharide branching units, such as PAMAM. Thus, in some embodiments, the effective amount of therapeutic, prophylactic or diagnostic agent required to treat or prevent injury, disorder or disease in the brain / CNS is up to 100 times less (100-fold less) than the amount required when the PAMAM dendrimer conjugate or therapeutic, prophylactic or diagnostic agent is used alone, for example, a quarter, a half, a fifth, a tenth, a twentieth, a thirtieth, a fortieth, a fiftieth, a sixtieth, a seventieth, a eightieth, a ninetieth, or a hundredth of the amount required when the PAMAM dendrimer conjugate or therapeutic, prophylactic or diagnostic agent is used alone.

[0199] D. Combination Treatments and Procedures The glucose dendrimer composition may be administered alone or in combination with one or more conventional therapies. In some embodiments, the conventional therapy includes administration of one or more of the compositions in combination with one or more additional therapeutic, preventive or diagnostic agents. The combination therapy may include administering the therapeutic, preventive or diagnostic agents together in the same mixture or as separate mixtures. Thus, in some embodiments, the pharmaceutical composition includes more than one therapeutic, preventive or diagnostic agent. Such formulations typically include an effective amount of the agent that targets the treatment site. The additional therapeutic, preventive or diagnostic agent(s) may have the same or different mechanisms of action. In some embodiments, the combination provides an additive effect on the treatment of a disease or condition. In some embodiments, the combination provides more than an additive effect on the treatment of a disease or disorder.

[0200] In some embodiments, the glucose dendrimer composition is administered prior to, together with, subsequent to, or alternating with treatment with one or more additional therapies or procedures. In some embodiments, the additional therapies are administered during a drug cycle or during a drug holiday that is part of the dosing regimen of the composition. For example, in some embodiments, the additional therapies or procedures are surgery, radiation therapy, or chemotherapy. Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder, or condition.

[0201] In the context of Alzheimer's disease, other therapeutic agents can include one or more of an acetylcholinesterase inhibitor (e.g., tacrine, rivastigmine, galantamine, or donepezil), a beta-secretase inhibitor, such as JNJ-54861911, an antibody, such as aducanumab, a 5-HT2A receptor agonist, such as pimavanserin, sargramostim, AADvac1, CAD106, CNP520, gantenerumab, solanezumab, and memantine.

[0202] In the context of Lewy body dementia, other therapeutic agents can include one or more of: acetylcholinesterase inhibitors, such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine; dopaminergic treatments, such as levodopa or selegiline; antipsychotics, such as olanzapine or clozapine; REM disorder treatments, such as clonazepam, melatonin, or quetiapine; antidepressant and anti-anxiety treatments, such as selective serotonin reuptake inhibitors (such as citalopram, escitalopram, sertraline, paroxetine) or serotonin and noradrenaline reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, e.g., Macijauskiene, et al., Medicina (Kaunas), 48(1):1-8 (2012)).

[0203] Exemplary neuroprotective agents are also known in the art and include, for example, glutamate antagonists, antioxidants, and NMDA receptor stimulants. Other neuroprotective agents and treatments include caspase inhibitors, trophic factors, anti-protein aggregation agents, therapeutic hypothermia, and erythropoietin.

[0204] Other common therapeutic, prophylactic or diagnostic agents for treating neurological dysfunction include amantadine and anticholinergics to treat motor symptoms, clozapine to treat psychosis, cholinesterase inhibitors to treat dementia, and modafinil to treat daytime sleepiness.

[0205] In the context of cancer treatment, other therapies include one or more of conventional chemotherapy, inhibition of checkpoint proteins, adoptive T cell therapy, radiation therapy, and surgical removal of the tumor.

[0206] E. Control The treatment results of the glucose dendrimer composition comprising one or more therapeutic, prophylactic or diagnostic agents can be compared to a control. Suitable controls are known in the art and include, for example, untreated subjects or placebo-treated subjects. A typical control is a comparison of the condition or symptom of the subject before and after administration of the glucose dendrimer composition. The condition or symptom can be a biochemical, molecular, physiological or pathological readout. For example, the effect of the composition on a particular symptom, pharmacological indicator, or physiological indicator can be compared to the condition of an untreated subject or the subject before treatment. In some embodiments, the symptom, pharmacological indicator, or physiological indicator is measured in the subject prior to treatment and measured again one or more times after treatment begins. In some embodiments, the control is a reference level or average determined based on the measurement of the symptom, pharmacological indicator, or physiological indicator in one or more subjects (e.g., healthy subjects) that do not have the disease or condition being treated. In some embodiments, the effect of the treatment is compared to a conventional treatment known in the art. In some embodiments, the untreated control subject suffers from the same disease or condition as the treated subject.

[0207] In some embodiments, the control comprises an equivalent amount of a therapeutic, prophylactic or diagnostic agent delivered alone or conjugated to a dendrimer of similar generation, molecular weight, and / or surface hydroxyl density that does not contain glucose-based branching units, such as a PAMAM dendrimer.

[0208] VI. Kit The composition can be packaged as a kit. The kit can include a single dose or multiple doses of a composition comprising one or more therapeutic, prophylactic or diagnostic agents encapsulated in, associated with or conjugated to a dendrimer (e.g., one or more glucose dendrimers as described in the Examples) and instructions for administering the composition. In particular, the instructions direct the administration of an effective amount of the dendrimer composition to an individual having a particular disease / disorder as indicated. The composition can be formulated as described above with reference to a particular treatment method and can be packaged in any convenient manner.

[0209] The invention will be further understood by reference to the following non-limiting examples. EXAMPLES

[0210] Example 1 Synthesis of glucose dendrimers material and method A.B. 4 Building Block Synthesis Synthesis of glucose-OAc-TEG-OT: A solution of peracetylated β-D-glucopyranoside (10 g, 25.6 mmol) was dissolved in 50 mL of anhydrous dichloromethane (DCM) followed by the addition of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.2 g, 17.9 mmol) and the reaction mixture was cooled to 0 °C. Boron trifluoride diethyl etherate (2.5 eq.) was added and the reaction was allowed to warm to room temperature. The reaction was monitored with the aid of TLC and quenched after 5 h by the addition of saturated sodium bicarbonate solution at 0 °C. After stirring for 10 min, DCM (300 mL) was added and the organic layer was washed three times with saturated sodium bicarbonate solution until the effervescence was quenched. The reaction mixture was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified by CombiFlash chromatography using ethyl acetate / hexane (70:30) mixture as eluent. The desired compound was obtained in 60% yield. The structure of Glucose-OAc-TEG-OT is shown below: [ka]

[0211] Glucose-OAc-TEG-N 3 Synthesis of Glucose-OAc-TEG-OT (6 g, 8.8 mmol) was dissolved in 40 mL of anhydrous DMF, followed by the addition of sodium azide (2 eq) and the reaction mixture was heated to 50° C. overnight. Upon completion, the reaction mixture was filtered and the DMF was evaporated. Once dry, the crude reaction mixture was passed through a Combi-Flash using ethyl acetate:hexane (70:30) as the eluent. Glucose-OAc-TEG-N 3 The structure of is shown below: [ka]

[0212] Synthesis of Glucose-OH-TEG-N3: Peracetylated β-D-glucopyranoside Tetraethylene glycol azide was dissolved in anhydrous methanol and sodium methoxide was added to adjust the pH to approximately 8.5-9. The reaction was stirred overnight at room temperature, then diluted with methanol and the pH was adjusted to approximately 6-7 with AMBERLIST® IR-120+. The reaction mixture was isolated by filtration and the solvent was removed by rotary evaporation. Glucose-OH-TEG-N 3 The structure is shown below. [ka]

[0213] Synthesis of D1-Glu6-OAc24 (compound 3a): The hexapropargylated compound (0.5 g, 1 mmol) and the azide derivative (1.2 eq. per (4.1 g, 7.4 mmol) acetylene) were suspended in a 1:1 mixture of DMF and water in a 20 mL microwave vial equipped with a magnetic stir bar. To this was added CuSO dissolved in a minimum amount of water. 4 5H 2 O (5 mol% / acetylene, 75 mg) and sodium ascorbate (5 mol% / acetylene, 60 mg) were added. The reaction was irradiated in a microwave at 50° C. for 6 h. The reaction mixture was dialyzed against DMF followed by water containing EDTA. EDTA was further removed by extensive water dialysis. The product was lyophilized to give D1-Glu6-OAc24. The structure of D1-Glu6-OAc24 is shown below. [ka]

[0214] Synthesis of D1-Glu6-OH24 (compound 3b): Peracetylated first generation glucose dendrimer (1 g, 0.26 mmol) was dissolved in anhydrous methanol and sodium methoxide was added to adjust the pH to approximately 8.5-9. The reaction was stirred overnight at room temperature, then diluted with methanol and the pH was adjusted to approximately 6-7 with AMBERLIST® IR-120+. The reaction mixture was separated by filtration and the solvent was removed by rotary evaporation, followed by water dialysis. D1-Glu 6 -OH 24 The structure is shown below. [ka]

[0215] Synthesis of D1-acetylene 24 (compound 4): D1-GLu6-OH24 (2 g, 0.721 mmol) was dissolved in anhydrous dimethylformamide (DMF, 50 mL) by sonication. To this stirred solution, sodium hydride [60% dispersion in mineral oil] (951 mg, 39.65 mmol) was added portionwise slowly at 0° C. The solution was further stirred at 0° C. for 15 min. Subsequently, propargyl bromide (3.85 mL, 34.608 mmol, 80% w / w solution in toluene) was added at 0° C. and stirring was continued at room temperature for another 6 h. The reaction mixture was quenched with ice and water, filtered, and dialyzed against DMF followed by water to give D1-acetylene 24. The structure of D1-acetylene 24 is shown below. [ka]

[0216] Synthesis of D2-Glu24-OAc96 (compound 5a): D1-acetylene dendrimer 24 (0.5 g, 0.13 mmol) and glucose-OAc-TEG-azide (2.2 g, 4 mmol) were suspended in a 1:1 mixture of DMF and water in a 20 mL microwave vial equipped with a magnetic stir bar. 4 5H 2O (5 mol% / acetylene, 5 mg) and sodium ascorbate (5 mol% / acetylene, 10 mg) were added. The reaction was irradiated in a microwave at 50° C. for 8 h. Upon completion, the reaction mixture was dialyzed against DMF followed by water containing EDTA. EDTA was further removed by extensive water dialysis. The product was lyophilized to give D2-Glu24-OAc96.

[0217] Synthesis of D2-Glu24-OH96 (compound 5b): Peracetylated second generation glucose dendrimer D2-Glu24-OH96 was dissolved in anhydrous methanol and sodium methoxide was added to adjust the pH to approximately 8.5-9.0. The reaction was stirred overnight at room temperature, then diluted with methanol and the pH was adjusted to approximately 6-7 with AMBERLIST® IR-120+. The reaction mixture was filtered to remove the resin and the filtrate was evaporated by rotary evaporation followed by dialysis against water to give the product as an off-white solid.

[0218] Synthesis of compound 6: D2-Glu24-OH96 (5b) (200 mg, 0.016 mmol) was dissolved in anhydrous dimethylformamide (DMF, 10 mL) by sonication. To this stirred solution, sodium hydride [60% dispersion in mineral oil] (22 mg, 0.934 mmol) was added portionwise slowly at 0° C. The solution was further stirred at 0° C. for 15 min. Subsequently, propargyl bromide (18.0 μL, 80% w / w solution in toluene) was added at 0° C. and stirring was continued at room temperature for another 6 h. The solvent was evaporated using a V10 evaporator system and the crude product was purified by passing through a PD10 Sephadex G25M column. The aqueous solution was lyophilized to give the product as an off-white solid.

[0219] Synthesis of Cy5-D2-Glu24-OH96 (compound 7): Compound 7 (200 mg, 0.016 mmol) and Cy5 azide (20.7 mg, 0.02 mmol) were suspended in a 1:1 mixture of DMF and water in a 25 mL round bottom flask equipped with a magnetic stir bar. To this was added CuSO dissolved in a minimum amount of water.4 5H2O (5 mol% / acetylene, 0.3 mg) and sodium ascorbate (10 mol% / acetylene, 0.5 mg) were added. The reaction was stirred at room temperature for 24 h. Upon completion, DMF was evaporated using a V10 and purification was carried out using a PD10 SEPHADEX® G25M column. The aqueous solution was lyophilized to give the product as a blue solid.

[0220] result Synthesis of glucose dendrimers The synthesis of glucose dendrimers was carried out using a hexapropargylated core (1) and AB-type dendrimer-based esters as described in Sharma, A. et al., Science Advances 2020, 6, (4), eaay8514; Sharma, R. et al., Chemical Communications 2014, 50(87), 13300-13303; and Sharma, R. et al., Polymer Chemistry 2015, 6(9), 1436-1444. 4 We started with the construction of the peracetylated β-glucose-PEG-azide building block. To construct the dendrimer, we used classical click reagents, catalytic amounts of copper sulfate pentahydrate, and sodium ascorbate to combine the hexapropargylated core (1) with the peracetylated β-glucose-PEG-azide building block. 4 -azide (2) was subjected to a CuAAC click reaction to give D1-GLU6-OAc24 (3a, Figure 1A). Treatment of the peracetylated dendrimer D1-GLU6-OAc24 (3a) under typical Zempren conditions afforded D1-GLU6-OH24 (3b). Complete acetic acid deprotection was then performed. 1 The terminal OH group in G1 dendrimer 3b was then modified using NaH and propargyl bromide to give D1-acetylene 24 (4). Successful propargylation was confirmed by the appearance of an alkyne peak corresponding to δ 2.4 ppm in NMR. The terminal alkyne group in dendrimer 4 was converted to peracetylated β-glucose-PEG by H NMR.4 -azide (2) to produce D2-GLU24-OAc96 (5a). 1 H NMR clearly confirmed the formation of the product with complete disappearance of 24 propargyl protons. Deprotection of acetate groups via Zempren reaction was performed to give the final dendrimer D2-GLU24-OH96 (5b). The disappearance of acetate peak in proton NMR confirmed the formation of the product. All intermediates and the final dendrimer were characterized via NMR, Mass and HPLC. The purity of D2-GLu24-OH96 is greater than 99% based on HPLC analysis. The size and zeta potential of the final dendrimer were measured using dynamic light scattering. It was demonstrated that D2-GLU24-OH96 has a size of 4.36 ± 0.38 nm and a nearly neutral zeta potential of -6.19 ± 0.56 mV. The physicochemical characterization of D2-GLU24-OH96 is shown in Table 1. Dendrimer D2-GLU24-OH96 is highly soluble in water / saline (>200 mg / mL).

[0221] [Table 1]

[0222] Synthesis of fluorescently labeled glucose dendrimers To evaluate the targeting ability of D2-GLU24-OH96 via confocal microscopy and fluorescence spectroscopy, the near-infrared dye cyanine 5 (Cy5) was conjugated to its surface. Two or three OH groups on the surface of D2-GLU24-OH96 (5b) were modified by reacting with propargyl bromide in the presence of sodium hydride to give compound 6 (Figure 1B), which was further reacted with Cy5-azide using a CuAAC click reaction to give fluorescently labeled Cy5-D2-GLU24-OH96 (7).

[0223] Dendrimer D2-GLU24-OH96 was highly stable in mouse and human plasma at physiological conditions for over 72 hours without any evidence of degradation via HPLC. Furthermore, liver and kidney extracts from mice (intranasal) and rabbits (intravenous) dosed with Cy5-D2-GLU24-OH96 showed intact dendrimer via HPLC 4 and 24 hours after dosing, demonstrating that the dendrimer is not degraded in vivo and is cleared intact.

[0224] Example 2 Synthesis of glucose dendrimer-drug conjugates 1) Glucose dendrimer-drug conjugates using copper-catalyzed alkyne-azide click (CuAAC) The synthesis of glucose dendrimer-drug conjugates begins with partial modification of OH groups to provide propargyl groups (Figure 2A). Meanwhile, the drug is modified using a releasable or non-releasable chemical bond to provide an azide end group via a polyethylene glycol linker. Some examples of glucose dendrimer-drug conjugates via CuAAC are described below.

[0225] i) Synthesis of glucose dendrimer-loperamide conjugates Procedure for the synthesis of glucose dendrimer-loperamide conjugate (Figure 2B): Loperamide hydrochloride was reacted with azide-PEG4-acid in the presence of DCC and DMAP in DCM at room temperature to give loperamide-PEG-azide. Meanwhile, partial modification of the OH groups of glucose dendrimer was carried out to give approximately 10 propargyl groups, which were reacted with loperamide-PEG-azide to give glucose dendrimer-loperamide conjugate. The structure confirmation and purity of the conjugate were confirmed by, respectively, 1 This is achieved by H NMR and HPLC.

[0226] ii) Synthesis of glucose dendrimer-rapamycin conjugates The synthesis of rapamycin-azide is achieved using Sharma, A. et al., Biomacromolecules 2020, 21, (12), 5148-5161.4. Meanwhile, partial modification of the OH groups of glucose dendrimer is carried out to provide about four propargyl groups, which are reacted with rapamycin-azide to obtain the glucose dendrimer-r conjugate (Figure 2C). The structural confirmation and purity of the conjugate are, respectively, 1 This is achieved by H NMR and HPLC.

[0227] iii) Synthesis of glucose dendrimer-valproic acid This was achieved by first attaching an enzyme-sensitive clickable linker to VPA (Figure 2D). The carboxylic acid groups of VPA (compound 8 in Figure 2D) were reacted with tetraethylene glycol azide to give VPA-azide (compound 9 in Figure 2D). Meanwhile, dendrimer D2-GLU24-OH96 was partially modified by reacting approximately 7–8 hydroxyl groups with hexynoic acid in the presence of coupling agents DCC, DMAP to give alkyne-terminated D2-acetylene 7 (10, Figure 2D), which was then converted to alkyne-terminated D2-acetylene 7 using a Cu(I)-catalyzed click (CuAAC) reaction with catalytic amounts of CuSO. 4 -5H 2 Further reaction with VPA-azide in the presence of O and sodium ascorbate yielded D2-VPA with an average of about 7–8 VPA molecules attached to the dendrimer surface (11, Figure 2D). Traces of copper were removed by dialysis against ethylenediaminetetraacetic acid (EDTA). The final D2-VPA conjugate was fully characterized by NMR and HPLC, with an HPLC purity of >98%. D2-VPA is highly stable at plasma conditions at pH (7.4) for up to 48 h, while the conjugate releases about 15% VPA in 1.5 h and about 33% in 48 h at intracellular conditions (pH 5.5 + esterase, Table 2), with the remainder released over time.

[0228] [Table 2]

[0229] 2) Glucose dendrimer conjugates using a combination of click and esterification / amidation reactions The synthesis is accomplished by partially modifying the OH groups of glucose dendrimers to give propargyl groups, which are reacted with linkers containing azide and amine termini to give surface amine groups (Figure 3A). Meanwhile, the drug is modified using a linker (hydrocarbon or PEG chains containing disulfide, ester, or amide bonds) with a carboxylic acid or -NHS ester terminus. The drug and dendrimer are reacted using an amidation reaction using coupling agents, e.g., EDC and DMAP.

[0230] i) Synthesis of glucose dendrimer-N-acetylcysteine ​​(NAC) conjugates: The synthesis of glucose dendrimer-NAC conjugates is achieved by partially modifying the OH groups of glucose dendrimers to give propargyl groups, which are reacted with linkers containing azide and amine termini to give surface amine groups (Figure 3B). On the other hand, SPDP-NAC is obtained by a published procedure. The glucose dendrimer-NAC conjugates are obtained by reacting NAC-SPDP and the dendrimer at pH 7.4.

[0231] 3) Glucose dendrimer conjugates using copper-free biorthogonal click chemistry Copper-free click reactions, e.g., TCO-triazine (Figure 4), strain-promoted azide-alkyne, Staudinger ligation, DBCO-azide click reactions, have been used to conjugate drugs, siRNA, peptides, mRNA, oligonucleotides, antibodies, and other biologics, where the presence of copper and reducing agents can reduce the efficacy of therapeutic, prophylactic or diagnostic agents.

[0232] Example 3 In vivo brain distribution of glucose dendrimers in a mouse stroke model Glucose dendrimers (GD) and PAMAM-GLU were further evaluated in a mouse seizure model to assess whether the specific colocalization with neurons could be extended to in vivo disease models. For this purpose, a pilocarpine-induced status epilepticus model was used. 5 μl of either GD or PAMAM-GLU (40 μg / μl) was injected into the right hemisphere of the brain using stereotaxic surgery. After delivery of the dendrimer, the mice were allowed to recover for 24 h. Pilocarpine was then administered (300 mg / kg, ip), resulting in behavioral seizures. Thirty minutes after the behavioral seizures, the mice were euthanized, perfused, fixed and histologically examined. Immunofluorescence detection of nuclei (DAPI), neurons (Thy-1 YFP), microglia (IBA1 antibody) and GD / PAMAM-GLU (Cy5-conjugate) facilitated colocalization studies, which were performed in the contralateral hemisphere to avoid the effects of mechanical brain injury induced by the stereotaxic injections. Both GD and PAMAM-GLU were able to target neurons and microglia in this seizure model. GD was mainly localized in the nuclei of neurons and microglia. However, significant levels of cytoplasmic GD were observed in microglia, unlike neurons. PAMAM-GLU was mainly distributed in the perinuclear cytoplasm of neurons and microglia.

[0233] These differences in GD and PAMAM-GLU distribution suggest that glucose dendrimers are novel and unique nanocarriers with the ability to translocate to the nucleus and may be a promising platform for targeting nuclear processes.

[0234] Example 4 In vivo distribution of glucose dendrimers in the retina of a mouse model of diabetic retinopathy (DR) Diabetes was induced in wild-type C57BL / 6J mice by daily intraperitoneal injection of 60 mg / kg streptozotocin (STZ) for 5 consecutive days. Blood glucose levels were measured 3 days after the last STZ injection, and if glucose levels were below 300 mg / dl, 3 more days of STZ treatment were administered. Animals were considered diabetic if blood glucose levels exceeded 350 mg / dL for 1 week. Four months (16–17 weeks) after diabetes induction, 20 μg of glucose dendrimer containing 1 μl (or 1 μl of sterile saline as control) was injected into the vitreous cavity of each mouse eye using a 30-gauge needle with a Hamilton syringe, and eyes were harvested 72 hours later. At the time of harvest, each retina was dissected from the eye and immediately processed for whole-mount preparation to visualize en face the innermost retinal layer, which is composed mainly of neuronal cells forming the optic nerve, i.e., retinal ganglion cell (RGC) bodies and their axons. Immunofluorescence staining of retinal whole mounts demonstrated the presence of Cy5-labeled GD in Tuj-positive RGCs, indicating robust neuronal uptake. Furthermore, the pattern of intracellular uptake clearly suggests nuclear colocalization of GD in RGCs. GD uptake was also observed in Iba1-labeled microglia, which are mostly present in deep synaptic layers of the retina but become activated in the DR.

[0235] Example 5 In vivo distribution of glucose dendrimers in the retina of a mouse model of oxygen-induced retinopathy (OIR) From the 7th to the 12th day after birth, the patient was kept in a high-oxygen environment (75% O 2Neonatal C57BL / 6J mice exposed to 100% erythrocytes (1:100, ... Z-stack images were processed using Imaris to construct 3D rendering images to demonstrate co-localization in microglia and ganglion cells. Tile-Z-stack images demonstrate that glucose dendrimers target and co-localize with both retinal neuronal cells (ganglion cells and bipolar cells) and retinal microglia / macrophages, confirming that they are distinct from our PAMAM dendrimers, which target and co-localize with activated microglia / macrophages in this model. Exposure to hyperoxia leads to activation of microglia and their distribution in all layers of the retina, whereas in normal retinas, microglia are only found in the outer retina.

[0236] Example 6 Glucose dendrimers are taken up by glutamate-injured neurons in primary neuronal cell cultures Primary neuronal cultures were incubated with 10 μM glutamate and 10 μg / ml dendrimer-Cy5 for 24 h. Cultures were then stained with an anti-tubulin antibody (tuj1) to identify neurons. Confocal images were analyzed for the presence of Cy5-dendrimer.

[0237] After 24 h of treatment, the second generation glucose dendrimer, Cy5-dendrimer, showed significant accumulation in neurons, with negligible hydroxyl PAMAM-OH uptake in neurons in this in vitro glutamate injury model.

[0238] Example 7 Intracranial administration of glucose dendrimers, but not hydroxyl PAMAM-OH dendrimers, targets neurons in the CA1 hippocampal pyramidal region in a mouse pilocarpine seizure model PAMAM-OH-Cy5 or GD2-Cy5 (50 μg / μl in 4 μl) was injected intracranially, followed by seizure induction 24 hours later with 300 mg / kg pilocarpine IP. Thirty minutes after inducing seizures (Racine scale 3 seizures), mice were sacrificed and brains were perfused for immunohistochemistry. The second generation glucose dendrimer (GD2) showed significant uptake in contralateral CA1 neurons. Meanwhile, PAMAM-OH uptake was negligible in contralateral CA1 neurons, but showed mostly diffuse microglial uptake in the ipsilateral side.

[0239] Neuronal fluorescence in contralateral CA1 neurons was more than 100-fold higher than that of the hydroxyl PAMAM dendrimer (Figure 5). In healthy animals (control mice without induced seizures), contralateral CA1 neurons did not show GD2 colocalization, indicating no neuronal uptake in these neurons.

[0240] Example 8 GD2 uptake is dependent on neuronal activity and GLUT transporters method 300 μm cortical brain sections were then frozen in Mg 2 + Free artificial cerebrospinal fluid (ACSF) (which increases neuronal firing) or Mg 2 + / ACSF containing NMDG (to suppress neuronal activity) or Mg containing cytochalasin B (5 μM) or glutol (10 μM) or phlorizin (10 μM)2 +-free ACSF for 30 min. After pretreatment, brain sections were incubated with GD2-Cy5 (10 μg / ml) for 30 min, followed by fixation in formalin and confocal imaging. The mean fluorescence intensity for GD-Cy5 in YFP-expressing cortical neurons was assessed. Confocal images of fixed sections showing DAPI (nuclei), YFP (neurons) and differently treated GD2-Cy5 were taken and analyzed.

[0241] result High metabolic activity by injured / hyperactive neurons may increase the need for glucose and thus the uptake of second generation glucose dendrimers (GD2). Therefore, we hypothesized that suppression of neuronal activity would inhibit GD2 dendrimer uptake. Indeed, N-methyl-D-glucamine (a substitute for NaCl) and high MgCl, which are known to suppress neuronal activity, were used to treat GD2 dendrimer uptake. 2 A significant decrease in GD2 colocalization in neurons was observed when neurons were incubated with a buffer solution containing 5 mM Glucocorticoid (Dribben, WH et al., Cell Death & Disease 2010, 1 (8), e63-e63;Stanojevic, M. et al., Journal of Elementology 2016, 21 (1), 221-230) (Figures 6A and 6B). Furthermore, blocking glucose transporters (GLUTs) using two different pharmacological antagonists: cytochalasin B (nonspecific GLUT inhibitor) and glutol (GLUT1-3 inhibitor) or blocking SGLT1 and 2 by phlorizin reduced GD2 uptake by neurons, suggesting the involvement of glucose transporters in the uptake (Figure 6B).

[0242] Example 9 GD2 targets selected neurons in acute brain slices ex vivo in a rabbit model of cerebral palsy When acute hippocampal brain slices from neonatal rabbits with cerebral palsy were incubated with GD2-Cy5 (20 μg / ml) in artificial cerebrospinal fluid (ACSF) for 45 minutes, GD2 was taken up as evidenced by confocal images (n=3 juvenile rabbits). Confocal images show the CA1 pyramidal neuronal layer (stained with PGP) with GD2-Cy5 accumulation in selected neurons. This model demonstrates delayed neuronal damage after in utero endotoxin insult (Balakrishnan, B. et al., Developmental neuroscience 2013, 35 (5), 396-405; and Kannan, S. et al., Sci Transl Med 2012, 4 (130), 130ra46-130ra46). Similar neuronal uptake has been seen in vivo in a juvenile rabbit model of controlled cortical impact-induced traumatic brain injury. This suggests that neuronal targeting occurs independent of injury mechanism and species.

[0243] Example 10 GD2 localizes in neurons upon intranasal delivery in a mouse model of pilocarpine-induced seizures Second generation glucose dendrimers labeled with Cy5 (GD2-Cy5) were administered intranasally (100 μg in 10 μl) after IP injection of 300 mg / kg pilocarpine. After 4 hours, mice dosed with GD2-Cy5 were perfused and fixed. Confocal images show the intensity of Cy5 localized in the olfactory bulb, cortex and neuronal layer of the hippocampal CA1 region, which are known to be affected by pilocarpine. This indicates that intranasal administration is a viable option for delivering glucose dendrimers to the brain.

[0244] Example 11 Treatment with GD2-VPA conjugate reduces the severity of seizures induced by pilocarpine injection Seizures were induced using pilocarpine. After visual verification of active seizures (Racine scale 3 and above), 100 μg of GD2-VPA (in 10 μl saline containing approximately 0.3 mg / kg VPA) was administered intranasally (15 min after pilocarpine injection). 1 μl of saline or GD2-VPA was administered into each nostril every 2 min. After seizure induction, GD2-VPA-treated mice showed a fast recovery. The characteristic loss of posture and tail stiffening during seizures were relatively lower in GD2-VPA-treated mice compared to saline-treated mice. Mice treated with GD2-VPA showed improved locomotion and activity earlier than saline-treated animals. When analyzed 2 h after intranasal drug administration, GD2-VPA-treated mice showed minimal splaying and tail stiffening. For GD2-VPA mice, improved motility was observed 1 hour after surgical induction when compared with saline and VPA treated mice.

[0245] In a pilot study, intranasal GD2-VPA treatment acutely reduced the frequency of spike-wave discharges and prevented seizure events following pilocarpine administration. Wireless electroencephalography (EEG) recording devices were implanted in adult mice (25-30 g) and allowed to recover for 3 days. Seizures were induced with pilocarpine and EEG was recorded simultaneously. Five-minute EEG recordings immediately before and 15 minutes after intranasal administration of saline or GD2-VPA were analyzed for electrographic events. Spike-wave discharges were increased in count and mean amplitude in saline-treated animals, whereas both count and mean amplitude of spike-wave depolarizations were decreased in mice treated with intranasal GD2-VPA at the same time. Twenty-four hours later, mice were administered a second dose of pilocarpine, after which EEG was recorded for 3 hours. Saline-treated mice showed seizure events with high frequency spikes lasting more than 150 seconds, whereas GD2-VPA-treated animals did not develop any seizure events at the same time. This indicates that a single dose of GD2-VPA demonstrated a sustained effect after 24 hours.

[0246] In summary, these data demonstrate that glucose dendrimers are primarily localized to neurons and that uptake appears to be mediated by glucose transporter(s). Furthermore, treatment with intranasal GD2-VPA leads to improved seizure frequency and motor function in the acute phase, indicating that this is a powerful platform for delivering drugs specifically to neurons. Experimental data show that glucose dendrimers are primarily taken up by injured neurons, unlike hydroxyl PAMAM dendrimers, which target "activated" microglia. Previously published studies have shown that hydroxyl PAMAM-OH dendrimers do not target neurons, but primarily target activated microglia / macrophages in the injured area in multiple models (Iezzi, R. et al., Biomaterials 2012, 33 (3), 979-988;Kambhampati, SP et al., Investigative ophthalmology & visual science 2015, 56 (8), 4413-4424;Kannan, S. et al., Science Translational Medicine 2012, 4 (130), 130ra46;Khoury, ES et al., Theranostics 2020, 10 (13), 5736-5748;Liaw, K. et al., Bioengineering & translational medicine 2021, 6 (2), e10205;Mishra, MK et al., ACS nano 2014, 8 (3), 2134-2147;Sharma, A. et al., Biomacromolecules 2020, 21 (9), 3909-3922;Sharma, A. et al., Theranostics 2018, 8 (20), 5529-5547;Sharma, R. et al., Journal of Controlled Release 2020, 323, 361-375).Hydroxyl PAMAM dendrimers are used as a "control" to establish the differential cell targeting of the two types of dendrimers.

[0247] When administered intranasally, both glucose and hydroxyl PAMAM dendrimers are transported to the brain, consistent with previous findings. However, in healthy brains, there is no uptake or retention at 8-24 hours. This suggests that the affinity of glucose dendrimers to GLUT / SGLT in healthy brain cells is not strong enough to allow uptake compared to their rapid diffusion rate. In contrast, in the presence of seizure-induced activity, cerebral palsy, or traumatic brain injury, increased activity of glucose transporters on injured neurons allows specific uptake and retention. This is unlikely to be a size or surface hydroxyl effect, as hydroxyl PAMAM dendrimers show no uptake in neurons or microglia in animals experiencing seizures. If hydroxyl PAMAM also shows uptake, then increased "nonspecific endocytosis" in activated neurons is also unlikely to be the reason for uptake. The presence of surface glucose in GD is important.

[0248] Example 12 Glucose dendrimers for targeted drug delivery to hyperactive neurons material and method GD2 and sodium-valproate (VPA).

[0249] Release studies of GD2-VPA conjugates under intracellular conditions pH 7.4: stable conjugate shows no release of VPA for up to 24 hours. Intracellular conditions: rapid release, about 15% release in 1-2 hours, 25% release in 24 hours.

[0250] mouse Thy1-YFP and wild-type C57BL / 6 mice were purchased from Jackson Laboratoeis and subsequently bred in the animal facility and housed under a 12-h light and 12-h dark cycle. Thy1-YFP mice were used to perform in vivo dendrimer localization studies following protocols approved by the Johns Hopkins University Animal Care and Use Committee (IACUC). Thy1-YFP mice were also used for acute brain slice experiments. Wild-type C57BL / 6 mice were used to perform pilocarpine-induced seizure studies as described in Arshad, Bio-protocol 10 (2020). Scopolamine methyl nitrate (2 mg / kg, Sigma-Aldrich S2250) was injected intraperitoneally, followed 30 min later by pilocarpine hydrochloride (ip; Sigma-Aldrich P6503, 300 mg / kg). Behavioral seizures were monitored and scored using a modified Racine scale (MN Arshad, JR Naegele, Bio-protocol 10 (2020)).

[0251] Isolation and culture of primary neurons from young rabbit brain tissue Primary hippocampal neuronal cultures were used in this study using standard procedures. Briefly, brain hippocampi from postnatal day 1 juvenile rabbits were microdissected, followed by removal of blood vessels and meninges in ice-cold dissection solution containing 1x Hank's balanced salt solution, 1x penicillin / streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 30 mM glucose. The hippocampi were then minced and digested using a papain dissociation kit according to the manufacturer's protocol (Worthington, USA). The digested tissue in the buffer was triturated with a sterile flame-polished glass pipette to separate tissue clumps and cells, and then centrifuged at 300 g for 5 min at 4 °C. The pellet was resuspended in Eagle's balanced salt solution containing ovomucoid protease inhibitor and bovine serum albumin and deoxyribonuclease. A discontinuous density gradient was then prepared by pipetting the cell suspension into a 5 ml layer of albumin-ovomucoid inhibitor solution and centrifuging at 70 g for 6 min at room temperature to remove the supernatant containing non-cellular debris. The resulting cell pellet was resuspended in neurobasal medium supplemented with 1× GlutaMAX, 2% B27, 1% penicillin-streptomycin, and 1% heat-inactivated horse serum. 50,000 cells were seeded on poly-D-lysine- and laminin-coated coverslips and incubated at 37°C. After 24 h, the medium was replaced with fresh medium containing 5 μM cytosine arabinoside. Half of the culture medium was changed weekly.

[0252] For immunocytochemistry, neuronal cultures were washed with PBS and fixed with formalin for 10 min, followed by additional PBS washes. Cultures were then blocked with 10% donkey serum for 30 min and then incubated overnight with anti-beta III tubulin antibody (1:1000, Abcam, MA.USA). When culture coverslips were washed, they were incubated with alexa fluorescent conjugated secondary antibody (donkey anti-rabbit AF488, 1:250) for 1 h. Coverslips were then treated with DAPI (1:5000) for 5 min, washed, and mounted.

[0253] Animal Models of Cerebral Palsy Pregnant New Zealand White rabbits (Robinson Services Inc) underwent laparotomy surgery on day 28 of gestation according to a protocol approved by the Johns Hopkins University Animal Care and Use Committee (IACUC). Briefly, during laparotomy, rabbits were injected along the uterine wall with a total of 1800 EU (endotoxin units) of LPS (Escherichia coli serotype O127:B8, Sigma-Aldrich, St. Louis, MO) (S. Kannan et al., Science translational medicine vol.4, 130ra146(2012)). Rabbits were induced G30 using an intravenous injection of Pitocin (0.5U / kg) (JHP Pharmaceuticals, Rochester, MI) and sacrificed for live brain slice experiments on the day of birth.

[0254] Behavioral seizures For efficacy studies, a single dose of pilocarpine 300 mg / Kg ip was used to induce status epilepticus (SE). Prior to inducing seizures, scopolamine methyl nitrate (2 mg / kg, Sigma-Aldrich S2250) was injected intraperitoneally to antagonize the peripheral effects of pilocarpine. Animals were videotaped in their individual cages and seizure activity was scored according to the modified Racine scale as follows (K. Borges et al., Experimental neurology 182, 21-34 (2003);CJ Mueller, et al, Experimental neurology vol. 219, 284-297 (2009)): Stage 0: Normal activity; Stage 1: Freezing, slight head tilt, Stage 3: Head shaking, wet dog shake, tail raising, Stage 4: Partial myoclonus, occasional jerks, body tremors, increased freezing or uncontrollable circling movements, Stage 4: Increased immobility and freezing, uncontrollable circling movements, Stage 5: Continuous tail raising, loss of limb control, followed by generalized tonic-clonic seizures, one episode of oral-gastrointestinal motility or rearing, Stage 6: Loss of balance, more than one episode of rearing followed by occasional falls, jumping and rolling, generalized tonic extension of the body, cardiopulmonary collapse and death. Continuous behavioral seizures were scored for 180 min.

[0255] Stages 1 and 2 were classified as low-grade seizures, stage 3 as intermediate-grade seizures, and stages 5-6 as high-grade seizures. Each episode lasted at least 30 seconds and typically varied in duration. A course between stages 3-6 was considered as continuous low-grade seizures when observed for 180 minutes in these animals. Repeated sufficiently long continuous seizures of intermediate grade or above, with short intervals (at least one such seizure within 5 minutes), were considered as the onset of status epilepticus (H. Shibley, BN Smith, Epilepsy research 49, 109-120 (2002);E. Trinka et al., Epilepsia 56, 1515-1523 (2015)). Behavioral seizures were monitored and scored using the revised Racine scale (MN Arshad, JR Naegele, Bio-protocol 10 (2020)).

[0256] Preparation of ex vivo brain slices CP juvenile rabbits or Thy1-YFP mice were deeply anesthetized with isoflurane and decapitated. The brains were removed and stored in oxygenated (95% O) bronchodilators. 2 / 5%CO 2 ), ice-cold N-methyl-D-glucamine (NMDG)-based buffer (Buffer 1, in mM: 92 NMDG, 2.5 KCl, 10 MgSO 4 , 0.5 CaCl 2 , 1.2 NaH 2 PO 4 , 30 NaHCO 3, 25 glucose, 20 HEPES, 5 sodium ascorbate, 3 sodium pyruvate, 2 thiourea; pH 7.4). Coronal brain sections (300 μM) were then obtained using a vibratome (VT1200, Leica). The sections were first incubated in the NMDG-based solution for 10 min at 34 °C and then transferred into the same solution and kept at room temperature for 1 h before starting the subsequent experiments. Dendrimer-Cy5 was dissolved in 5 ml of buffer 2 (in mM: 125 NaCl, 2.5 KCl, 1 MgCl) containing the brain sections. 2 , 2 CaCl 2 , 1.25 NaH 2 PO 4 , 26NaHCO 3 , 20 Glucose; pH 7.4) and incubated for 30 min while oxygenating at room temperature. Brain sections were fixed in formalin, immunostained for neuronal (MAP2 or PGP) proteins, and visualized under an upright microscope equipped with laser scanning confocal optics (LSM880, Zeiss).

[0257] Immunohistochemistry For immunohistochemistry, animals were perfused transcardially with saline and postfixed in 10% formalin for 24 h, followed by cryoprotection in 30% sucrose solution for 2 days. Coronal sections (30 μm, 1:6 serial) were taken and blocked by 5% normal donkey serum in 0.1 M PBS. Sections were then incubated with goat anti-IBA1 (1:500, Abcam, MA.USA) overnight at 4 °C, followed by incubation with secondary antibody for 2 h at room temperature. After incubation with DAPI (1:1000, Invitrogen) for 15 min, slides were washed, dried and coverslipped with mounting medium. Similarly, for acute CP brain section immunostaining, sections were fixed overnight, washed and incubated with primary antibody anti-PGP antibody (1:100, Abcam, MA.USA), followed by incubation in secondary antibody solution for 2 h. Confocal images were acquired with a Zeiss ZEN LSM 710 (Zeiss, CA, USA) and processed with ZEN software.

[0258] statistics Data were summarized using mean ± SEM, and for between-group comparisons, either t-tests or one-way-ANOVA were used. Where appropriate, Bonferroni correction was used to adjust for multiple comparisons. Analyses were performed using GraphPad Prism software. Statistical significance was set as P<0.05, and all tests were two-sided.

[0259] result Synthesis and characterization of glucose dendrimers (GD) Using a highly efficient click chemistry approach, a synthetic scheme was designed and standardized for the preparation of second-generation glucose dendrimers (GD2) (Figure 7). This glucose dendrimer platform was validated in approximately 1 g quantities. The synthesis of GD2 was accomplished in a rapid manner and began by reacting a hexapropargylated core (1) with an AB4β-D-glucose-PEG4-azide building block (2) via click reaction to give the first-generation glucose dendrimers (GD1, 3, Figure 7). The hexapropargylated core (1) and AB4β-D-glucose-PEG4-azide (2) building blocks were synthesized using protocols by Sharma et al., Sci Adv 6, eaay8514 (2020);A. Sharma et al., Biomacromolecules 21, 5148-5161 (2020);R. Sharma et al., Biomacromolecules 22, 3574-3589 (2021). The hydroxyl groups of GD1 were further propargylated to give GD1-acetylene 24 (4), which was reacted again with AB4β-D-glucose-PEG4-azide (2) to give the second generation glucose dendrimer (GD2, 5) bearing 24 glucose molecules containing 96 surface hydroxyl groups. Near-infrared fluorescent tag Cy5 was attached to GD2 by propargylating approximately 2–3 hydroxyl groups to give alkyne-containing dendrimer (6), which was further reacted with Cy5-azide to give fluorescently labeled GD2-Cy5 (7). The dendrimer was purified using tangential flow filtration technique to give a highly pure product. The final dendrimer and intermediates were characterized using 1H and 13C NMR for structure and HPLC for purity. The physicochemical properties of GD2 are shown in Table 1. The HPLC purity of the final dendrimer was greater than 99%. GD2 is highly soluble (>200 mg / mL) in water / saline. GD2 was highly stable in mouse and human plasma at physiological conditions for 72 h and showed no evidence of degradation via HPLC.

[0260] GD2 was highly stable in mouse and human plasma for at least 72 hours at demonstrated physiological conditions and did not show any signs of degradation (HPLC). Furthermore, liver and kidney extracts from mice (intranasally) and rabbits (intravenously) dosed with GD2 showed intact dendrimers via HPLC 4 and 24 hours after dosing, demonstrating that the dendrimers are not degraded in vivo and are cleared intact.

[0261] Neuronal uptake of glucose dendrimers (GD2) in in-vitro primary cultures and ex-vivo brain slices Glutamate excitotoxicity is ubiquitous in many pathological brain conditions. To mimic similar injury conditions, primary rabbit neuronal cells were cultured and exposed to 10 μM glutamate for 24 h, which results in neuronal excitotoxicity. Simultaneous exposure to 10 μg / ml dendrimer for 24 h demonstrated significant accumulation of GD2 in neurons. Sister neuronal cultures exposed to 10 μM glutamate and 10 μg / ml PAMAM-OH showed negligible accumulation of PAMAM-OH dendrimer in neurons in this in-vitro glutamate injury model. The data demonstrate intraneuronal uptake of GD2 in an in-vitro glutamate injury model. Briefly, 3-4 week old primary neuronal cultures were incubated with 10 μM glutamate and 10 μg / ml dendrimer-Cy5 for 24 h. Cultures were then immunostained with an anti-tubulin antibody to identify neurons. Confocal images were acquired and analyzed for the presence of dendrimers in the Cy5-channel (633-666 nm). The data showed that GD2 was localized in pyramidal neurons, whereas PAMAM-OH was not.

[0262] Unique neuronal targeting propensity of GD2 in ex vivo acute brain slices from neonatal rabbits with brain injury caused by maternal systemic LPS-induced inflammation (rabbit model of cerebral palsy). This ex vivo brain slice model offers a unique advantage over primary neuronal cultures because it largely preserves the intrinsic synaptic organization of neurons. When acute hippocampal brain slices from neonatal rabbits with cerebral palsy were incubated with GD2-Cy5 (20 μg / ml) for 45 min. Treated acute brain slices were formalin fixed (10% formalin) and immunostained for neuronal markers: ubiquitin carboxy-terminal hydrolase L1 (PGP) or MAP2. Confocal microscopy images showed the abundance of GD2-Cy5 in hippocampal pyramidal neurons (n=3 juvenile rabbits) (data not shown). Significant levels of GD2-Cy5 were observed in the CA1 pyramidal cell layer when hippocampal slices were incubated with GD2-Cy5 (20 μg / ml) in standard artificial cerebrospinal fluid (ACSF) for 45 minutes. These data demonstrate that GD2 targets selected CA1 neurons in acute hippocampal brain slices taken from a rabbit model of cerebral palsy.

[0263] In vivo GD2-Cy5 uptake in neurons depends on neuronal hyperactivity. Intracranial injection of Cy5-conjugated PAMAM-OH or GD2 (50 μg / μl in 4 μl) followed by seizure induction 24 h later with 300 mg / kg pilocarpine IP promoted GD2 uptake by contralateral CA1 neurons. PAMAM-OH uptake was negligible in contralateral CA1 neurons. Thirty minutes after seizure induction (Racine scale 3 seizures), mice were sacrificed and brains were perfused for immunohistochemistry. These data demonstrate the feasibility of the GD2-dendrimer platform for specific delivery of drugs to hyperactive neurons.

[0264] Glucose transporters mediate GD2-Cy5 uptake High metabolic activity by injured / hyperactive neurons may increase the need for glucose and therefore increase GD2 uptake. We hypothesized that suppression of neuronal activity would inhibit GD2 dendrimer uptake. We found a significant decrease in GD2 colocalization in neurons when we treated them with N-methyl-D-glucamine (a substitute for NaCl) and high MgCl, which is known to suppress neuronal activity. 2 Blockade of glucose transporters (GLUTs) using two different pharmacological antagonists, cytochalasin B (a nonspecific GLUT inhibitor) and glutol (a GLUT1-3 inhibitor), reduced GD2 uptake by neurons, indicating the involvement of GLUT-dependent uptake.

[0265] GD2 localizes in neurons upon intranasal delivery in a mouse model of pilocarpine-induced seizures Cy5-labeled glucose dendrimers (GD2-Cy5) were administered intranasally (100 μg in 10 μl) after IP injection of 300 mg / kg pilocarpine. After 4 hours, mice dosed with GD2-Cy5 were perfused and fixed. Confocal images show that Cy5 intensity was localized in the olfactory bulb, cortex, and both neuronal layers of the hippocampal CA1 region. These data indicate that GD2-Cy5 localizes in the olfactory bulb and CA1 neuronal cell layer upon intranasal administration. This indicates that intranasal administration is a viable option for delivering GD2 to the brain.

[0266] Synthesis and validation of GD2-VPA conjugates Motivated by the neuronal targeting by GD2-Cy5, valproate (VPA) was conjugated to the GD2 dendrimer (Figure 7). The synthesis of GD2-VPA was achieved by first attaching an enzyme-sensitive clickable linker to VPA (Figure 7). The carboxylic acid group of VPA (8) was reacted with tetraethylene glycol azide to give VPA-azide (9). Meanwhile, GD2 was partially modified by reacting approximately 7–8 hydroxyl groups with hexynoic acid in the presence of coupling agents DCC, DMAP to give alkyne-terminated GD2-acetylene 7 (10), which was then converted to alkyne-terminated GD2-acetylene 7 using a Cu(I)-catalyzed click (CuAAC) reaction with a catalytic amount of CuSO. 4 -5H 2 Further reaction with VPA-azide in the presence of O and sodium ascorbate afforded GD2-VPA (11) with an average of about 7–8 VPA molecules attached to the dendrimer surface (Figure 7). Click chemistry makes the synthesis facile and robust, thus providing good control over synthesis and ligand loading. Traces of copper were removed by dialysis with ethylenediaminetetraacetic acid (EDTA). The final GD2-VPA conjugate was fully characterized by NMR and HPLC, with an HPLC purity of more than 98%. GD2-VPA is highly stable at plasma conditions at pH (7.4), while the conjugate releases about 15% of VPA in 1.5 h and about 25% in 24 h at intracellular conditions (pH 5.5 + esterase, Figure 7).

[0267] GD2-VPA protects against pilocarpine-induced behavioral seizures To evaluate the efficacy of GD2-VPA, a pilocarpine mouse model of status epilepticus was used. Pilocarpine is a potent muscarinic agonist and can produce continuous behavioral and electrographic seizures. Pilocarpine IP injection (300 mg / kg) was administered, followed by visual verification of active seizures (Racine scale 3 and above) before intranasal administration of GD2-VPA (0.3 mg / kg VPA base) (15 min after pilocarpine injection). 1–2 μl of saline or GD-VPA solution (10 μg / μl) was administered into each nostril every 2 min. Mice treated with saline showed increased postural broadening and tail stiffening compared to GD2 VPA 1 h after pilocarpine treatment. Furthermore, exploratory behavior was significantly increased with GD2-VPA treatment (Figure 8C). GD2-VPA prolonged the latency to the first episode of both intermediate- and high-grade seizures after pilocarpine administration and reduced the total duration of high-grade seizures.

[0268] Uptake of GD2-Cy5 dendrimers by selected neurons (Syngap mouse seizure model) Experiments were performed as shown in the experimental timeline in Figure 12A. The modified Racine scale was used as shown in Table 3 below. Category 3-5 seizures were typically of 15-90 s duration and were separated by periods of relative inactivity or by other phases of differing duration. Periods between category 3-6 seizure events were usually marked by consecutive category 1 and 2 type seizure activity. Mice that experienced at least 3 categories of 3-6 seizure events within 2 h after pilocarpine injection were considered to have experienced SE (Shibley et al, 2002).

[0269] [Table 3]

[0270] result The results are shown in Figures 12B-12I. Figures 12B-12I show the uptake of GD2-Cy5 dendrimers by selected neurons (Syngap mouse seizure model). Figures 12B and 12C are bar graphs of the seizure duration scores and the latency to high grade seizures on day 1. Figures 12D-12F are bar graphs showing the seizure duration scores for low grade seizures (Figure 12D), medium grade seizures (Figure 12E), and high grade seizures (Figure 12F). Figures 12G-12I are bar graphs showing the seizure duration and the latency to high grade seizures on day 2 for low grade seizures (Figure 12G), medium grade seizures (Figure 12H), and high grade seizures (Figure 12I).

[0271] Using different models of increasing complexity, it was demonstrated that GD2-Cy5 is internalized in neurons under hyperactive conditions. Pharmacological blocker experiments suggest that glucose dendrimers are predominantly localized in neurons and that uptake is likely mediated by glucose transporters. Furthermore, treatment with intranasal GD2-VPA resulted in improved seizure frequency and motor function in the acute phase, indicating that this is a powerful platform for delivering drugs specifically to neurons.

[0272] Neurons maintain a negative membrane potential in the resting state and temporarily depolarize and repolarize during action potential activation (Alle, et al., Science 325, 1405-1408 (2009)). Transient membrane potential fluctuations also occur during subthreshold synaptic neurotransmission (Harris, et al., Neuron 75, 762-777 (2012)). Protecting neuronal membrane polarization is an active process that requires cellular ATP, which is required for action potential generation, restoration of ion concentrations, or vesicle recycling (H. Alle, et al., Science 325, 1405-1408 (2009);JJ Harris, et al., Neuron 75, 762-777 (2012)). Upon neuronal stimulation, glucose can be directly transported into cells via glucose transporters (LK Bak et al., J. Neurochem. 109, 87-93 (2009);Diaz-Garcia et al., Cell metabolism 26, 361-374. e364 (2017);Lundgaard et al., Nat Commun 6, 6807 (2015)) unlike neuron-astrocyte lactate shuttle used during resting state glucose metabolism (Yellen, J. Cell Biol. 217, 2235-2246 (2018)). In neurons, glucose can bypass glycolysis and pass through the pentose phosphate pathway to reduce oxidative stress (A. Herrero-Mendez et al., Nature Cell Biology 11, 747-752 (2009)). Increased activity and expression of neuronal GLUT transporters during neuronal activity can result in the binding of GD2 to the transporters.Based on molecular simulation studies, GD2 cannot pass through the GLUT3 transporter due to steric hindrance (DS Dwyer, Proteins: Structure, Function, and Bioinformatics 42, 531-541 (2001)). However, GD2 in close proximity to the neuronal membrane through GLUT receptor interactions can promote internalization of GD2-Cy5 through other mechanisms.

[0273] GLUT3 transporters are expressed in neuronal dendrites and axons (BS McEwen, LP Reagan, Eur J Pharmacol 490, 13-24 (2004)). During acute brain injury, synaptic neurotransmission is increased, making synaptic vesicle recycling more necessary. In the presence of nearby GD2, nanoparticles can be internalized via synaptic vesicle recycling (SO Rizzoli, The EMBO journal 33, 788-822 (2014)).

[0274] Previous neuron-targeting inorganic nanoparticles were limited to surface interactions and were not internalized (Dante et al., ACS nano 11, 6630-6640 (2017)). However, GD2 can internalize into the cytoplasm and target intracellular organelles and macromolecules. The selectivity of GD2 to target only neurons in brain-injury conditions, e.g., a seizure mouse model, is demonstrated in Figures 8A and 8B, and does not accumulate in healthy neurons, providing much-needed selectivity in the development of neurotherapeutics. Overall, a dendrimer that inherently targets hyperactive neurons and localizes intracellularly has been developed in this study.

[0275] In summary, the data demonstrate that Cy5-labeled glucose dendrimers (GD2-Cy5) localize in hyperexcitable neurons in vitro and in vivo, (2) glucose transporters regulate neuronal GD2 uptake, and (3) intranasally delivered glucose dendrimer-valproate conjugates (GD2-VPA) significantly reduce seizure severity in a mouse model of pilocarpine-induced epilepsy.

[0276] Modifications and variations of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims. All references cited herein are incorporated by reference.

Claims

1. (a) central core and (b) One or more branched units, which are glucose-based branched units, wherein the glucose may be mono, di, or oligosaccharide. A branch unit having a linker conjugated to it as needed; (c) One or more therapeutic, prophylactic or diagnostic agents and A glucose dendrimer containing, A glucose dendrimer in which one or more branched units are conjugated to the central core, and the surface groups of the dendrimer contain monosaccharide glucose molecules.

2. The glucose dendrimer according to claim 1, wherein the central core is dipentaerythritol or a hexapropargyl derivative thereof.

3. The glucose dendrimer according to claim 1, wherein the branched units are conjugated to the central core via linkers selected from hydrocarbons and oligoethylene glycol chains.

4. The branched unit is a β-D-glucopyranoside tetraethylene glycol azide having the following structure, 【Chemistry 17】 The glucose dendrimer according to claim 1, or a peracetylated derivative thereof.

5. The glucose dendrimer according to claim 1, wherein the dendrimer is a first-generation, second-generation, third-generation, fourth-generation, fifth-generation, or sixth-generation dendrimer.

6. The aforementioned dendrimer has the following structure: [Chemistry 18] The glucose dendrimer according to claim 1, which is a first-generation dendrimer having

7. The aforementioned dendrimer has the following structure: 【Chemistry 19】 The glucose dendrimer according to claim 1, which is a second-generation dendrimer having the following characteristics.

8. The glucose dendrimer according to claim 1, wherein the one or more therapeutic, prophylactic, or diagnostic agents encapsulated, associated, and / or conjugated in the dendrimer are selected from the group consisting of therapeutic agents, prophylactic agents, and diagnostic agents.

9. The glucose dendrimer according to claim 1, wherein the one or more prophylactic, therapeutic, and / or diagnostic agents encapsulated, associated, and / or conjugated in the dendrimer are concentrated in a concentration of about 0.01% to about 30% by weight, preferably about 1% to about 20% by weight, and more preferably about 5% to about 20% by weight.

10. The aforementioned dendrimer Small molecules, antibodies or their antigen-binding fragments, nucleic acids, and polypeptides The glucose dendrimer according to claim 1, conjugated with one or more therapeutic, prophylactic, or diagnostic agents selected from the group consisting of the following.

11. The glucose dendrimer according to claim 8, wherein the therapeutic agent is selected from the group consisting of anti-inflammatory agents, antioxidants, and immunomodulators.

12. The glucose dendrimer according to claim 8, wherein the diagnostic agent is selected from the group consisting of fluorescent dyes, near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes.

13. The glucose dendrimer according to claim 1, comprising one or more linkers or coupling agents between the dendrimer and the therapeutic, prophylactic, or diagnostic agent.

14. The glucose dendrimer according to claim 13, wherein the one or more linkers or coupling agents between the dendrimer and the therapeutic, prophylactic, or diagnostic agent are one or more hydrocarbons or oligoethylene glycol chains.

15. The glucose dendrimer according to claim 13, wherein the therapeutic, prophylactic, or diagnostic agent is conjugated to the dendrimer via one or more bonds selected from the group consisting of disulfide, ester, ether, thioester, and amide bonds.

16. A pharmaceutical formulation comprising a dendrimer according to any one of claims 1 to 11 and 13 to 15 and a pharmaceutically acceptable carrier or excipient.

17. The pharmaceutical formulation according to claim 16, wherein the formulation is formulated for systemic administration.

18. The pharmaceutical formulation according to claim 16, wherein the formulation is formulated for enteral or parenteral administration.

19. The pharmaceutical formulation according to claim 16, wherein the formulation is formulated for intramuscular, intraperitoneal, intravenous, or subcutaneous injection.

20. A pharmaceutical formulation according to claim 16 for treating or preventing one or more diseases, conditions, and / or injuries of the eye, brain and / or nervous system (CNS) in a subject requiring treatment or prevention of such diseases, conditions, and / or injuries of the eye, brain and / or nervous system (CNS).

21. The pharmaceutical formulation according to claim 20, wherein the one or more diseases, conditions, and / or injuries of the eye, the brain, and / or the nervous system are diseases, conditions, and injuries related to neurons and / or activated microglia.

22. The pharmaceutical formulation according to claim 20, wherein the one or more diseases, conditions, and / or injuries of the eye are ocular diseases related to retinal ganglion cells, selected from the group consisting of glaucoma, diabetic retinopathy, acute retinal ischemia, traumatic optic nerve injury, optic nerve atrophy, and Leber hereditary optic neuropathy.

23. The pharmaceutical formulation according to claim 22, wherein the one or more therapeutic agents encapsulated, associated, and / or conjugated in the dendrimer are selected from the group consisting of ROCK inhibitors, α-2 adrenergic receptor agonists, and caspase inhibitors.

24. The pharmaceutical formulation according to claim 20, wherein the one or more diseases, conditions, and / or injuries of the brain and / or nervous system are neurological and / or neurodegenerative diseases selected from the group consisting of traumatic brain injury, demyelinating disease, epilepsy, neuralgia, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, cerebral palsy, autism, multiple sclerosis, spinal muscular atrophy, neuronal ceroid lipofuscinosis, and neuropathic Gaucher disease.

25. The pharmaceutical formulation according to claim 24, wherein the one or more therapeutic agents encapsulated, associated, and / or conjugated in the dendrimer are selected from the group consisting of calpain inhibitors, GPR52 antagonists, NMDA antagonists, mTOR inhibitors, LLRK2 inhibitors, nuclear factor erythroid 2-related factor 2 activators, and SMN-2 promoters.

26. The pharmaceutical formulation according to claim 20, wherein the one or more diseases, conditions, and / or injuries of the brain and / or nervous system are neurological disorders related to motor neurons.

27. The pharmaceutical preparation according to claim 26, wherein the neurological disorder is a motor neuron disorder selected from the group consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, and Kennedy disease.

28. The pharmaceutical preparation according to claim 26, wherein the neurological disorder is spinal muscular atrophy.

29. The pharmaceutical formulation according to claim 28, wherein the one or more therapeutic agents encapsulated, associated, and / or conjugated in the dendrimer are HDAC inhibitors or antisense oligonucleotides.

30. The pharmaceutical formulation according to claim 29, wherein the antisense oligonucleotide is nusinersene.

31. The pharmaceutical formulation according to claim 20, characterized in that the dendrimer formulation is administered orally, intravenously, intraperitoneally, or intravitreously.

32. The pharmaceutical formulation according to claim 20, wherein the amount of a therapeutic, prophylactic, or diagnostic agent effective in treating or preventing one or more of the aforementioned symptoms is less than the amount of the same therapeutic, prophylactic, or diagnostic agent administered in the absence of the glucose dendrimer or as a formulation combined with the dendrimer in the absence of surface glucose molecules.

33. A pharmaceutical formulation comprising the dendrimer according to claim 12 and a pharmaceutically acceptable carrier or excipient.

34. The pharmaceutical formulation according to claim 33, wherein the formulation is formulated for systemic administration.

35. The pharmaceutical formulation according to claim 33, wherein the formulation is formulated for oral administration, intravenous administration, or intraperitoneal administration.

36. A pharmaceutical formulation according to any one of claims 33 to 35 for labeling one or more neurons and / or activated microglia associated with one or more diseases, conditions, and / or injuries of the eye, brain and / or nervous system (CNS) in a subject, A pharmaceutical formulation characterized in that the formulation is administered in an amount effective to label one or more cells associated with one or more diseases, conditions, and / or injuries of the eye, the brain, and / or the nervous system (CNS).

37. The pharmaceutical formulation according to claim 36, wherein the labeling is used to diagnose or identify one or more diseases, conditions, and / or injuries of the eye, brain and / or nervous system (CNS) in the subject.

38. The pharmaceutical formulation according to claim 36, wherein the labeling is used to monitor or guide treatment and / or surgical procedures.

39. The pharmaceutical formulation according to claim 36, characterized in that the dendrimer formulation is administered orally, intravenously, intraperitoneally, or intravitreously.

40. A pharmaceutical formulation comprising a dendrimer according to any one of claims 1 to 11 and 13 to 15 and a pharmaceutically acceptable carrier or excipient for delivering one or more therapeutic, prophylactic or diagnostic agents to one or more neurons of a subject that requires delivery of one or more therapeutic, prophylactic or diagnostic agents to one or more neurons, or a pharmaceutical formulation according to any one of claims 33 to 35.

41. The pharmaceutical formulation according to claim 40, wherein the one or more neurons are selected from the group consisting of cerebral cortical neurons, motor neurons, dopaminergic neurons, hypothalamic neurons, thalamic neurons, brainstem neurons, raphe nucleus neurons, Purkinje neurons, retinal ganglion cells, and other neurons in the central nervous system.

42. The pharmaceutical formulation according to claim 40, wherein the amount of the one or more therapeutic, prophylactic, or diagnostic agents accumulated in the one or more neurons is at least 5, 10, 20, 30, 40, or 50 times greater than the amount of the same therapeutic, prophylactic, or diagnostic agent administered in the absence of the dendrimer or administered as a formulation combined with the dendrimer in the absence of surface glucose molecules.

43. The pharmaceutical preparation according to claim 40, characterized in that the preparation is administered systemically to the subject.

44. The pharmaceutical preparation according to claim 43, characterized in that the preparation is administered orally, intravenously, intraperitoneally, or intravitreously.

45. A method for producing a dendrimer having high-density surface glucose groups, (a) Preparing a hypercore by propargylating a central core, wherein the central core contains two or more reactive groups for propargylation; (b) A first hypermonomer AB is obtained from the branched unit having (n+1) reactive groups by conjugating protecting groups to n of the reactive groups of the branched unit and conjugating one azide group to one of the reactive groups of the branched unit. ４ The preparation of a solution, where n is equal to or greater than 2; (c) Mix the hypercore and hypermonomer for copper(I) catalyzed alkyne azido click chemistry to obtain a first-generation dendrimer. A method that includes this.

46. (d) Propargylation of the first generation dendrimer; (e) Conjugate protecting groups to n of the reactive groups of the branched unit and conjugate one azide group to one of the reactive groups of the branched unit, thereby obtaining a second hypermonomer AB from the branched unit having (n+1) reactive groups. ４ The preparation of a solution, where n is equal to or greater than 2; (f) Mix the propargylated first-generation dendrimer from step (d) with the second hypermonomer from step (e) for copper(I)-catalyzed alkyne azido-click chemistry to obtain a second-generation dendrimer. The method according to claim 45, further comprising:

47. The method according to claim 45 or 46, wherein the central core is dipentaerythritol or a derivative thereof.

48. First hypermonomer AB ４ and the second hypermonomer AB ４ However, the structure is as follows: 【Chemistry 20】 The method according to claim 45 or 46, wherein the peracetylated β-D-glucopyranoside tetraethylene glycol azide has the following properties.

49. The method according to claim 45 or 46, further comprising the step of deprotecting one or more functional groups of the dendrimer.

50. The method according to claim 49, wherein one or more functional groups of the dendrimer are hydroxyl groups.

51. The method according to claim 45 or 46, wherein the dendrimer is further complexed and / or conjugated with one or more therapeutic, prophylactic, and / or diagnostic agents.

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