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WO2025193538A1 - Dendrimer compositions for targeted delivery of gcpii inhibitors - Google Patents

Dendrimer compositions for targeted delivery of gcpii inhibitors

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Publication number
WO2025193538A1
WO2025193538A1 PCT/US2025/018902 US2025018902W WO2025193538A1 WO 2025193538 A1 WO2025193538 A1 WO 2025193538A1 US 2025018902 W US2025018902 W US 2025018902W WO 2025193538 A1 WO2025193538 A1 WO 2025193538A1
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WO
WIPO (PCT)
Prior art keywords
dendrimer
generation
gcpii
dendrimers
inhibitor
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PCT/US2025/018902
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French (fr)
Inventor
Barbara Slusher
Yu Su
Kannan Rangaramanujam
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Johns Hopkins University
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Johns Hopkins University
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Publication of WO2025193538A1 publication Critical patent/WO2025193538A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • Sarcopenia has been defined as an age related, involuntary loss of skeletal muscle mass and strength. Beginning as early as the 4 th decade of life, evidence suggests that skeletal muscle mass and skeletal muscle strength decline in a linear fashion, with up to 50% of mass being lost by the 8 th decade of life. Age-related loss of muscle mass and strength (sarcopenia) significantly impairs quality of life in the elderly yet lacks effective treatments. Sarcopenia has a multifactorial cause, with declines in activity and nutrition, disease states, inflammation, degeneration or deterioration in neuromuscular junctions, and aging related changes in mitochondria, apoptosis, and the angiotensin system recently found to be contributory.
  • the primary symptoms of sarcopenia include low muscle function, low muscle strength, and low muscle mass.
  • the loss of muscle mass is accompanied by the accumulation of fat in the muscles, which contributes to decreased muscle strength.
  • the total estimated cost of hospitalizations in individuals with sarcopenia was USD $40.4 billion with an average per person cost of USD $260.
  • the only treatments for sarcopenia include nutritional therapy and exercise therapy.
  • An intervention combining nutritional therapy with a comprehensive exercise program that includes resistance training is more effective than a single intervention in improving sarcopenia, however there remains a significant need for effective therapeutic interventions that do not rely upon physical or nutritional activity. It is an object of the present invention to provide compositions for the effective treatment of sarcopenia. 45718416.1 It is also an object of the present invention to provide effective treatment regimens reducing or preventing one or more symptoms of sarcopenia. SUMMARY OF THE INVENTION It has been discovered that GCPII inhibitors can be covalently conjugated to hydroxylated dendrimers to produce therapeutic and/or prophylactic agents to reduce muscle function decline and/or decrease deterioration of neuromuscular junctions.
  • Methods for treating or preventing one or more symptoms associated with sarcopenia in a subject in need thereof include administering to a subject a pharmaceutically acceptable composition including hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor.
  • GCPII glutamate-carboxypeptidase-II
  • This is formulated into a dosage form to provide an effective amount for administration to a human for reducing, delaying, inhibiting or reversing one or more symptoms associated with sarcopenia in the subject as compared to an untreated control subject.
  • the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to reduce glutamate and/or increase NAAG in muscle macrophage and/or around neuromuscular junctions (NMJ) in the subject.
  • the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is provided in an amount effective to reduce the amount of one or more markers of sarcopenia in the subject.
  • markers are selected from follistatin (FST), adiponectin, leptin, decrease interleukin-6 (IL-6), and tumor necrosis factor ⁇ (TNF- ⁇ ).
  • the methods are effective to increase myostatin (MSTN), decrease follistatin (FST), increase irisin, increase brain-derived neurotrophic factor (BDNF), decrease adiponectin, decrease leptin, increase insulin-like growth factor-1 (IGF-1); increase dehydroepiandrosterone sulphate (DHEAS), decrease C-reactive protein (CRP); decrease interleukin-6 (IL-6), or decrease tumor necrosis factor ⁇ (TNF- ⁇ ) in the subject, or combinations thereof.
  • the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to increase serum creatinine to serum cystatin C ratio in the subject.
  • Exemplary symptoms of sarcopenia that can be treated or prevented by the conjugate include lack of strength, such as lack of hand-grip or limb strength, need for assistance in walking, difficulty rising from a chair, difficulty climbing stairs, increased number of falls, reduced muscle mass, reduced balance, and reduced muscular control.
  • the subject has a score of 4 or more on the SARC-F scale.
  • Exemplary subjects are at least 40 years of age, 50 years of age, 60 years of age, or 70 years of age.
  • the subject does not have and/or has never been diagnosed as having one or more diseases or disorders selected from Alzheimer’s disease (AZ), Parkinson’s disease (PD) amyotrophic lateral sclerosis (ALS), dementia, Schizophrenia, multiple sclerosis, and depression.
  • the subject has, or is identified as being at risk of having, 45718416.1 one or more diseases or disorders selected from type II diabetes mellitus, albuminuria, sarcopenic obesity, sarcopenic arterial stiffness, and peripheral neuropathy.
  • diseases or disorders selected from type II diabetes mellitus, albuminuria, sarcopenic obesity, sarcopenic arterial stiffness, and peripheral neuropathy.
  • Methods for reducing, preventing or delating neuromuscular junction (NMJ) denervation or deterioration in a subject are also provided.
  • NMJ neuromuscular junction
  • the methods include administering to a human subject a pharmaceutically acceptable composition including hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor in an effective amount for reducing, delaying, inhibiting or reversing glutamate and/or increasing NAAG from muscle macrophage and/or around neuromuscular junctions (NMJ) in the subject.
  • a pharmaceutically acceptable composition including hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor in an effective amount for reducing, delaying, inhibiting or reversing glutamate and/or increasing NAAG from muscle macrophage and/or around neuromuscular junctions (NMJ) in the subject.
  • the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor that is administered to the subject is effective to provide binding of the GCPII inhibitor to the GCPII enzyme on the surface of or inside target cells.
  • target cells include neuron
  • the dendrimer includes a generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, or generation 8 dendrimer.
  • the dendrimer is a poly(amidoamine) (PAMAM) dendrimer, preferably having greater than 40% or 50% free hydroxyl surface groups.
  • the dendrimer is a generation 4 PAMAM dendrimer.
  • the dendrimer includes one or more surface-bound monosaccharides. Exemplary monosaccharides include glucose, galactose, glucosamine, galactose, mannose, and fructose. In some forms the dendrimer includes ten or more surface monosaccharide moieties.
  • the dendrimer is linked to the GCPII inhibitor via a spacer.
  • the spacer includes a cleavable linkage to the dendrimer.
  • An exemplary cleavable linkage is selected from the group including ester, disulfide, phosphodiester, tri-glycyl peptide, and hydrazine linkages.
  • the spacer includes a non-cleavable linkage to the dendrimer.
  • An exemplary non-cleavable linkage is selected from the group including amide, ether, and amino alkyl linkages.
  • the spacer linking the dendrimer and the GCPII inhibitor includes a hydrocarbon such as an alkylene, a diethylene glycol moiety, and/or oligoethylene glycol chain.
  • An exemplary spacer includes a triazole moiety.
  • Exemplary GCPII inhibitors include 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA), ZJ-43, ZJ-11, ZJ-17, ZJ-38, VA-033, quisqualic acid, 2- [[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (GPI-5232), 2- (3-mercaptopropyl)pentanedioic acid (2-MPPA), 3-(2-carboxy-5-mercaptopentyl)benzoic acid, 3- (1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid, N- substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, phenylalkylphosphonamidates, 45718416.1 NAAG peptide analogs, and glutamate derivatives.
  • the GCPII inhibitor includes 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA).
  • the composition provides sustained release of the GCPII-dendrimer conjugate to yield an effective amount in the subject for a period of 24 hours.
  • the composition is formulated for systemic administration.
  • the composition is formulated for enteral or parenteral administration.
  • Exemplary compositions are formulated for oral, mucosal (intranasal, buccal, rectal, vaginal, sublingual, pulmonary), intramuscular, intravenous, or subcutaneous.
  • the composition is in a form such as a hydrogel, nanoparticles or microparticles, suspensions, powders, tablets, capsules, or solutions.
  • FIG 1 is a schematic depicting a synthetic route for producing an exemplary GCPII inhibitor conjugate with an enzyme sensitive ester linkage, showing conjugation of GCPII inhibitor, 2-PMPA (1), to a cleavable PEG linker in presence of Azido-PEG-11-alcohol (EDC/DMAP/DMF for 24 hours at room temperature) to form 2-PMP-PEG-N3(2).
  • FIG 2 is a schematic showing conjugation of hydroxylated (OH) dendrimer (d) (D-OH; (3)) to the 2-PMP-PEG-N3 of FIG.1, by first conjugating a 56-hexynoic acid to (3) in presence of EDC, DMAP, DMF for 24 hours at room temperature to form D-hexyne (4), then click chemistry-based conjugation of (4) with (2) in the presence of CuSO4.5H2O Na Ascorbate (DMF/THF/H2O for 6 hours at 50 ⁇ C in a microwave) to form D-2-PMPA (5), having a cleavable ester linkage (indicated).
  • DMF/THF/H2O CuSO4.5H2O Na Ascorbate
  • FIGs 3A-3D are graphs of the comparative IC50s determined using human recombinant GCPII, showing %inhibition (-25%-125%) over concentration (Log M) for each of 2-PMPA alone (Fig.3A), 2-PMPA-PEG-Azide (Fig.3B), D-2-PMPA conjugate (Fig.3C), and D-OH alone (Fig. 3D), respectively.
  • the structures of each respective molecule used in the assay, as well as the molecular weight are depicted at left of each graph, and the calculated IC50 value for each molecule is indicated under each curve, respectively.
  • FIG 4 is a histogram of GCPII enzyme activity in mice muscle CD11b+ macrophage cells, showing GCPII Activity (0-600 fmol/mg/h) for each of young, old vehicle (controls) and Old D-2- PMPA, samples, respectively.
  • Aged mice muscle showed increased GCPII activity specifically in CD11b+ enriched macrophage cells (138.6 ⁇ 3.0 vs.336.6 ⁇ 52.9; p ⁇ 0.05), but not in CD11b- cells.
  • Systemic D-2-PMPA therapy (20 mg/kg 2-PMPA equivalent; IP 3 ⁇ /week) completely inhibited the elevated GCPII activity (336.5 ⁇ 52.8 vs.21.8 ⁇ 9.2 fmol/mg/h; p ⁇ 0.001).
  • FIG 5 is a graph of mice muscle volume preservation after 5 months’ treatment, showing 20-month calf muscle volume relative to 15 month old (%) in each of groups treated with D- 2PMPA, and vehicle (control), respectively.5-months of D-2-PMPA therapy initiated with 15- 45718416.1 month-old mice led to significant preservation of calf muscle volume (95.0 ⁇ 0.8% vs.90.4 ⁇ 0.7%; p ⁇ 0.001).
  • FIGs 6A-6B are graphs of mice muscle isometric force (Stim Frequency at 80 Hz), showing isometric force (0-400 mN) for each of groups treated with D-2-PMPA or vehicle (control), respectively, based on assays in female (Fig.6A) and male (Fig.6B) test animals, respectively.
  • FIG 7 is a graph of mice all limb grip strength preservation after 5 months’ treatment, showing 20-month all limb grip strength relative to 15 month old (%) in each of groups treated with D-2PMPA, and vehicle (control), respectively.5-months of D-2-PMPA therapy initiated with 15- month-old mice led to significant preservation of calf muscle volume (95.0 ⁇ 0.8% vs.90.4 ⁇ 0.7%; p ⁇ 0.001).
  • FIGs 8A-8B are graphs of mice rotarod latency after 5 months’ treatment, showing rotarod latency (0-200 seconds) for each of groups treated with D-2-PMPA or vehicle (control), respectively, based on assays in female (Fig.8A) and male (Fig.8B) test animals, respectively.
  • D-2- PMPA enhanced rotarod latency (i.e., from 119.0 ⁇ 6.6 vs.93.5 ⁇ 9.3 s, female p ⁇ 0.05; and 104.5 ⁇ 5.2 vs.79.3 ⁇ 4.2 s, male p ⁇ 0.001).
  • FIGs 9A-9B are graphs depicting improved compound muscle action potential (CMAP) latency, showing CMAP latency (1.25-2.00 ms) (Fig.9A) and CMAP amplitude voltage (0-20 mV) (Fig.9B), respectively, for each of groups treated with D-2-PMPA or vehicle (control), respectively.
  • FIG 10 is a graph of single fiber EMG (Jitter) of gastrocnemius (GTN) muscle after 5 months’ treatment, showing mean consecutive difference (2.5-12.5 ⁇ s) in each of groups treated with D-2PMPA, and vehicle (control), respectively. NMJ integrity was preserved with treatment as demonstrated by single fiber jitter (5.4 ⁇ 0.3 vs.7.5 ⁇ 0.5 ⁇ s; p ⁇ 0.01).
  • FIG 11 is a schematic showing experimental design for D-2PMPA treatment of mice from 15-20 months of age from baseline (15 month of age) to endpoint MRI (isometric force), with Motor function monitored monthly measurements of compound muscle action potentials (CMAP) grip strength and rotarod performance.
  • CMAP compound muscle action potentials
  • FIGs 12A-12B are histograms showing rotarod latency to fall (s) over age (months) for each of vehicle ( ⁇ ) and D2PMPA ( ⁇ ), respectively (Fig.12A); and showing grip strength (% of initial) over age (months) for each of vehicle ( ⁇ ) and D2PMPA ( ⁇ ), respectively (Fig.12B).
  • FIGs 13A-13C are histograms showing calf muscle volume preservation (%) for each of vehicle and D2PMPA, respectively, in each of male (Fig.13A), female (Fig.13B), and combined (Fig.13C), groups, respectively. *P ⁇ 0.05, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIGs 13D-13F are histograms showing isometric force (mN) for each of vehicle and D2PMPA, respectively, in each of male (Fig.13D), female (Fig.13E), and combined (Fig.13F), groups, respectively. *P ⁇ 0.05.
  • FIGs 14A-14D are histograms showing ankle stimulation CMAP amplitude (mV) (Fig.14A); Hip stimulation CMAP amplitude (mV) (Fig.14B); ankle stimulation CMAP latency (s) (Fig.14C); and Hip stimulation CMAP latency (s) (Fig.14D), respectively, over age (months) for each of vehicle ( ⁇ ) and D2PMPA ( ⁇ ), respectively.
  • active agent or “biologically active agent” are used interchangeably to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, which may be prophylactic, therapeutic, or diagnostic. These may be a nucleic acid, a nucleic acid analog, a small molecule having a molecular weight less than 2 kD, more typically less than 1 kD, a peptidomimetic, a protein or peptide, carbohydrate or sugar, lipid, or a combination thereof.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of agents, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs.
  • the term “therapeutic agent” refers to an agent that can be administered to treat one or more symptoms of a disease or disorder.
  • diagnostic agent generally refers to an agent that can be administered to reveal, pinpoint, and define the localization of a pathological process.
  • the diagnostic agents can label target cells that allow subsequent detection or imaging of these labeled target cells.
  • “Analog” as relates to a given compound refers to another compound that is structurally similar, functionally similar, or both, to the specified compound. Structural similarity can be determined using any criterion known in the art, such as the Tanimoto coefficient that provides a quantitative measure of similarity between two compounds based on their molecular descriptors.
  • the molecular descriptors are 2D properties such as fingerprints, topological indices, and maximum common substructures, or 3D properties such as overall shape, and molecular fields. Tanimoto coefficients range between zero and one, inclusive, for dissimilar and identical pairs of 45718416.1 molecules, respectively.
  • a compound can be considered an analog of a specified compound, if it has a Tanimoto coefficient with the specified compound between 0.5 and 1.0, inclusive, preferably between 0.7 and 1.0, inclusive, most preferably between 0.85 and 1.0, inclusive.
  • a compound is functionally similar to a specified compound, if it induces the same pharmacological effect, physiological effect, or both, as the specified compound.
  • “Analog” can also refer to a modification including, but not limited to, hydrolysis, reduction, or oxidation products, of the compounds. Hydrolysis, reduction, and oxidation reactions are known in the art.
  • the term “therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and/or onto dendrimers, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.
  • the term “effective amount” refers to an amount of a therapeutic agent or prophylactic agent to reduce or diminish the symptoms of one or more diseases.
  • dendrimer compositions including one or more inhibitors may inhibit or reduce the activity and/or quantity of diseased neurons by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from the activity and/or quantity of the same cells in equivalent tissues of subjects that did not receive, or were not treated with, the dendrimer compositions.
  • the inhibition and reduction are compared at levels of mRNAs, proteins, cells, tissues, and organs. For example, an inhibition and reduction in the rate of neural loss, in the rate of decrease of muscle weight, or in the rate of decrease of muscle volume, as compared to an untreated control subject.
  • treating means to ameliorate, reduce or otherwise stop a disease, disorder or condition from occurring or progressing in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by 45718416.1 administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully “treated” if one or more symptoms associated with sarcopenia are mitigated or eliminated, including, but are not limited to, reducing the level of anxiety, agitation, or restlessness, improving feelings of sadness, tearfulness, emptiness or hopelessness, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease.
  • phrases “pharmaceutically acceptable” or “biocompatible” refers to compositions, polymers, and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to pharmaceutically acceptable materials, compositions, or vehicles, such as a liquid or solid filler, diluent, solvent, or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body.
  • biodegradable generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted in vivo. The degradation time is a function of composition and morphology.
  • dendrimer includes, but is not limited to, a molecular architecture with an interior core, interior layers, or “generations” of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation.
  • a molecule may be functionalized by the introduction of a molecule that makes the molecule a strong nucleophile or strong electrophile.
  • targeting moiety refers to a moiety that localizes to or away from a specific location.
  • the moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule.
  • the location may be a tissue, a particular cell type, a subcellular compartment, or a molecule such as a receptor.
  • Prolonged residence time refers to an increase in the time required for an agent to be cleared from a patient’s body, or organ or tissue of that patient.
  • Prolonged residence time refers to an agent that is cleared with a half-life that is 10%, 20%, 50% or 75% longer than a standard of comparison such as a comparable agent without conjugation to a delivery vehicle such as a dendrimer.
  • prolonged residence time refers to an agent that is cleared with a half-life of 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 target specific cell types.
  • incorporated and “encapsulated” refer to incorporating, formulating, or otherwise including an agent into and/or onto a composition that allows for release, such as sustained release, of such agent in the desired application.
  • the agent or other material can be incorporated into a dendrimer, by binding to one or more surface functional groups of such dendrimer (by covalent, ionic, or other binding interaction), by physical admixture, by enveloping the agent within the dendritic structure, and/or by encapsulating the agent inside the dendritic structure.
  • central nervous system includes the brain and spinal cord.
  • peripheral nervous system (“PNS”) refers to the nerves other than in the brain and spinal cord.
  • “Sugar-based,” as relates to dendrimers refers to dendrimers that contain a sugar moiety (such as a saccharide moiety) in their core, or their core and on their surface.
  • compositions Compositions of dendrimers conjugated or complexed with one or more inhibitors of glutamate carboxypeptidase II (GCPII) have been developed.
  • the compositions are particularly suited for treating and/or ameliorating one or more symptoms of age related reduction of muscle mass and strength.
  • the dendrimers are glucose dendrimers or hydroxyl terminated dendrimers such as hydroxyl terminated PAMAM or sugar modified dendrimers
  • the GCPII inhibitor is 2-phosphonomethyl-pentanedioic acid (2-PMPA).
  • Glutamate carboxypeptidase II Inhibitor Active Agents Compositions including active agents that reduce, prevent or otherwise inhibit the biological functions of the Glutamate carboxypeptidase II (GCPII) enzyme are described.
  • the GCPII inhibitor 45718416.1 is typically selectively specific to the target GCP enzymes.
  • the GCPII inhibitors inhibit the biological function of GCPII in vivo.
  • the inhibitor of GCPII is a small molecule, a peptide, a lipid, a nucleic acid, a synthetic polymer, or combinations thereof.
  • the GCPII inhibitor is a small molecule drug, for example, that binds to one or more active sites of biologically active GCPII and prevents, reduces or otherwise mediates the function of the GCPII relative to a GCPII in the absence of the inhibitor.
  • the GCPII inhibitors and/or their derivatives bind to the GCPII enzyme on the surface of the target cell or inside the target cell.
  • the inhibitors of GCPII include a structure that is a glutarate moiety that binds to the C-terminal glutamate recognition site of GCPII, and or a zinc chelating group to coordinate the divalent zinc atoms at the enzyme’s active site, or both. 1.
  • Glutamate carboxypeptidase II Glutamate carboxypeptidase II
  • Glutamate carboxypeptidase II Glutamate carboxypeptidase II
  • PSMA prostate-specific membrane antigen
  • NAALADase N-acetylated- ⁇ -linked acidic dipeptidase
  • NAAG N-acetylaspartyl- glutamate
  • FOLH1 folate hydrolase
  • GCPII is an extracellular, glial enzyme with its active site in the extra-synaptic space, making it ideally positioned to control whether NAAG functions to block or drive glutamatergic transmission. Under basal conditions, GCPII activity appears low, permitting NAAG to function as an intact dipeptide. However, under conditions of high synaptic activity, NAAG release and its cleavage by GCPII is enhanced, serving to liberate glutamate that subsequently activates extra- synaptic glutamatergic receptors on surrounding neurons and glia. GCPII inhibitors appear to reverse this activated state, decreasing glutamate release and increasing NAAG, returning the system to its basal state.
  • Glutamate is the primary excitatory neurotransmitter of the central nervous system (CNS), which has a crucial role in a complex communication network established between all residential brain cells.
  • CNS central nervous system
  • An excess release of glutamate leads to the activation of ionotropic and metabotropic receptors, resulting in the accumulation of toxic cytoplasmic Ca2+ and neuronal cell death, as observed in multiple neurological disorders like neuropathic pain, stroke, diabetic neuropathy, schizophrenia, addiction, multiple sclerosis, and traumatic brain injury.
  • GCPII is implicated in inflammatory bowel disease (IBD), as well as in the preclinical model of ovarian cancer.
  • GCP-II inhibitors have demonstrated the ability of GCP-II inhibitors to be therapeutically beneficial in cases of glutamate-mediated neuronal damage from brain injuries and neurological.
  • elevated levels of glutamate and NAAG flood the synapse.
  • NAAG that reaches the extra-synaptic space is rapidly cleaved by GCPII to liberate glutamate.
  • the excess glutamate and NAAG activate both GluN2A- and GluN2B-rich NMDA receptors, increasing excitatory postsynaptic currents (EPSCs).
  • EPCs excitatory postsynaptic currents
  • extra-synaptic NMDARs typically are enriched in GluN2B subunits, and are thought to constitute a major signaling pathway that triggers neuronal death.
  • Blockade of GCPII under activated conditions prevents the breakdown of NAAG lowering overall glutamate levels.
  • the resulting increased NAAG further decreases glutamate release through feedback inhibition via presynaptic mGlu3 receptors and induces trophic effects via activation of glial mGlu3 receptors.
  • Overall, inhibition of GCPII increases NAAG and lowers glutamate, returning the system toward its basal state. Numerous studies have also shown that an increase in NAAG is neuroprotective against NMDA receptor mediated neurotoxicity without adverse side effects.
  • the GCPII inhibitor is 2-Phosphonomethyl-pentanedioic acid (2-PMPA).
  • 2-PMPA is a phosphonate analogue of glutamate and is highly selective for GCPII with characteristics of low-molecular-weight and high aqueous solubility, having an IC50 value for GCPII of 0.3 nM.
  • the pentanedioic acid portion of the inhibitor was designed to interact with the glutarate recognition site of GCPII while the phosphonate group was utilized to chelate to the active site zinc ions.
  • 2-PMPA was characterized as a competitive inhibitor with an IC 50 value of 300 pM with extraordinarily selectivity having no activity at over 100 different transporter, enzymes and receptors, including several glutamate targets.
  • a recently published study has reported a noninvasive method for delivering GCPII inhibitors to the brain via intranasal (i.n.) administration on rodents and nonhuman primates, where i.n. administration of 2-PMPA exhibited the highest level of brain penetration compared to intraperitoneal administration.
  • the inhibitor of GCPII is one or more inhibitors including, but not limited to, a thiol- based inhibitor compound such as 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI-5693); 2-[[hydroxy[2,3,4,5,6 pentafluorophenyl)methyl]phosphinyl] methyl] pentanedoic acid (also known as GPI-5232); a 2-PMPA derivative/urea-based inhibitor compound such as ZJ-43, ZJ 11, ZJ 17, and ZJ 38; a 2-PMPA derivative compound such as 3-(2-carboxy-5- mercaptopentyl)benzoic acid, 3-(1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4- mercaptobutyl)thio]benzoic acid, N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxy
  • the inhibitor of GCPII is 2-(3-mercaptopropyl)pentanedioic acid, also known as 2-MPPA, also known as GPI-5693, having an IC 50 for the GCPII of 90 nM, and having a structure as set forth in STRUCTURE B, below.
  • 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI-5693).
  • the inhibitor of GCPII is 3-(2-carboxy-5-mercaptopentyl)benzoic acid, having an IC 50 for the GCPII of 15 nM, and having a structure as set forth in STRUCTURE C, below.
  • the inhibitor of GCPII is 3-(1-carboxy-4-mercaptobutoxy) benzoic acid, having an IC 50 for the GCPII of 14 nM, and having a structure as set forth in STRUCTURE D, below.
  • the inhibitor of GCPII is 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid, having an IC50 for the CGPII of 32 nM, and having a structure as set forth in STRUCTURE E, below.
  • the inhibitor of GCPII is 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, having an IC50 for the CGPII of 22 nM, and having a structure as set forth in STRUCTURE F, below. 3-(2- acid.
  • the inhibitor of GCPII is 2- [[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (also known as GPI-5232), having an IC50 for the GCPII of 82 nM, and having a structure as set forth in STRUCTURE G, below.
  • 2-[[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid also known as GPI-5232.
  • the inhibitor of GCPII is VA-033, having an IC50 for the GCPII of 12 nM, and having a structure as set forth in STRUCTURE H, below.
  • the inhibitor of GCPII is a prodrug of and/or derivative of 2-PMPA, such as ZJ-43, having a Ki for the GCPII of 0.8 nM, and having a structure as set forth in STRUCTURE J, below. ZJ-43.
  • the inhibitor of GCPII is a hydroxamate-based inhibitor having a core structure as set forth in STRUCTURE K, below. JHU 241. 45718416.1 B.
  • Dendrimers are three-dimensional, hyperbranched, monodispersed, globular and polyvalent macromolecules including surface end groups (Tomalia, D. A., et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)).
  • the term “dendrimer” includes, but is not limited to, a molecular architecture with an interior core (“G0”) and layers (or “generations”) of repeating units which are attached to and extend from this interior core, each layer having one or more branching points, and an exterior surface of terminal groups attached to the outermost generation.
  • dendrimers have regular dendrimeric or “starburst” molecular structures.
  • the dendrimers have a diameter between about 1 nm and about 60 nm, more preferably between about 1 nm and about 50 nm, between about 1 nm and about 40 nm, between about 1 nm and about 30 nm, 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.
  • the diameter is between about 1 nm to about 2 nm.
  • the preferred size of the dendrimers for crossing the blood brain barrier (“BBB”) is less than 5 nm, whereas those for not crossing the BBB and staying in the peripheral circulation are greater than 5 nm.
  • the dendrimers have a diameter effective to penetrate BBB and to be retained close to or within target neural and/or glial cells for delivery of the agents conjugated thereto.
  • the dendrimers have a diameter effective to penetrate a BBB and to be internalized into target neural and/or glial cells for delivery of the agents conjugated thereto, such as for example, neurons, oligodendrocytes, astrocytes, microglial, and neuroglial support cells.
  • the dendrimers have a diameter effective to penetrate a barrier interface, such as a blood nerve barrier (“BNB”), and to be internalized into neural and/or glial cells of the peripheral nervous system for delivery of the agents conjugated thereto such as for example, motoneurons, activated macrophage cells, Schwann cells, satellite cells, and neuroglial support cells.
  • BNB blood nerve barrier
  • the dendrimers have a diameter effective to be retained in the peripheral circulation for delivery of the agents conjugated thereto to target cells of the peripheral nervous system e.g., perisynaptic Schwann cells and glia, neuromuscular junctions (NMJ), and musculature.
  • dendrimer conjugates A major benefit of the use of dendrimer conjugates is the ability of the dendrimer to enhance the binding of the inhibitor of GCPII to its target receptor on target cells, for example, binding compounds to the GCPII on motoneurons and activated macrophage cells in the affected areas of the CNS, NMJ, and surrounding musculature. 45718416.1
  • dendrimers have a molecular weight between about 500 Daltons and about 100,000 Daltons inclusive, between about 500 Daltons and about 50,000 Daltons inclusive, or between about 1,000 Daltons and about 20,000 Daltons inclusive. Dendrimer sizes of less than 30,000 Da are preferred for transport across the BBB, and sizes of greater than 50,000 Da are preferred for confinement to the periphery.
  • the dendrimers have a hypercore (e.g., dipentaerythritol) and one or more monosaccharide branching units.
  • the monosaccharide branching units are conjugated to the core or the prior layer of monomers via linkers such as polyethylene glycol chains.
  • the hypercore is dipentaerythritol and the monosaccharide branching unit is glucose-based branching unit, such as shown in Structures II-IV.
  • the dendrimers are made entirely of glucose building blocks. Exemplary dendrimers are G1 to G3 glucose dendrimers, such as G1, G2, and/or G3 glucose dendrimers.
  • Suitable dendrimers scaffolds for use in the conjugates include, but are not limited to, poly(amidoamine), also known as PAMAM, or STARBURSTTM dendrimers; polypropylamine (POPAM), polyethylenimine, polylysine, polyester, iptycene, aliphatic poly(ether), aromatic polyether dendrimers, dendrimer of a sugar (e.g., glucose, galactose, mannose, fructose, etc.), and copolymers thereof, such as a copolymer of a sugar and an alkylene glycol (e.g., a dendrimer formed by glucose and ethylene glycol building blocks).
  • POPAM polypropylamine
  • POPAM polyethylenimine
  • polylysine polylysine
  • polyester e.g., iptycene
  • aliphatic poly(ether) aliphatic poly(ether)
  • aromatic polyether dendrimers
  • the dendrimers can have a plurality of surface functional groups, such as carboxylic, amine, hydroxyl, and/or acetamide.
  • surface functional groups and “terminal groups” are used interchangeably herein.
  • the dendrimers have surface hydroxyl groups.
  • one or more of these surface functional groups are further modified with other molecules, such as further modified with a sugar (e.g., glucose, galactose, mannose, fructose, etc.) and/or a polyalkylene glycol, for example, polyethylene glycol, and thus have sugar molecules and/or polyalkylene glycols as terminal moieties/molecules.
  • a sugar e.g., glucose, galactose, mannose, fructose, etc.
  • polyalkylene glycol for example, polyethylene glycol
  • Dendrimers can be any generation including, but not limited to, generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, generation 8, generation 9, or generation 10.
  • the dendrimers are PAMAM dendrimers used as a platform and modified with functional groups for increased number of surface hydroxyl groups.
  • Preferred PAMAM dendrimers include hydroxylated PAMAM dendrimers, particularly G3 to G6 hydroxyl-terminated PAMAM dendrimers, such as G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers.
  • the dendrimer-active agent conjugates can be confined to the peripheral circulation and specifically target a particular tissue region and/or cell type, e.g., 45718416.1 activated macrophages and Schwaan cells in the affected areas of the NMJ, and surrounding musculature, by using higher generation dendrimer (such as generation 4, 5, or 6 PAMAM dendrimer, generation 2, 3, or higher glucose-based dendrimers). Additionally, or alternatively, the dendrimer-active agent conjugates can be confined to the peripheral circulation by appropriate functionalization of the dendrimer (such as PEGylation).
  • higher generation dendrimer such as generation 4, 5, or 6 PAMAM dendrimer, generation 2, 3, or higher glucose-based dendrimers.
  • the dendrimer-active agent conjugates can be confined to the peripheral circulation by appropriate functionalization of the dendrimer (such as PEGylation).
  • the dendrimers can specifically target a particular tissue region and/or cell type of the central nervous system (CNS), the peripheral nervous system (PNS), and/or the periphery, especially activated macrophages and Schwann cells by using dendrimers of a certain generation, such as PAMAM dendrimers and/or glucose dendrimers of generation 2 (G2), G3, G4, and G5.
  • dendrimers of a certain generation such as PAMAM dendrimers and/or glucose dendrimers of generation 2 (G2), G3, G4, and G5.
  • the branching units include monosaccharides.
  • the monosaccharide branching units are conjugated to the core or the prior layer of monomers via linkers such as polyethylene glycol chains.
  • the monosaccharide branching units are glucose-based branching units.
  • the branching units can include PEG and/or alkyl chain linkers between different dendrimer generations.
  • the glucose layers are connected via PEG linkers and triazole rings.
  • the branching units are the same for each generation of dendrimers generated from the core. Therefore, for example, the branching units are glucose-based branching units for generating generation 1 dendrimers, for generating generation 2 dendrimers, and for generating generation 3 dendrimers.
  • the dendrimers have a hypercore such as dipentaerythritol and one or more monosaccharide branching units.
  • the hypercore is dipentaerythritol and the monosaccharide branching unit is glucose-based branching unit.
  • spacer molecules can also be alkyl (CH2)n–hydrocarbon-like units.
  • dendrimers synthesized using glucose building blocks, with a surface made predominantly of glucose moieties specifically targets cells including injured neurons, ganglion cells, and other neuronal cells in the brain, the eye, and/or in peripheral nervous system.
  • the glucose-based dendrimer selectively targets or is enriched inside target neural and/or glial cells.
  • the glucose-based dendrimer selectively targets or enriches the surface of target neural and/or glial cells.
  • the glucose-based dendrimer selectively targets or is enriched inside target neuronal cells and on the surface of the target neural and/or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched inside and/or on the surface of injured, diseased, and/or hyperactive neurons and/or glial cells. 45718416.1 In some cases, the dendrimers include an effective number of sugar molecules and terminal groups, for example, glucose and/or hydroxyl groups, for targeting to one or more neurons and/or glia of the CNS, PNS, and/or the eye.
  • the terminal hydroxyl groups of these dendrimers may be part of terminal glucose molecules or extra hydroxyl groups that are not part of the glucose molecules, or a combination thereof. In some embodiments, all the terminal hydroxyl groups are part of the terminal glucose molecules. In some embodiments, the number of sugar molecules on the termination of dendrimer is determined by the generation number. In some embodiments, dendrimers are made of glucose and oligoethylene glycol building blocks. Exemplary glucose dendrimers are shown in Structures V and VII.
  • Some exemplary glucose dendrimers include a generation 1 glucose dendrimer having 24 hydroxyl (-OH) end groups, a generation 2 glucose dendrimer having 96 hydroxyl (-OH) end groups, a generation 3 glucose dendrimer having 396 hydroxyl (-OH) end groups, and generation 4 glucose dendrimer having 1584 hydroxyl (-OH) end groups.
  • the glucose dendrimer is a generation 2 glucose-based dendrimer that has 24 glucose molecules at the periphery and 6 embedded glucose molecules in the backbone held together by PEG segments.
  • Dendrimer compositions that can selectively accumulate inside neurons, particularly in the nucleus of injured and/or hyperactive neurons, referred to as “glucose dendrimers” also accumulate at a high level inside activated microglia. However, compared to hydroxyl dendrimers which primarily accumulate in microglia, these dendrimers primarily go to neurons. Glucose dendrimers are described in PCT/US23/17548 “Dendrimer Compositions for Targeted Delivery of Therapeutics to Neurons” by The Johns Hopkins University.
  • Glucose dendrimers include (a) a central core, (b) one or more branching units, wherein the branching units are monosaccharide glucose-based branching units, optionally with a linker conjugated thereto; and optionally (c) one or more therapeutic, prophylactic and/or diagnostic agents.
  • the one or more branching units are conjugated to the central core, and the surface groups of the dendrimer are monosaccharide glucose molecules.
  • the central core is dipentaerythritol, or a hexa-propargylated derivative thereof.
  • the branching unit is conjugated to the central core via a linker such as a hydrocarbon or an oligoethylene glycol chain.
  • the branching units are ⁇ -D- Glucopyranoside tetraethylene glycol azide having the structure of STRUCTURE L, or peracetylated derivatives thereof. 45718416.1 STRUCTURE L: ⁇ -D-Glucopyranoside tetraethylene glycol azide
  • the glucose dendrimer is a generation 1, generation 2, generation 3, generation 4, generation 5, or generation 6 dendrimer.
  • the dendrimer is a generation 1 dendrimer having the following structure:
  • the dendrimer is a generation 2 dendrimer having the following structure: 45718416.1
  • RESULTURE N Exemplary G2 Glucose dendrimer.
  • GCPII inhibitor agents, and/or diagnostic agents are encapsulated, associated, and/or conjugated in the dendrimer, at a concentration of between about 0.01% to about 30%, preferably about 1% to about 20%, more preferably about 5% to about 20% by weight.
  • the dendrimers may also be conjugated to one or more diagnostic agents such as fluorescent dyes, near infra-red dyes, SPECT imaging agents, PET imaging agents, and radioisotopes.
  • the dendrimer and the agent(s) are conjugated via one or more linkers or coupling agents such as one or more hydrocarbon or oligoethylene glycol chains.
  • dendrimers are prepared using methods in which the dendrimer is assembled from a multifunctional core, which is extended outward by a series of reactions.
  • a multifunctional core moiety allows stepwise addition of branching units (i.e., generations) around the core.
  • Exemplary chemical structures suitable as core moieties 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
  • the core moiety is chitosan.
  • azide-modified chitosan, or alkyne-modified chitosan are suitable for conjugating to branching units using click chemistry.
  • the central core is dipentaerythritol or a hexa- propargylated derivative thereof.
  • the core moiety is ethylenediamine, or tetra(ethylene oxide).
  • the core moiety is dipentaerythritol.
  • Exemplary chemical structures suitable for use as core moieties are shown in Table 1 below. Table 1. Structural representation of various building blocks (cores, branching units, surface functional groups, monomers) for the synthesis of dendrimers.
  • Branching Units Exemplary chemical structures suitable as branching units include monosaccharides. In some embodiments, the monosaccharide branching units are conjugated to the core or the prior layer of monomers via linkers such as polyethylene glycol chains. In some forms, the monosaccharide branching units are glucose-based branching units. Exemplary glucose-based branching units are shown in Structures II-IV.
  • the branching units are the PEG or alkyl chain linkers between different dendrimer generations, for example, the glucose layers are connected via PEG linkers and triazole rings. 45718416.1
  • the branching units are the same for each generation of dendrimers generated from the core. Therefore, in one embodiment, the branching units are glucose-based branching units for generating generation 1 dendrimers as shown in Structures V-VII.
  • the branching units are hyper-monomers i.e., ABn building blocks. Exemplary hyper-monomers include AB4, AB5, AB6, AB7, AB8 building blocks.
  • Hyper-monomer strategy drastically increases the number of available end groups.
  • An exemplary AB4 hypermonomer is peracetylated ⁇ -D-Glucopyranoside tetraethylene glycol azide as shown in Structure III.
  • the chemical structures listed in Table 1, are also suitable as building blocks to form the branching units of the dendrimer.
  • the branching units of the dendrimers are formed by 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-hydroxye))
  • branching units of the dendrimers include, but are not limited to, sugar moieties, such as glucose, galactose, mannose, and fructose, and alkylene glycol, such as ethylene glycol, and combinations thereof.
  • the branching unit is chitosan.
  • azide- modified chitosan, or alkyne-modified chitosan are suitable for conjugating to the core moiety or additional same or different branching units using click chemistry.
  • the branching unit is methyl acrylate or ethylenediamine, or a combination thereof.
  • the branching unit is polyethylene glycerol linear or branched.
  • the branching unit is a copolymer of an alkylene glycol (such as ethylene glycol) and a sugar moiety, such as glucose, galactose, mannose, and/or fructose. 45718416.1 4.
  • Surface Functional Groups Surface functional groups/molecules of the dendrimers are not limited to a primary amine end group, a hydroxyl end group, a carboxylic acid end group, an acetamide end group, a sugar molecule, an oligo- or poly-alkylene glycol, and/or a thiol end group.
  • the desired terminal functional groups can be added via one of the conjugation methods for the core and branching unit.
  • the surface functional groups are hydroxyl groups, for example those of PAMAM dendrimers, of generation 2 OEG dendrimer as shown in Structure I, or of the terminal glucose of dendrimers prepared with glucose-based branching units as shown in Structures V and VII.
  • desired surface functional groups can be modified or added via one of the conjugation methods for the core and branching unit.
  • Exemplary surface functional groups include hydroxyl end groups, amine end groups, carboxylic acid end groups, acetamide end group, and thiol end groups, and combinations thereof.
  • the dendrimers can specifically target a particular tissue region and/or cell type, such as the cells and tissues of the central nervous system (CNS), the peripheral nervous system (PNS).
  • the dendrimers specifically target neurons and/or glia of the CNS. In some embodiments, the dendrimers specifically target neurons and/or glia of the PNS. In some embodiments, the dendrimers specifically target non-neural and/or non-glial cells such as activated macrophage cells in and around neuromuscular junctions.
  • the glucose dendrimers are those of generation 1 (G1), G2, G3, G4, and G5. In some embodiments, the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more neurons and/or glia of the CNS, the PNS, and/or the eye.
  • the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more non-neural and/or non-glial cells such as activated macrophage cells in and around neuromuscular junctions.
  • dendrimers are made of glucose and oligoethylene glycol building blocks. Exemplary generation 1 glucose dendrimer is shown in Structure VI, and generation 2 glucose dendrimers is shown in Structure VIII.
  • the dendrimers have a plurality of surface functional groups, such as hydroxyl (-OH) groups, amine groups, acetamide groups, and/or carboxyl groups on the periphery of the dendrimers (also referred to herein as surface functional groups or peripheral functional groups).
  • the surface density of such peripheral functional groups is at least 1 group/nm 2 (number of the surface functional groups/surface area in nm 2 ).
  • the surface density of the surface functional groups, such as hydroxyl groups is more 45718416.1 than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm 2 , such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm 2 .
  • the volumetric density of surface functional groups, such as hydroxyl groups is between about 1 and about 50 groups/nm 3 , between about 5 and about 30 groups/nm 3 , or between about 10 and about 20 groups/nm 3 .
  • the surface density of the surface functional groups is between about 1 and about 50, preferably 5-20 group/nm 2 (number of surface functional groups/surface area in nm 2 ), while each surface functional moiety has a molecular weight of between about 100 Da and about 10 kDa, preferably between about 100 Da and 1000 Da.
  • the amount of the surface functional groups, such as any one of those described above, e.g., hydroxyl groups, of the dendrimer is at least 30%, at least 40%, at least 50%, more than 40%, more than 50%, or in a range from more than 30% to 100%. %.
  • the amount of surface hydroxyl groups of the dendrimer is preferably more than 35%.
  • one or more of the surface functional groups, such as any one of those described above, on the periphery of the dendrimers are further modified by conjugating with one or more carbohydrate molecules and/or more or more polyalkylene glycols, such as polyethylene glycols.
  • the surface density of the terminal carbohydrate moieties/molecules and/or polyalkylene glycols can have any of the ranges described above for hydroxyl groups.
  • PAMAM dendrimer modified on the surface with sugar moieties (with >10% of surface groups modified by sugars, especially by glucose, and glucose dendrimers (where the dendrimers are made of glucose building blocks are preferred).
  • sugar moieties with >10% of surface groups modified by sugars, especially by glucose, and glucose dendrimers (where the dendrimers are made of glucose building blocks are preferred.
  • constructs with a total molecular weight of ⁇ 30,000 Da are preferred.
  • constructs with a total molecular weight of >50,000 Da are preferred.
  • the terminal hydroxyl groups of these dendrimers may be part of the terminal sugar moieties/molecules or extra hydroxyl groups that are not part of the sugar moieties/molecules, or a combination thereof. In some embodiments, all of the terminal hydroxyl groups are part of the terminal sugar moieties/molecules. a. Hydroxyl-terminated Dendrimers In some embodiments, the dendrimers include a plurality of hydroxyl groups.
  • Some exemplary high-density hydroxyl groups-containing dendrimers include commercially available polyester dendritic polymer such as hyperbranched 2,2-Bis(hydroxyl- methyl)propionic acid polyester polymer (for example, hyperbranched bis-MPA polyester-64- hydroxyl, generation 4), dendritic polyglycerols.
  • the hydroxyl terminated 45718416.1 dendrimers include hydroxyl-terminated PAMAM dendrimers, particularly G3 to G6 hydroxyl- terminated PAMAM dendrimers, such as G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers.
  • the high-density hydroxyl groups-containing dendrimers are oligo ethylene glycol (OEG)-like dendrimers.
  • OEG oligo ethylene glycol
  • D2-OH-60 generation 2 OEG dendrimer
  • Highly dense polyol dendrimer at very low generation in minimum reaction steps can be achieved by using an orthogonal hypermonomer and hypercore strategy, for example as described in WO2019094952.
  • the dendrimer backbone has non-cleavable polyether bonds throughout the structure to avoid the disintegration of dendrimer in vivo and to allow the elimination of such dendrimers as a single entity from the body (non-biodegradable). dendrimer. 45718416.1
  • the dendrimers have a plurality of hydroxyl (-OH) groups on the periphery of the dendrimers.
  • the surface density of hydroxyl (-OH) groups is at least 1 OH group/nm 2 (number of surface hydroxyl groups/surface area in nm 2 ).
  • the surface density of hydroxyl groups, per nm 2 is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm 2 , such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm 2 .
  • the volumetric density of hydroxyl groups is between about 1 and about 50 groups/nm 3 , between about 5 and about 30 groups/nm 3 , or between about 10 and about 20 groups/nm 3 .
  • the surface density of hydroxyl (-OH) groups is between about 1 and about 50, or between 5 and 20 OH group/nm 2 (number of surface hydroxyl groups/surface area in nm 2 ) while having a molecular weight of between about 100 Da and about 1000 Da.
  • the amount of the surface hydroxyl groups of the dendrimer is preferably greater than 35%, at least 40%, at least 50%, more than 40%, more than 50%, or in a range from more than 40% to 100.
  • the dendrimers may have a fraction of the hydroxyl groups exposed on the outer surface, with the others in the interior core of the dendrimers.
  • the dendrimer specifically targets a particular tissue region and/or cell types following administration into the body. In some embodiments, the dendrimer specifically targets a particular tissue region and/or cell type without a targeting moiety. In some embodiments, the dendrimers include an effective number of hydroxyl groups for targeting CNS cells and/or PNS cells, such as microglial, astrocytes, and/or neurons associated with a disease, disorder, or injury of the central nervous system or the peripheral nervous system.
  • the dendrimer specifically targets a particular tissue region and/or cell type without a targeting moiety and the active agent conjugated thereto bind directly to a receptor on the surface and/or interior of target neural and/or glial cells, such as activated macrophage cells in and around neuromuscular junctions.
  • Unmodified PAMAM dendrimers with hydroxyl end groups do not enrich in the neurons of brain and/or retinal ganglion cells (RGCs) in the eye as much as glucose dendrimers.
  • the glucose dendrimers with terminal glucose monosaccharide and a high density of hydroxyl functional groups effectively target the neurons in a generation dependent manner.
  • Generation 2 (G2), and G3 and G4 should be efficacious. G5 and above are more difficult to use.
  • the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more motoneurons.
  • the hydroxyl groups on the dendrimer surface are part of glucose molecules. There are no extra hydroxyls in addition to the glucose molecules on the surface. The number of sugar molecules on the surface is determined by the generation number. All generations are expected to target neurons.
  • glucose dendrimers include a generation 1 glucose dendrimer having 24 hydroxyl (-OH) end groups, a generation 2 glucose dendrimer having 96 hydroxyl (-OH) end groups, a generation 3 glucose dendrimer having 396 hydroxyl (-OH) end groups, and generation 4 glucose dendrimer having 1584 hydroxyl (-OH) end groups.
  • the glucose dendrimer is a generation 2 glucose based dendrimer that has 24 glucose molecules at the periphery and 6 embedded glucose molecules in the backbone held together by PEG segments.
  • the dendrimers contain one or more carbohydrate molecules at the termination.
  • terminal carbohydrate molecules can be prepared by conjugating one or more surface functional groups of a dendrimer, such as amine groups, carboxyl groups, or hydroxyl groups, with one or more carbohydrate molecules.
  • a dendrimer such as amine groups, carboxyl groups, or hydroxyl groups
  • the dendrimers prior to carbohydrate conjugation, are hydroxyl-terminated dendrimers such as hydroxyl-terminated PAMAM dendrimers and one or more of the hydroxyl groups are conjugated with one or more carbohydrate molecules.
  • hydroxyl-terminated dendrimers modified with surface glucose molecules selectively target activated macrophage cells in and around neuromuscular junctions in vitro and in vivo; and/or selectively accumulate on the surface and/or within these targets, such that the active agent(s) conjugated thereto bind to one or more receptors on/in the target activated macrophage cells in and around neuromuscular junctions.
  • hydroxyl- terminated dendrimers modified with surface glucose molecules selectively target immune system cells, such as activated macrophage cells in and around neuromuscular junctions in vitro and in vivo; and/or selectively accumulate on the surface and/or within these targets, such that the active agent(s) conjugated thereto bind to one or more receptors on/in the target activated macrophage cells in and around neuromuscular junctions.
  • the carbohydrate moieties used to modify one or more surface functional groups of the dendrimers are monosaccharides.
  • Exemplary monosaccharides suitable for modifying the dendrimers include glucose, glucosamine, galactose, mannose, fructose, dehydroascorbic acid, urate, myo-inositol.
  • the dendrimers are conjugated to glucose and thus contain glucose as terminal moieties/molecules.
  • hydroxyl- terminated dendrimers are modified with one or more glucose moieties to the dendrimer (“D-Glu”).
  • D-Glu glucose moieties to the dendrimer
  • the dendrimers are conjugated to galactose.
  • the dendrimers are conjugated to mannose.
  • the dendrimers are conjugated to fructose.
  • the dendrimers are conjugated to one or more monosaccharides other than glucose, such as galactose, mannose, and/or fructose.
  • the carbohydrate 45718416.1 moieties are oligosaccharides which terminate in one or more monosaccharides including glucose, glucosamine, mannose, fructose, thus exposing these sugar moieties on the surface for binding.
  • the glucose or hydroxyl-terminated PAMAM dendrimers, or carbohydrate-functionalized dendrimers are conjugated to one or more active agents that have affinity to and are suitable for binding directly or indirectly, to GCPII at the surface of, or within target cells.
  • the dendrimers are conjugated to one or more carbohydrates moieties that have affinity to and are suitable for binding directly or indirectly to one or more of AMPA receptors, NMDA receptors, EGFR1 receptors, EGFR2 receptors, histamine (H1) receptors, GABA receptors, and trace amine-associated receptor 1 (TAAR1).
  • AMPA receptors AMPA receptors
  • NMDA receptors NMDA receptors
  • EGFR1 receptors EGFR1 receptors
  • EGFR2 receptors histamine (H1) receptors
  • GABA receptors GABA receptors
  • trace amine-associated receptor 1 TAAR1
  • the dendrimers are conjugated to one or more active agents that have affinity to and are suitable for transport via one or more of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, and GLUT14.
  • active agents that have affinity to and are suitable for transport via one or more of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, and GLUT14.
  • the dendrimers are conjugated to one or more glucose and/or glucosamine moieties.
  • the dendrimers contain carbohydrate moieties which enable transport of the active agent to target cells/receptors, wherein the activity at the target cell or receptor is driven by the active agent.
  • the carbohydrates and glucose moieties enable better drug targeting to cells and/or receptors of interest.
  • the dendrimers are conjugated to one or more glucose and/or glucosamine moieties.
  • the dendrimers are conjugated to one or more oligosaccharides terminating in glucose and/or glucosamine moieties, i.e., glucose and/or glucosamine moieties are exposed on the surface of the dendrimer conjugates suitable for binding to one or more of the GLUTs, 5HT receptors, NE receptors, DA receptors and/or transporters.
  • the dendrimers have a plurality of carbohydrate moieties/molecules such as monosaccharides, e.g., glucose, on the periphery of the dendrimers.
  • the surface density of carbohydrate molecules such as monosaccharides, e.g., glucose, is at least 1 carbohydrate molecule/nm 2 (number of surface carbohydrate groups/surface area in nm 2 ).
  • the surface density of carbohydrate molecules, per nm 2 is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm 2 , such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm 2 .
  • surface density of carbohydrate molecules, per nm 2 is more than 10.
  • the volumetric density of surface carbohydrate molecules is between about 1 45718416.1 and about 50 groups/nm 3 , between about 5 and about 30 groups/nm 3 , or between about 10 and about 20 groups/nm 3 .
  • the surface density of carbohydrate molecules is between about 1 and about 50, between about 5 and about 20, per nm 2 (number of surface carbohydrate molecules/surface area in nm 2 ) while each carbohydrate moiety having a molecular weight of between about 100 Da and about 1000 Da.
  • the terminal hydroxyl groups may be part of the terminal sugar moieties/molecules or extra hydroxyl groups that are not modified with sugar moieties/molecules and thus are not part of the sugar moieties/molecules, or a combination thereof.
  • carbohydrate molecules such as monosaccharides, e.g., glucose
  • carbohydrate molecules are present in an amount by weight that is between about 1% and 40% of the total weight of the glycosylated dendrimer, for example, between about 2% and 20%, between about 5% and 15%, or between 9 % and 12 % of the total weight of the glycosylated dendrimer.
  • the carbohydrate moieties are present in an amount that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the glycosylated dendrimer following conjugation.
  • conjugation of carbohydrate molecules through one or more surface functional groups occurs via about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimers prior to the conjugation.
  • the conjugation of carbohydrate molecules occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40% of total available surface functional groups of the dendrimers prior to the conjugation.
  • the dendrimers contain one or more polyalkylene glycols at the termination. These terminal polyalkylene glycols can be prepared by conjugating one or more of surface functional groups of the dendrimers, such as hydroxyl groups, with a polyalkylene glycol, such as PEG.
  • the dendrimers prior to conjugation, are hydroxyl-terminated dendrimers such as hydroxyl-terminated PAMAM dendrimers and at least a portion of the surface hydroxyl groups are conjugated with PEG.
  • the dendrimers have a plurality of polyalkylene glycols such as PEG on the periphery of the dendrimers.
  • the surface density of polyalkylene glycols such as PEG is at least 1 polyalkylene glycol/nm 2 (number of surface polyalkylene glycol/surface area in nm 2 ).
  • the surface density of polyalkylene glycols, per nm 2 is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 polyalkylene glycol/nm 2 , such as at least 10, 15, 20, 45718416.1 25, 30, 35, 40, 45, 50, or more than 50 polyalkylene glycol/nm 2 .
  • surface density of polyalkylene glycols, per nm 2 is more than 10.
  • the volumetric density of surface polyalkylene glycols is between about 1 and about 50 groups/nm 3 , between about 5 and about 30 groups/nm 3 , or between about 10 and about 20 groups/nm 3 .
  • the surface density of polyalkylene glycols such as PEG is between about 1 and about 50, between about 5 and about 20, per nm 2 (number of surface polyalkylene glycols/surface area in nm 2 ) while having a molecular weight of between about 100 Da and about 1000 Da.
  • the polyalkylene glycol molecules such as PEG can be present in an amount by weight that is between about 1% and 40% of the total weight of the pegylated dendrimer, for example, between about 2% and 20%, between about 5% and 15%, or between 9 % and 12 % of the total weight of the pegylated dendrimer.
  • the polyalkylene glycol molecules such as PEG are present in an amount that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the pegylated dendrimer following conjugation.
  • conjugation of polyalkylene glycol molecules such as PEG through one or more surface functional groups of the dendrimer occurs via about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimers prior to the conjugation.
  • the conjugation of polyalkylene glycol molecules such as PEG occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40% of total available surface functional groups of the dendrimers prior to the conjugation.
  • Dendrimer-GCPII inhibitor agent conjugates can be formed from one or more active agents covalently conjugated or non-covalently attached to a dendrimer.
  • the one or more active agents are covalently conjugated to the dendrimer.
  • the one or more active agents are conjugated to the dendrimer via one or more spacers.
  • spacer includes chemical moieties and functional groups used for linking an active agent to the dendrimer.
  • the spacer can be either a single chemical entity or two or more chemical entities linked together.
  • the spacer can include any small chemical entity, peptide or polymers having sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone, carbonate, etc.
  • the spacer via which the active agent is conjugated to the dendrimer contains different linkages such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazides, ether, and amide linkages.
  • the spacer between a dendrimer and an active agent can be designed to provide a releasable or non-releasable form of the dendrimer conjugate in vivo.
  • the conjugation between active agent and dendrimer is via an appropriate spacer that contains an ester bond between the active agent and the dendrimer.
  • one or more spacers between a dendrimer and active agents can provide desired and effective release kinetics in vivo.
  • spacers may contain cleavable linkages (e.g., ester, disulfide, phosphodiester, triglycyl peptide, and hydrazine) or non-cleavable linkages (e.g., amide, ether, and amino alkyl).
  • the conjugation between active agents and dendrimers can be performed using reaction known in the art, such as click chemistry, acid-amine coupling, Steglich esterification, etc.
  • the conjugation between active agent and dendrimer is via a spacer that contains disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, ether, or amide linkages, or a combination thereof.
  • the conjugation between active agent and dendrimer is via an appropriate spacer that contains an ester linkage or an amide linkage between the agent and the dendrimer depending on the desired release kinetics of the agent.
  • the spacer can be chosen from among a class of compounds terminating in sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone and carbonate group.
  • the spacer can include thiopyridine terminated compounds such as dithiodipyridine, N-Succinimidyl 3-(2-pyridyldithio)- propionate (SPDP), Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or Sulfo-LC-SPDP.
  • the spacer can also include peptides wherein the peptides are linear or cyclic, having sulfhydryl groups 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).
  • RGDC arg-gly- asp-cys
  • c(RGDfC) cyclo(Arg-Gly-Asp-D-Tyr-Cys)
  • cyclo(Arg-Ala-Asp-d-Tyr-Cys cyclo(Arg-Ala-Asp-d-Tyr-Cys).
  • the spacer can be a mercapto acid derivative such as 3 mercapto propionic acid, mercapto acetic acid, 4 mercapto butyric acid, thiolan-2-one, 6 mercaptohexanoic acid, 5 mercapto valeric acid and other mercapto derivatives such as 2 mercaptoethanol and 2 mercaptoethylamine.
  • a mercapto acid derivative such as 3 mercapto propionic acid, mercapto acetic acid, 4 mercapto butyric acid, thiolan-2-one, 6 mercaptohexanoic acid, 5 mercapto valeric acid and other mercapto derivatives such as 2 mercaptoethanol and 2 mercaptoethylamine.
  • the spacer can be thiosalicylic acid and its derivatives, (4- succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridithio]propionyl hydrazide,
  • the spacer can have maleimide terminations wherein the spacer includes polymer or small chemical entity such as bis-maleimido diethylene glycol and bis-maleimido triethylene glycol, Bis- Maleimidoethane, bismaleimidohexane.
  • the spacer can include vinylsulfone such as 1,6-Hexane- bis-vinylsulfone.
  • the spacer can include thioglycosides such as thioglucose.
  • the spacer can be reduced proteins such as bovine serum albumin and human serum albumin, any thiol terminated compound capable of forming disulfide bonds.
  • the spacer can include polyethylene glycol having maleimide, succinimidyl, and/or thiol terminations.
  • D. Dendrimer-Agent Conjugates or Complexes Conjugate molecules including a dendrimer coupled and/or complexed with one or more GCPII inhibitor(s) are provided.
  • Dendrimer-active agent conjugates can be formed of GCPII 45718416.1 inhibitor agents covalently conjugated or non-covalently attached to a dendrimer, a dendritic polymer, or a hyperbranched polymer.
  • conjugation to the dendrimer may further improve safety and efficacy of these agents.
  • dendrimer conjugation may change specific receptor activity and/or modify biodistribution.
  • use of higher generation dendrimers and/or dendrimers with molecular weights greater than 24 kDa can confine these agents to the peripheral nervous system in order to preclude their effects elsewhere, such as in the brain.
  • one or more GCPII inhibitor agents are covalently conjugated to one or more terminal groups of the dendrimer such as terminal hydroxyl groups.
  • dendrimer conjugates include one or more GCPII inhibitor agents conjugated to the dendrimer via one or more spacers. The spacer between a dendrimer and an active agent can be designed to provide a releasable or non-releasable form of the dendrimer conjugate in vivo.
  • the spacer can be cleavable or contain a chemical linkage that is cleavable, for example, by exposure to the intracellular compartments of target neural and/or glial cells or upon binding to the receptor on the surface or in the interior of the target neural and/or glial cells in vivo.
  • cleavable linkages that can be used in a spacer of the dendrimer-GCPII inhibitor agent conjugates include, esterase sensitive ester bond, glutathione sensitive disulfide bond, phosphatase-sensitive phosphodiester bond, oligopeptide such as triglycyl peptide linker capable of lysosomal release, acid cleavable hydrazine linkage etc.
  • the spacer between a dendrimer and active agents can provide desired and effective release kinetics in vivo.
  • the spacer between the dendrimer and the active agent can be non-cleavable or contain a chemical linkage that is non-cleavable, such as amide, ether, and amino alkyl linkages.
  • the spacer between the dendrimer and active agent has a length sufficient for the active agent conjugated thereto to reach and bind to the target receptor on the surface and/or inside of the target cell.
  • the spacer between the dendrimer and active agent has a length in a range from 50 Da to 2,000 Da, depending on the release kinetics desired, and the receptor binding flexibility desired.
  • the length of the spacer can vary, depending on the location of the target receptor (for example, on the cell surface, in the cytoplasm of the cell, or in an intercellular compartment of the cell) and/or density of the receptor when located on the cell surface.
  • the dendrimer can be a generation 2, generation 3, generation 4, generation 5, generation 6, and up to generation 10.
  • the dendrimer is conjugated to one or more active 45718416.1 agents via spacers containing cleavable (ester, disulfide, phosphodiester, triglycyl peptide, and hydrazine) or non-cleavable (amide, ether, and amino alkyl) linkages.
  • the density of active agents covalently conjugated to or non-covalently attached to the dendrimer can be adjusted based on the specific GCPII inhibitor agent being delivered, the target receptors, the target neural and/or glial cells, the location of the target neural and/or glial cells, etc.
  • a plurality of active agents conjugated to the dendrimer are on the periphery of the dendrimer and the surface density of the active agent is at least 1 active agent/nm 2 (number of active agent conjugated/surface area in nm 2 ).
  • the surface density of active agent per nm 2 is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH/nm 2 , such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH/nm 2 .
  • the volumetric density of active agent is between about 1 and about 50 groups/nm 3 , between about 5 and about 30 groups/nm 3 , or between about 10 and about 20 groups/nm 3 .
  • the dendrimer-active agent conjugates have a hydrodynamic volume in the nanometer range.
  • the glucose dendrimer-active agent conjugates including one or more GCPII inhibitor agents conjugated to the dendrimer have a diameter of about 2 nm to about 100 nm, or more than 100 nm, up to 500 nm, depending upon the generation of dendrimer, the chemical composition and amount of active agent conjugated thereto.
  • a dendrimer-active agent conjugate including one or more GCPII inhibitor agents conjugated to the dendrimer has a diameter effective to penetrate nervous-system tissues and to retain on the surface and/or in target neural and/or glial cells for a period of time sufficient for the active agent to bind to the targeted receptors on the surface and /or in the target neural and/or glial cells.
  • a dendrimer-active agent conjugate including one or more GCPII inhibitor agents conjugated to the dendrimer has a diameter effective to remain in the peripheral circulation and to retain on the surface and/or in target neural and/or glial cells for a period of time sufficient for the active agent to bind to the targeted receptors on the surface and/or in the target neural and/or glial cells such as for example, neural and/or glial cells of the gastrointestinal system.
  • the dendrimer-active agent conjugates can be neural, have a positive charge or a negative charge.
  • the dendrimer-GCPII inhibitor agent conjugates are neutral. The presence of GCPII inhibitor agents can affect the surface charge of the dendrimer conjugates.
  • the surface charge of the dendrimer conjugated to GCPII inhibitor agents is between -100 mV and 100 mV, between -50 mV and 50 mV, between -25 mV and 25 mV, between -20 mV and 20 mV, between -10 mV and 10 mV, between -10 mV and 5 mV, between -5 mV and 5 mV, or between -2 mV and 2 mV.
  • the range above is inclusive of all values from -100 mV to 100 45718416.1 mV.
  • the surface charge of the dendrimer-GCPII inhibitor agent conjugates is neutral or near-neutral, i.e., from about -10 mV to about 10 mV, inclusive.
  • An exemplary dendrimer-GCPII inhibitor agent conjugate is represented by Formula (I).
  • the dendrimer of the exemplary conjugate contains surface hydroxyl groups, wherein one or more of the surface hydroxyl groups are conjugated to one or more active agents via one or more spacers as shown in Formula (I), below: wherein D can be a or 2 to generation 10 dendrimer, such as any one of those described above, for example, PAMAM (such as hydroxyl- terminated PAMAM dendrimer) or a glucose-based dendrimer; each occurrence of L can be any suitable chemical moiety, preferably containing a triazole moiety; Y can be a bond or a linkage selected from secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonate (-O-C(O)-O-), ureas (-
  • the dendrimer can be PAMAM (such as hydroxyl-terminated PAMAM) or a glucose dendrimer, which is 100% hydroxyl.
  • m and n depend on the size of the dendrimer D, n should be such that the weight percent of the drug in the total conjugate is 5-20%. This range is also appropriate for binding and internalization.
  • the oxygen atom shown in Formula (I) is from the surface functional group of the dendrimer, such as a surface hydroxyl group, where the surface hydroxyl group may or may not be part of a terminal sugar moiety/molecule (e.g., glucose).
  • one 45718416.1 or more hydroxyl groups of the dendrimer that are not conjugated to active agents may be modified with one or more carbohydrates and/or polyalkylene glycols, such as PEG.
  • the GCPII inhibitor agent (“X” of Formula (I)) can bind to a target receptor on the surface of the target cell or inside the target cell.
  • the agent X when the GCPII inhibitor agent X binds to the target receptor, the agent X remains conjugated to the dendrimer.
  • the agent X may be released from the dendrimer or remain conjugated to the dendrimer as an intact dendrimer-active agent conjugate.
  • each occurrence of L can be represented by -A’-L1-B’-L2-, wherein A’ can be a carbonyl (-C(O)-) or a bond (including single, double, and triple bonds, for example a single bond); B’ can be a bond (including single, double, and triple bonds, for example a single bond), an amide, an ester, an ether, a thiol, a dithiol, an aryl, a heteroaryl, a polyaryl, a heteropolyaryl, or a heterocyclic; and L1 and L2 can be independently a bond, an alkylene, a heteroalkylene, an aryl, an aralkyl, an ether, a polyether, a thiol, a dithiol, a thiol
  • L1-B’-L2- together form a chemical moiety selected from an -alkylene-triazole-di(alkylene glycol)-, a -di(alkylene glycol)-triazole-alkylene-, -alkylene- triazole-oligo(alkylene glycol)-, an -oligo(alkylene glycol)-triazole-alkylene-, an -alkylene-triazole- poly(alkylene glycol)-, -poly(alkylene glycol)-triazole-alkylene-, an -alkylene-triazole-ether-, an - alkylene-triazole-alkylene-, an -alkylene-amide-alkylene-, and combinations thereof.
  • B’ can be a bond (including single, double, and triple bonds, for example a single bond), an amide group, or a heterocyclic group, such as a triazole group.
  • L1 can be a bond; an alkylene, such as a C 1 -C 10 alkylene, a C 1 -C 8 alkylene, a C1-C6 alkylene, a C1-C5 alkylene, a C1-C4 alkylene, or a C1-C3 alkylene; or an oligo- or poly-(alkylene glycol), such where p is an integer from 1 to 20, from 1 to 18, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or 1 or 2.
  • L2 can be a bond; an alkylene, such as a C1-C10 alkylene, a C1-C8 alkylene, a C1-C6 alkylene, a C1-C5 alkylene, a C1-C4 alkylene, or a C1-C3 alkylene; an oligo- or poly-(alkylene glycol), such where p is an integer from 1 to 20, from 1 to 18, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 4, or or an oligo- or poly- peptide, such as a triglycyl peptide; a thiol; or a dithiol; or L2 is composed of a combination of two or more of alkylene, oligo- or poly-(alkylene glycol), oligo- or poly-peptide, thiols, and dithiols.
  • an alkylene such as a C1-C10 alkylene,
  • L2 is represented by , to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2, such as 0, 1, or 2; and G’ is a thiol, a dithiol, an oligo- peptide such as a triglycyl peptide, or a poly-peptide.
  • Y is a linkage that is minimally cleavable in vivo. In some embodiments, Y is a linkage that is cleavable in vivo.
  • Y is an amide (- CONH-), an ester (-C(O)-O-), an ether (-O-), a phosphodiester, or a disulfide group.
  • L and Y are both a single bond, and D is directly conjugated to X (an active agent or analog thereof) via an ether linkage.
  • D is a generation 2 PAMAM dendrimer, a generation 3 PAMAM dendrimer, a generation 4 PAMAM dendrimer, a generation 5 PAMAM dendrimer, a generation 6 PAMAM dendrimer, a generation 1 glucose dendrimer, a generation 2 glucose dendrimer, a generation 3 glucose dendrimer, a generation 4 glucose dendrimer, a generation 5 glucose dendrimer, or a generation 6 glucose dendrimer.
  • the GCPII inhibitors and/or their derivatives bind to a target receptor on the surface of the target cell or inside the target cell.
  • the agent when GCPII inhibitors and/or their derivatives bind to the target receptor, the agent remains conjugated to the dendrimer. In these embodiments, following binding, the agent may be released from the dendrimer or remain conjugated to the dendrimer as an intact dendrimer-active agent conjugate. In some embodiments, the GCPII inhibitors and/or their derivatives are released from the dendrimer at close proximity to the target receptor and then binds to the target receptor on the target neural and/or glial cell. 45718416.1 III. Methods of Making Dendrimer Conjugates Methods of synthesizing dendrimers and making dendrimer-GCPII inhibitor conjugate nanoparticles are also described. A.
  • Dendrimers can be prepared via a variety of chemical reaction steps. Dendrimers are usually synthesized according to methods allowing controlling their structure at every stage of construction. The dendritic structures are mostly synthesized by two main different approaches: divergent or convergent. In some embodiments, dendrimers are prepared using divergent methods, in which the dendrimer is assembled from a multifunctional core, which is extended outward by a series of reactions, commonly a Michael reaction. The strategy involves the coupling of monomeric molecules that possesses reactive and protective groups with the multifunctional core moiety, which leads to stepwise addition of generations around the core followed by removal of protecting groups.
  • PAMAM-NH2 dendrimers are first synthesized by coupling N-(2-aminoethyl) acryl amide monomers to an ammonia core.
  • dendrimers are prepared using convergent methods, in which dendrimers are built from small molecules that end up at the surface of the sphere, and reactions proceed inward, building inward, and are eventually attached to a core.
  • the core of the dendrimer, one or more branching units, one or more spacers, and/or one or more surface functional groups can be modified to allow conjugation to further functional groups (branching units, spacers, surface functional groups, etc.), monomers, and/or agents via click chemistry, employing one or more Copper-Assisted Azide-Alkyne Cycloaddition (CuAAC), Diels-Alder reaction, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules.2015 May 20;20(5):9263-94).
  • CuAAC Copper-Assisted Azide-Alkyne Cycloaddition
  • Diels-Alder reaction Diels-Alder reaction
  • thiol-ene and thiol-yne reactions thiol-yne reactions
  • azide-alkyne reactions Arseneault M et al., Mol
  • pre-made dendrons are clicked onto high-density hydroxyl polymers.
  • lick chemistry involves, for example, the coupling of two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface functional group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or equivalent thereof) on the surface of the first moiety and an azide moiety (e.g., present on a triazine composition or equivalent thereof), or any active end group such as, for 45718416.1 example, a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.
  • one or more hydroxyl groups on the surface of the dendrimer are modified to contain an alkyl group and a drug is modified to contain an azide group.
  • one or more hydroxyl groups on the surface of the dendrimer are modified to contain an azide group and a drug is modified to contain an alkyne group. The azide and alkyne are then reacted via a 1,3-dipolor cycloaddition reaction to form a triazole moiety.
  • dendrimer synthesis relies upon one or more reactions such as thiol- ene click reactions, thiol-yne click reactions, CuAAC, Diels-Alder click reactions, azide-alkyne click reactions, Michael Addition, epoxy opening, esterification, silane chemistry, and a combination thereof.
  • methods involve one or more protection and deprotection steps of the function groups (e.g., hydroxyl groups) on the central core, branching units, and/or therapeutic, prophylactic or diagnostic agents to facilitate addition of branching units to generate desired dendrimer molecules, or addition of therapeutic, prophylactic or diagnostic agents to generate desired dendrimer conjugates.
  • hydroxyl groups may be protected by formation of an ether, an ester, or an acetal.
  • Other exemplary protection groups include Boc and Fmoc. Any existing dendritic platforms can be used to make dendrimers of desired functionalities, i.e., with a high-density of surface hydroxyl groups by conjugating high-hydroxyl containing moieties such as 1-thio-glycerol or pentaerythritol.
  • Dendritic platforms such as polyamidoamine (PAMAM), poly (propylene imine) (PPI), poly-L-lysine, melamine, poly (etherhydroxylamine) (PEHAM), poly (esteramine) (PEA) and polyglycerol can be synthesized and explored.
  • Dendrimers also can be prepared by combining two or more dendrons. Dendrons are wedge- shaped sections of dendrimers with reactive focal point functional groups. Many dendron scaffolds are commercially available. They come in 1, 2, 3, 4, 5, and 6th generations with, respectively, 2, 4, 8, 16, 32, and 64 reactive groups. In certain embodiments, one type of agents is linked to one type of dendron and a different type of agent is linked to another type of dendron.
  • the two dendrons are then connected to form a dendrimer.
  • the two dendrons can be linked via click chemistry i.e., a 1,3- dipolar cycloaddition reaction between an azide moiety on one dendron and alkyne moiety on another to form a triazole linker.
  • click chemistry i.e., a 1,3- dipolar cycloaddition reaction between an azide moiety on one dendron and alkyne moiety on another to form a triazole linker.
  • glucose-based dendrimers are assembled from a multifunctional core, which is extended outward by a series of reactions.
  • the strategy involves the coupling of monomeric molecules that possess reactive and protective groups with the multifunctional core moiety, which leads to stepwise addition of generations around the core followed by removal of protecting groups.
  • glucose dendrimers are synthesized by coupling AB 4 peracetylated ⁇ -D glucose-PEG4-azide monomers to hexapropargylated core.
  • the hypercore is prepared from dipentaerythritol, for example by performing propargylation of dipentaerythritol to achieve the hexa-propargylated core.
  • the branching units are hypermonomers i.e., AB n building blocks.
  • Exemplary hypermonomers include AB3, AB4, AB5, AB6, AB7, AB8 building blocks.
  • Hypermonomer strategy drastically increases the number of available end groups.
  • An exemplary hypermonomer is AB4 orthogonal hypermonomer including one azide functional group and four allyl groups prepared from dipentaerythritol with five allyl groups reacted with monotosylated triethylene glycol azide.
  • the branching unit is polyethylene glycerol linear or branched e.g., as shown by Formula III.
  • hypermonomer AB 4 is based on glucose molecules.
  • the hypermonomer AB 4 is conjugated to a polyethylene glycerol, for example, tetraethylene glycol (PEG4).
  • the hypermonomer AB4 is peracetylated ⁇ -D-Glucopyranoside tetraethylene glycol azide.
  • the synthesis of glucose-Oac-TEG-Ots involves the following steps: a solution of peracetylated ⁇ -D-glucopyranoside (10g, 25.6mmol) was dissolved in 50mL of anhydrous dichloromethane (DCM) followed by addition of 2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.2g, 17.9mmol) and the reaction mixture was cooled to 0°C. Boron trifluoride diethyl etherate (2.5 eq.) was added and the reaction was allowed to come to room temperature.
  • DCM anhydrous dichloromethane
  • the reaction was monitored with the help of TLC and quenched after 5hrs by the addition of saturated sodium bicarbonate solution at 0°C. After 10 minutes of stirring, DCM (300mL) was added and the organic layer was washed with saturated sodium bicarbonate solution 3 times 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 / hexanes (70:30) mixture as eluents. The desired compound was achieved in 60% yield.
  • glucose-Oac (“acetoxy”)- triethylene glycol (“TEG”)-tosylate (“Ots”) is shown below:
  • the synthesis of glucose-Oac-TEG-N 3 involves the following steps: a solution of glucose-Oac-TEG-Ots (6g, 8.8mmoles) is dissolved in 40 mL of anhydrous DMF followed by the addition of sodium azide (2eq) and the reaction mixture is heated to 50 o C for overnight. Upon completion, the reaction mixture is filtered and DMF is evaporated. Once dried, the crude reaction mixture is passed through combiflash using ethyl acetate:hexane (70:30) as eluent.
  • glucose-Oac-TEG-N3 Structure of glucose-Oac-TEG-N3 is shown below: 45718416.1
  • the synthesis of glucose-OH-TEG-N3 involves the following steps: the peracetylated ⁇ -D-Glucopyranoside tetraethylene glycol azide is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH around 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with Amberlist IR- 120+ around 6-7. The reaction mixture is separated by filtration and the solvent removed by rotary evaporation. Structure of glucose-OH-TEG-N 3 is shown below. b.
  • glucose dendrimers are synthesized by coupling AB 4 peracetylated ⁇ -D glucose-PEG4-azide monomers to hexapropargylated core.
  • the hexapropargylated core is linked to AB 4 ⁇ -D-glucose-PEG4-azide building block (2) via click reaction to obtain generation 1 dendrimer.
  • generation one dendrimer D1-Glu6-Oac24 is prepared according to the following: Hexapropargylated compound (0.5g, 1mmoles) and an azido derivative ((4.1g, 7.4mmoles) 1.2 eq.
  • generation one dendrimer D1-Glu6-OH24 is prepared according to the following: the peracetylated generation 1 glucose dendrimer (1g, 0.26mmoles) is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH to around 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with AMBERLIST ® IR-120+ around 6-7. The reaction mixture is separated by filtration and the solvent removed by rotary evaporation, followed by water dialysis. Structure of generation one glucose dendrimer, D1-Glu6-OH24, is shown below. 45718416.1
  • D1-Glu 6 -OH 24 generation one glucose dendrimer D1-Glu6-OH24 is propargylated to provide D1-Acetylene24 according to the following: D1-Glu 6 -OH 24 (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) is slowly added in portions at 0°C to the solution with stirring. The solution is stirred for an additional 15 minutes at 0°C.
  • DMF dimethylformamide
  • D1-acetylene24 In some embodiments, generation one dendrimer D1-acetylene24 is further reacted with AB 4 ⁇ -D-glucose-PEG4-azide to provide generation 2 dendrimer with 24 glucose molecules containing 96 surface hydroxyl groups.
  • An exemplary generation two dendrimer D2-Glu24-Oac96 is prepared according to the following: D1-acetylene dendrimer24 (0.5g, 0.13 mmoles) and glucose-Oac-TEG-azide (2.2g, 4mmoles) are suspended in a 1:1 mixture of DMF and water in a 20 mL microwave vial equipped with a magnetic stir bar.
  • generation two dendrimer D2-Glu 24 -OH 96 is prepared according to the following: the peracetylated generation 2 glucose dendrimer D2-Glu24-OH96 is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH around 8.5-9.0. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with 45718416.1 AMBERLIST® IR-120+ around 6-7. The reaction mixture is filtered to remove the resin and the filtrate is evaporated by rotary evaporation followed by water dialysis to obtain the product as off- white solid. Structure of generation two glucose dendrimer, D2-Glu 24 -OH 96 , is shown below.
  • generation two dendrimer D2-Glu 24 -OH 96 is propargylated at one or more terminal hydroxyl groups suitable for further conjugation to one or more therapeutic, prophylactic or diagnostic agents.
  • one or more terminal hydroxyl groups of generation two dendrimer D2-Glu24-OH96 is propargylated according to the following: D2-Glu24- OH96 (5b) (200 mg, 0.016 mmol) is dissolved in anhydrous dimethylformamide (DMF, 10 mL) by sonication. To this stirring solution, sodium hydride [60% dispersion in mineral oil] (22 mg, 0.934 mmol) is slowly added in portions at 0°C.
  • the solution is additionally stirred for 15 minutes at 0°C. This is followed by the addition of propargyl bromide (18.0 ⁇ L, 80% w/w solution in toluene) at 0°C and the stirring is continued at room temperature for another 6h.
  • the solvent is evaporated 45718416.1 using V10 evaporator system and the crude product is purified by passing through PD10 SEPHADEX ® G25 M column.
  • the aqueous solution is lyophilized to afford the product as off- white solid.
  • one or more fluorescent dyes such as infrared fluorescent Cy5 dyes are conjugated to generation two dendrimer D2-Glu24-OH96.
  • Cy5-D2-Glu24- OH96 (compound 7 of FIG.1B) is prepared according to the following: 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 25mL round bottom flask equipped with a magnetic stir bar. To this, CuSO4 ⁇ 5H2O (5mol%/acetylene, 0.3 mg) and sodium ascorbate (10 mol%/acetylene, 0.5 mg) dissolved in the minimum amount of water are added. The reaction is stirred at room temperature for 24 h. Upon completion, the DMF is evaporated using V10 and the purification is performed using PD10 Sephadex G25 M column.
  • the aqueous solution is lyophilized to afford the product as blue solid.
  • 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 out of total 96 available hydroxyl groups of the exemplary generation 2 dendrimer with 24 glucose molecules containing 96 surface hydroxyl groups.
  • one or more GCPII inhibitor agents are covalently attached to the dendrimers.
  • the agents are attached to the dendrimer via a spacer that is designed to be non-cleavable in vivo.
  • the agents are attached to the dendrimer via a spacer that is designed to be cleaved in vivo.
  • the spacer can be designed to be cleaved hydrolytically, enzymatically, or combinations thereof, so as to provide for the sustained release of the GCPII inhibitor agents in vivo.
  • both the chemical structure of the spacer and its point of attachment to the GCPII inhibitor agent can be selected so that cleavage of the spacer releases either an agent, or a suitable prodrug thereof.
  • the chemical structure of the spacer can also be selected in view of the desired release rate of the agents.
  • the conjugation between the GCPII inhibitor agent and dendrimer is via one or more of disulfide, ester, ether, phosphodiester, triglycyl peptide, hydrazine, amide, or amino alkyl linkages.
  • the conjugation between the GCPII inhibitor agent and dendrimer is via an appropriate spacer that provides an ester bond or an amide bond between the 45718416.1 agent and the dendrimer depending on the desired release kinetics of the agent.
  • an ester or disulfide bond is introduced for releasable form of GCPII inhibitor agents.
  • Spacers generally contain one or more organic functional groups.
  • suitable organic functional groups contained in the spacers include secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonate (-O-C(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-), wherein R is
  • the identity of the one or more organic functional groups within the spacer is chosen in view of the desired release rate of the GCPII inhibitor agents.
  • the one or more organic functional groups can be selected to facilitate the covalent conjugation of the agents to the dendrimers.
  • the conjugation between the GCPII inhibitor agent and dendrimer is via an appropriate spacer that provides a disulfide bridge between the agent and the dendrimer.
  • the dendrimer-active agent conjugates are capable of rapid release of the agent in vivo by thiol exchange reactions, under the reduced conditions found in body.
  • the spacer contains one or more of the organic functional groups described above in combination with a linking group.
  • the linking group can be composed of any assembly of atoms, including oligomeric and polymeric chains; for example, the total number of atoms in the linking group is between 3 and 200 atoms, between 3 and 150 atoms, between 3 and 100 atoms, or between 3 and 50 atoms.
  • suitable linking groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains.
  • Variation of the linking group provides additional control over the release of the GCPII inhibitor agents in vivo.
  • the spacer includes a linking group
  • one or more organic functional groups will generally be used to connect the linking group to both the GCPII inhibitor agent and the dendrimers.
  • Appropriate methods for the covalent conjugation of a given agent can be selected in view of the linking moiety desired, as well as the structure of the agents and dendrimers as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds.
  • the amount of active agent in the dendrimer-active agent conjugates depends on many factors, including the choice of active agent, dendrimer structure and size, and tissues to be treated.
  • the one or more GCPII inhibitor agents are conjugated to the dendrimer at a concentration between about 0.01% and about 45%, inclusive; between about 0.1% and about 30%, inclusive; between about 0.1% and about 20%, inclusive; between about 0.1% and about 10%, inclusive; between about 1% and about 10%, inclusive; between about 1% and about 5%, inclusive; between about 3% and about 20% by weight, inclusive; or between about 3% and about 10% by weight, inclusive.
  • specific drug loading for any given active agent, dendrimer, and site of target can be determined by routine methods, such as those described herein.
  • the conjugation of GCPII inhibitor agents/spacers occurs via about 1%, 2%, 3%, 4%, or 5% of the total available surface functional groups, such as hydroxyl groups, of the dendrimers prior to the conjugation. In other embodiments, the conjugation of agents/spacers occurs on 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% total available surface functional groups of the dendrimers prior to the conjugation with active agents.
  • dendrimer-GCPII inhibitor agent conjugates retain an effective amount of surface functional groups for targeting to activated macrophage cells in and around motoneuron/muscle junctions, whilst conjugated to an effective amount of GCPII inhibitor agents to treat, or prevent age-related muscular decline.
  • dendrimer-GCPII inhibitor agent conjugates retain an effective amount of GCPII inhibitor agents for targeting to target cells and binding to target receptors on the surface or in the interior of the target cells. More specific methods for preparing exemplary dendrimer-GCPII inhibitor agent conjugates are described in the Examples below. IV.
  • compositions including dendrimer-GCPII inhibitor agent conjugates may be formulated in a conventional manner using one or more physiologically acceptable carriers, optionally including excipients which facilitate processing of the GCPII inhibitor compounds into preparations which can be used pharmaceutically.
  • excipients include aqueous buffers, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof.
  • Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS) and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • pharmaceutically acceptable salts of the actives can be prepared by reaction of the free acid or base forms of an agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Pharmaceutically acceptable salts include salts of an agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p.704. Examples of ophthalmic drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac.
  • the compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • the phrase “dosage unit form” refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total single administration of the compositions will be decided by the attending physician within the scope of sound medical judgment.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs, or extrapolated from human data.
  • Therapeutic efficacy and toxicity of conjugates can be determined by standard pharmaceutical procedures in experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and is expressed as the ratio, LD50/ED50.
  • compositions which exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use.
  • the compositions of dendrimer-GCPII inhibitor agent conjugates can be formulated together with one or more additional active agents, for example, agents that are known or contemplated to exert some beneficial effect to a subject. 45718416.1 V. Methods of Use
  • the compositions can be administered to treat or alleviate one or more symptoms of reduction in muscle mass and/or strength, for example, associated with sarcopenia.
  • the compositions are typically administered in an amount effective to provide a therapeutic effect, but insufficient to provide an adverse effect.
  • the dendrimer compositions selectively target specific cells and specific receptors on the cells to address a variety of diseases, disorders and conditions.
  • the methods include administering to a subject in need, the compositions in an amount effective to increase binding of the GCPII enzyme inhibitor compound to GCPII on the surface of target cells or at specific receptors in specific cells, particularly activated immune cells, such as activated macrophages in the muscles, and Schwaan cells at the neuromuscular junctions (NMJ), and surrounding synapses.
  • A. Methods of Treatment It has been established that inhibition of glutamate-carboxypeptidase-II in and around the neuromuscular junctions (NMJ) of aged subjects can increase muscle mass and thereby strength.
  • hydroxylated dendrimers conjugated with glutamate-carboxypeptidase-II (GCPII) inhibitors are administered to a subject in an amount effective to reduce, delay or reverse one or more symptoms or markers of sarcopenia in the subject.
  • an amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is administered in an amount effective to reduce glutamate and/or cytoplasmic Ca2+ concentrations within and/or around neuromuscular junctions (NMJ) in the subject and thereby prevent or reduce neuronal cell damage and death in the subject.
  • NMJ neuromuscular junctions
  • the amount of dendrimers conjugated with GCPII inhibitors is effective to bind to GCPII at the surface of target cells, including neuronal cells, glial cells, Schwann cells, and activated macrophages.
  • the compositions can be administered to prevent, treat, and/or manage the symptoms of ofther disorders, diseases, and conditions in additiona to age-related decrease in muscle mass, age- related decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and related disorders.
  • the compositions can be administered to reduce, prevent, delay or reverse one or more neurological disorders such as a decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), neuromuscular transmission defects, and related disorders in a subject in need thereof.
  • the dendrimer-GCPII inhibitor conjugates may be administered to a subject prophylactically, e.g., to prevent, delay or otherwise mediate the onset or rate of age-related decrease in effective 45718416.1 neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and related disorders. 1.
  • compositions and methods are designed to circumvent existing challenges in selective drug delivery to specific target cells and/or tissues, motoneurons, perisynaptic Schwann cells, and activated macrophage cells within and around neural synapses and neuromuscular junctions (NMJ) and are administered peripherally.
  • Glutamate-carboxypeptidase-II (GCPII) inhibitors conjugated to hydroxylated dendrimers can be selectively delivered to specific cell types in vivo to reduce, prevent or delay neuromuscular junction (NMJ) denervation in a subject.
  • GCPII Glutamate-carboxypeptidase-II
  • the hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor is administered in an effective amount to provide binding of the GCPII inhibitor to one or more receptors on the surface of or inside target cells such as neuronal cells, glial cells, Schwann cells, and activated macrophages.
  • the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is administered in an amount effective for reducing, delaying, inhibiting or reversing glutamate and/or cytoplasmic Ca2+ concentrations within and/or around neuromuscular junctions (NMJ) in the subject.
  • NMJ neuromuscular junctions
  • Macrophage influx specifically in the muscle tissue of a test animal model for neurodegenerative disease was found to destabilize the perisynaptic Schwann cells, which are important for NMJ health.
  • reducing macrophage activation was shown to prolong the NMJ integrity, indicating that activated macrophages can be detrimental to perisynaptic Schwann cells (Van Dyke, et al., Experimental neurology.2016; (277) pp.275–282).
  • One mechanism for infiltrating macrophages to exacerbate the axonal withdrawal is through the upregulation of GCPII activity and increased glutamate production.
  • acetylcholine is the canonical neurotransmitter at the neuromuscular synapse
  • glutamate also plays an importantrole during neuromuscular synapse development.
  • Activated macrophages may, in part, elicit neurotoxic effects through enhanced release of glutamate.
  • PAMAM-OH and PEG-OL dendrimers may target microglia/macrophages ), and glucose dendrimers that target both neurons and microglia.
  • the role of upregulated GCPII activity on infiltrating macrophages is likely where activated macrophages have detrimental effects on Schwann cells and axonal integrity such as Charcot-Marie-Tooth disease or even the general aging process of axonal withdrawal.
  • GCPII inhibition may be altering the immune response and affecting disease progression by reducing harmful immune cell activation.
  • Chronic immune cell activation in the muscle can be pathological and immune cells are known to modulate the extracellular matrix, which is a key 45718416.1 component of nerve regeneration and repair. Therefore, administering dendrimer-GCPII inhibitor conjugates to selectively target activated macrophages at or near the NMJ may prevent, reduce or reverse the effects of increased/deleterious GCPII receptor activity at or near motoneurons and the NMJ.
  • the dendrimer-GCPII inhibitor conjugates increase drug bioavailability in and around motoneurons and neuromuscular junctions by one or more of the following: (i) increasing drug solubility, (ii) facilitating target engagement i.e., increasing site-specific binding, (iii) improving drug pharmacokinetics, and (iv) targeting to activated immune cells at an near the neuromuscular junctions.
  • the methods permit selective delivery of compounds to the neuromuscular junctions, thereby increasing the potential of the compositions to be used to selectively treat neuromuscular impairment and related diseases and disorders.
  • the compositions are used to prevent, treat, and/or manage symptoms of sarcopenia, including age-related decrease in muscle mass, age-related decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and sarcopenic obesity and arterial stiffness.
  • sarcopenia including age-related decrease in muscle mass, age-related decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and sarcopenic obesity and arterial stiffness.
  • the methods reduce, prevent, delay or reverse one or more symptoms of sarcopenia in a subject in need thereof.
  • Sarcopenia has been defined as a generalized disease that causes decreased muscle mass and muscle function; it is a chronic, progressive skeletal muscle disease characterized by low muscle strength and quantity or quality, leading to reduced physical performance and increased physical frailty.
  • Symptoms of Sarcopenia significantly worsens prognosis of, and is related to a higher risk of polypharmacy, impaired quality of life, falls and fractures, loss of physical independence, and in can lead directly to death.
  • Symptoms of Sarcopenia include dynapenia (low muscle strength), reduced multisensory integration (balance), reduced muscle mass, increased physical frailty, such as falls and difficulties in lifting and carrying load (e.g., 4.5 kg or more), moving, getting up from a chair/bed, or going up a flight of stairs.
  • symptoms of sarcopenia are determined using one or more of a maximal handgrip strength test (upper limbs), total body mass and/or body mass index (BMI; e.g., calculated by the ratio between body mass and height (meters) squared (body mass/height 2 ), and based on 45718416.1 responses to self-assessment of strength, walking aids, difficulty getting up from a chair, difficulty climbing stairs, and falls (e.g., based on the SARC-F score determined from a 5-item questionnaire, described below); or ability in the Senior Fitness Test (SFT; to evaluate physical function in healthy elderly people including six tests: the 30-s Chair Stand Test (CST), the 30-s arm curl test (ACT), the chair sit and reach test, the back-scratch test (BST), the 8-foot up-and-go test (FUG), and the 6- min walk test (6MWT)).
  • a maximal handgrip strength test upper limbs
  • BMI body mass and/or body mass index
  • ACT
  • an amount of a dendrimer conjugated to one or more GCPII inhibitors is administered in an amount effective to treat one or more symptoms of sarcopenia such as lack of strength, need for assistance in walking, difficulty rising from a chair, difficulty climbing stairs, increased number of falls, reduced muscle mass, reduced balance and reduced muscular control in a subject in need thereof.
  • the conjugates are effective to increase strength, reduce the need for assistance in walking, reduce difficulty rising from a chair, reduce difficulty climbing stairs, reduce the number of falls, increase muscle mass, increase balance and/or increase muscular control in a subject relative to an untreated control subject.
  • the methods are effective to slow or stop the progression of sarcopenia in a subject, relative to an untreated control subject.
  • the methods are effective to reduce the rate of loss of strength, reduce the rate of loss of muscle mass, reduce the rate or extent of loss of balance and/or reduce the rate of loss of muscular control in a subject as compared to an untreated control subject.
  • the methods increase the speed of walking, increase the speed of muscular contractions, increase vigor, increase energy and/or reduce body fat, increase lifespan and/or decrease mortality in a subject identified as having sarcopenia.
  • SARC-F Test Score In some forms, symptoms of sarcopenia are determined using the SARC-F test.
  • a subject has a SARC-F score of between 1 and 10, inclusive before administration of the dendrimer-GCPII inhibitor conjugates. In some forms, the subject has a SARC-F score of 2, 3, 4, 5, 6 , 7, 8, 9 or 10 before administration of the dendrimer-GCPII inhibitor conjugates. Data suggests that a SARC-F score of ⁇ 4 best predicts the need for further, more comprehensive evaluation.
  • the methods administer to a subject an amount of the dendrimer-GCPII inhibitor conjugates effective to reduce the SARC-F score of the subject by 1 or more points, such as by 2, 3, 4 ,56, 7, 8, 9 or 10 points, relative to an untreated control.
  • the dendrimer-GCPII inhibitor conjugates reduce the SARC-F score of a subject from 4 or more to less than 4.
  • Biomarkers of Sarcopenia Thedendrimer-GCPII inhibitor conjugates may be effective to alter the presence of one or more biomarkers of sarcopenia in the subject.
  • biomarkers that are associated with sarcopenia are known in the art, for example, as described in Gugliucci, et al., J Clin Med.2024 Feb; 13(4): 1107, which is incorporated herein by reference in its entirety.
  • biomarkers related to sarcopenia can be divided into markers evaluating musculoskeletal status (biomarkers specific to muscle mass, markers of the neuromuscular junction, or myokines), and markers assuming causal factors (adipokines, hormones, and inflammatory markers).
  • biomarkers associated with assessment of sarcopenia include: myostatin (MSTN, also known as GDF-8; decreased in Sarcopenia); follistatin (FST; increased in Sarcopenia); irisin (decreased in Sarcopenia); brain-derived neurotrophic factor (BDNF; decreased in Sarcopenia); procollagen type III N-terminal peptide (PIIINP; P3NP – varies depending upon other factors, but typically changed in sarcopenia); sarcopenia index (serum creatinine to serum cystatin C ratio, decreased in Sarcopenia); adiponectin (increased in Sarcopenia); leptin (increased in Sarcopenia); insulin-like growth factor-1 (IGF-1; decreased in Sarcopenia); dehydroepiandrosterone sulphate (DHEAS; decreased in Sarcopenia); C-reactive protein (CRP; increased in Sarcopenia); interleukin-6 (IL-6; increased in Sarcopenia), and tumor necrosis factor
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered in an amount effective to increase the relative amount of myostatin (MSTN, also known as GDF-8) in a subject. It may be that myostatin muscle protein turnover decreases during the progression/development of sarcopenia. Methods of measuring myostatin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. Therefore, in some forms, the methods administer one or more dendrimer-GCPII inhibitor conjugates to a subject in an amount effective to increase myostatin muscle protein turnover in a subject identified as having sarcopenia, as compared to an untreated control. ii.
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of follistatin (FST) in a subject. It may be that follistatin muscle protein turnover is acutely increased with exercise during the progression/development of sarcopenia. Methods of measuring follistatin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. Therefore, in some forms, the methods administer one or more dendrimer-GCPII inhibitor conjugates to a subject in an amount effective to decrease follistatin muscle protein in a subject identified as having sarcopenia, as compared to an untreated control. iii.
  • Irisin Theone or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of irisin in a subject. It may be that irisin muscle protein turnover is decreased during the progression/development of sarcopenia. Methods of measuring irisin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. iv.
  • Brain-derived neurotrophic factor (BDNF) and/or Glial cell line-derived neurotrophic factor (GDNF) may be administered to a human subject in an amount effective to increase the relative amount of brain-derived neurotrophic factor (BDNF) and/or glial cell line-derived neurotrophic factor (GDNF) in a subject.
  • Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are markers associated with the neuromuscular junction and neuroinflammation. These are neurotrophins released from both neurons and muscles and play an important role in muscle development and metabolism and the regulation of synapse function.
  • BDNF and/or GDNF is decreased due to remodeling of the neuromuscular junctions (NMJ) during the progression/development of 45718416.1 sarcopenia.
  • Methods of measuring BDNF and/or GDNF serum levels in a subject are known in the art, including immunoassay measurement methods.
  • PIIINP Procollagen type III N-terminal peptide
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to alter the relative amount of procollagen type III N-terminal peptide (PIIINP; P3NP) in a subject.
  • PIIINP is produced during type III collagen synthesis, and PIIINP levels increase with higher BMI and age.
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative Sarcopenia index (SI; serum creatinine to serum cystatin C ratio) in a subject.
  • SI is a formula for evaluating muscle mass; Serum creatinine concentrations reflect muscle protein turnover but are also determined by renal function. That is why, in this formula, creatinine level is related to another marker of renal function—cystatin C.
  • SI has been correlated with CT skeletal muscle cross-sectional surface area, calf circumference, BMI, handgrip strength, and gait speed.
  • Adiponectin The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of adiponectin in a subject.
  • Adiponectin could play a role in a compensatory mechanism for mitigating sarcopenia resulting from chronic inflammation and oxidative stress. It may be that the anti-inflammatory factor adiponectin is increased due to increased muscle-fat crosstalk during the progression/development of sarcopenia.
  • Methods of measuring adiponectin serum levels in a subject are known in the art, including immunoassay measurement methods. 45718416.1 viii.
  • Leptin The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of leptin in a subject.
  • Leptin is an adipokine that, among other things, improves immune response and induces lipid catabolism; leptin serum levels are positively correlated with muscle and fat mass and negatively correlated with muscle strength—increased leptin levels are linked to a higher risk of dynapenia (low muscle strength). It may be that the adipokine factor leptin is increased due to increased fat deposition during the progression/development of sarcopenia. Methods of measuring leptin serum levels in a subject are known in the art, including immunoassay measurement methods. ix.
  • IGF-1 Insulin-like growth factor-1
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of insulin-like growth factor-1 (IGF-1) in a subject.
  • Age-related decline in IGF-1 levels is considered a possible factor contributing to the development of sarcopenia and it may be that serum levels of IGF-1 are decreased due to age- related anabolic metabolism during the progression/development of sarcopenia.
  • Methods of measuring IGF-1 serum levels in a subject are known in the art, including immunoassay measurement methods. x.
  • DHEAS Dehydroepiandrosterone sulphate
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of dehydroepiandrosterone sulphate (DHEAS) in a subject.
  • DHEAS dehydroepiandrosterone sulphate
  • Its role in sarcopenia has long been studied, and the age-related decrease in its levels is perceived as a causal factor for muscle loss; DHEAS concentrations correlate positively with older patients’ skeletal muscle mass and strength and seem to be decreased in sarcopenic individuals. It may be that levels of serum DHEAS are decreased due to age-related anabolic metabolism during the progression/development of sarcopenia.
  • C-reactive protein The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of C-reactive protein (CRP) in a subject.
  • CRP C-reactive protein
  • One of the crucial mechanisms that may be related to sarcopenia pathogenesis is chronic inflammation. Inflammatory cytokines have been linked to muscle wasting, promoting protein catabolism and suppressing muscle tissue synthesis. It may be that the C-reactive protein (CRP) is increased due to increased chronic inflammation during the progression/development of sarcopenia.
  • Methods of 45718416.1 measuring C-reactive protein (CRP) levels in a subject are known in the art, including immunoassay measurement methods.
  • xii. Interleukin-6 (IL-6)
  • the one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of Interleukin-6 (IL-6) in a subject. It may be that IL-6 is increased due to increased low-grade chronic inflammation associated with reduced muscle mass during the progression/development of sarcopenia.
  • Methods of measuring C-reactive protein IL-6 levels in a subject are known in the art, including immunoassay measurement methods.
  • Tumor necrosis factor ⁇ The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of tumor necrosis factor ⁇ (TNF- ⁇ ) in a subject. It may be that tumor necrosis factor ⁇ (TNF- ⁇ ) is increased due to increased low-grade chronic inflammation associated with reduced muscle mass during the progression/development of sarcopenia. Methods of measuring C-reactive protein TNF- ⁇ levels in a subject are known in the art, including immunoassay measurement methods.
  • Sarcopenia is an age-related disease and affects approximately 10% to 27% of the population aged ⁇ 60 years in the USA.
  • sarcopenia in nursing homes is higher than in community-dwelling older adults and is estimated at 51% in men and 31% in women.
  • a subject has one or more symptoms of sarcopenia, or is identified as being at risk of developing sarcopenia.
  • Systems and methods for the diagnosis of sarcopenia are known in the art, and the method can include selecting a subject having or at risk of having sarcopenia based on any one or more of the known systems or methods for diagnosing sarcopenia known in the art.
  • Exemplary methods for diagnosis of sarcopenia include methods proposed by the European Working Group on Sarcopenia in Older People (EWGSOP), methods proposed by the Asia Working Group for Sarcopenia (AWGS), methods proposed by the International Working Group on Sarcopenia (IWGS), methods proposed by Society for Sarcopenia Cachexia and Wasting Disorders (SCWD), and methods proposed by the Foundation for the National Institutes of Health (FNIH) Sarcopenia Project.
  • EWGSOP European Working Group on Sarcopenia in Older People
  • AGS Asia Working Group for Sarcopenia
  • IWGS International Working Group on Sarcopenia
  • SCWD Society for Sarcopenia Cachexia and Wasting Disorders
  • FNIH National Institutes of Health
  • identification of sarcopenia in a subject is based on one or more procedures including Screening assessment using the SARC-F scale; measuring hand grip strength (or performing a Chair Stand Test) to diagnose low muscle strength; estimating muscle quantity with dual-energy X-ray absorptiometry (DXA); bioelectrical impedance analysis (BIA); magnetic resonance imaging (MRI); and computed tomography (CT) of one or more regions of the body of 45718416.1 the subject.
  • the methods include one or more additional physical performance tests, such as the Timed-Up-and-Go test (TUG), Short Physical Performance Battery (SPPB), gait speed measurement, and a defined distance Walk Test, such as a 400 meter walk test.
  • these methods identify a subject as having sarcopenia.
  • the subject may have one or more additional or underlying medical indications in addition to one or more symptoms of sarcopenia.
  • a subject has type II diabetes mellitus, albuminuria, sarcopenic obesity, sarcopenic arterial stiffness and or peripheral neuropathy.
  • the subject does not have an underlying indication and is otherwise a healthy subject.
  • the subject does not have and/or has never had been diagnosed with a neurological disease.
  • the subject has not had or been diagnosed as having Alzheimer’s disease (AZ), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS).
  • AZ Alzheimer’s disease
  • PD Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • the subject has not had or has never been diagnosed as having dementia, depression, or mental illness. Because of several shared pathways between the two diseases, sarcopenia is also a risk factor for developing type 2 diabetes mellitus (T2DM) in older patients. Therefore, in some forms, the subject has or is identified as being at risk of having type 2 diabetes mellitus (T2DM). In Exemplary forms, the subject has a score of 4 or more on the SARC-F scale, such as a score of 5, 6, 7, 8, 9, or 10 on the SARC-F scale. Exemplary subjects are at least 40 years of age, or at least 50 years of age, or at least 60 years of age, or at least 70 years of age.
  • the subject does not have and/or has never been diagnosed as having one or more diseases or disorders selected from Alzheimer’s disease (AZ), Parkinson’s disease (PD) amyotrophic lateral sclerosis (ALS), dementia, Schizophrenia, multiple sclerosis, and depression.
  • AZ Alzheimer’s disease
  • PD Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • ALS dementia
  • Schizophrenia multiple sclerosis
  • depression depression.
  • Dosage and Effective Amounts Dosage and Dosing regimens are dependent on the severity and location of the disorder and/or methods of administration, as well as the specific agent being delivered. This can be determined by those skilled in the art.
  • an effective amount of dendrimer complexes including a combination of a dendrimer with one or more therapeutic GCPII inhibitor active agents are administered to an individual in need thereof.
  • the dendrimers may also include a targeting agent, but as demonstrated by the examples, these are not required for delivery to activated macrophage cells associated with the nervous system, neuromuscular junctions and muscles surrounding synapses.
  • the dosage of dendrimer complexes include an effective amount of one or more GCPII inhibitors attached or conjugated to a dendrimer, which are capable of preferentially releasing the drug at the target receptor.
  • the GCPII inhibitor agent can be either covalently attached or intra-molecularly 45718416.1 dispersed or encapsulated.
  • the amount of dendrimer complexes administered to the subject is selected to deliver an effective amount to reduce, prevent, or otherwise alleviate one or more clinical or molecular symptoms of the disease or disorder to be treated, such as sarcopenia, as compared to a control, for example, a subject treated with the active agent without dendrimer.
  • dosages are expressed in mg/kg, particularly when the expressed as an in vivo dosage of composition(s) of dendrimer-GCPII inhibitor conjugates.
  • the preferred route of administration is oral or subcutaneous injection
  • doses are in the range from microgram/kg up to about 100 mg/kg of body weight of the recipient.
  • Exemplary dosages are, for example 0.01 mg/kg to about 1,000 mg/kg, or 0.5 mg/kg to about 1,000 mg/kg, or 1 mg/kg to about 1,000 mg/kg, or about 10 mg/kg to about 500 mg/kg, or about 20 mg/kg to about 500 mg/kg per dose, or 20 mg/kg to about 100 mg/kg per dose, or 25 mg/kg to about 75 mg/kg per dose, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg/kg per dose. Efficacy has been shown at 20 mg/kg administered orally.
  • the compositions of dendrimer-GCPII inhibitor agents do not target or otherwise impact non-activated immune cells, or cells that not within or associated with impacted tissues/motoneurons, or do so at a reduced level compared to activated immune cells and motoneurons associated with a disease or disorder such as a sarcopenia.
  • dendrimer compositions are administered in an amount that leads to an improvement, or enhancement, function in an individual with a disease or disorder, such as sarcopenia or an associated disorder.
  • the actual effective amounts of the composition can vary according to factors including the specific agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder. Generally, for intravenous injection or infusion, the dosage will be lower than for oral administration. In general, the timing and frequency of administration will be adjusted to balance the efficacy of a given treatment or diagnostic schedule with the side effects of the given delivery system. In some embodiments, dosages are administered daily, biweekly, weekly, every two weeks or less frequently in an amount to provide a therapeutically effective increase in the blood level of the therapeutic agent.
  • the compositions may be delivered over a period of more than one hour, e.g., 3-10 hours, to produce a therapeutically effective dose within a 24-hour period.
  • the compositions can be formulated for controlled release, wherein the composition is administered as a single dose that is repeated on a regimen of once a week, or less frequently.
  • a 45718416.1 dosing regimen can be any length of time sufficient to treat the disorder in the subject.
  • the regimen includes one or more cycles of a round of therapy followed by a drug holiday (e.g., no drug).
  • the drug holiday 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. Any of the therapeutic effects of the described compositions including one or more dendrimer-GCPII inhibitor agents can be compared to a suitable control.
  • a control includes an equivalent amount of GCPII inhibitor agents delivered alone, or bound to dendrimers without a similar generation, molecular weight, and/or surface group density (e.g., hydroxyl groups).
  • the kit can include a single dose or a plurality of doses of a composition including one or more GCPII inhibitors associated with or conjugated to a dendrimer (e.g., one or more hydroxyl-terminated PAMAM dendrimers or glucose dendrimers as described in the Examples), and instructions for administering the compositions.
  • a dendrimer e.g., one or more hydroxyl-terminated PAMAM dendrimers or glucose dendrimers as described in the Examples
  • the instructions direct that an effective amount of the dendrimer composition be administered to an individual with 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. The present invention will be further understood by reference to the following non-limiting examples.
  • Example 1 Synthesis of Hydroxyl-Polyamidoamine (PAMAM-OH) dendrimer 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA) conjugates NAAG is synthesized pre-synaptically by NAAG synthetase and packaged into vesicles by sialin. After release, intact NAAG interacts with mGlu3 and NMDA receptors and/or is hydrolyzed by glial GCPII to release glutamate outside the synaptic cleft. Under “basal”/non-pathological conditions, GCPII activity and synaptic NAAG and glutamate concentrations are relatively low.
  • PAMAM-OH Hydroxyl-Polyamidoamine
  • 2-PMPA Phosphonomethyl)-pentanedioic Acid
  • NAAG modulates synaptic activity by activating presynaptic mGlu3 receptors and postsynaptic GluN2A-rich NMDA receptors. Further, NAAG that reaches the extra-synaptic space inhibits GluN2B-rich NMDA receptor-mediated EPSCs and stimulates glial mGlu3 receptors which induces trophic effects. Under pathologic conditions with increased synaptic activity, elevated levels of glutamate and NAAG flood the synapse. NAAG that reaches the extra-synaptic space is rapidly cleaved by GCPII to liberate glutamate. The excess glutamate and NAAG activate both GluN2A- and GluN2B-rich NMDA receptors, increasing EPSCs.
  • Blockade of GCPII under activated conditions prevents the breakdown of NAAG lowering overall glutamate levels.
  • the resulting increased NAAG further decreases glutamate release through feedback inhibition via presynaptic mGlu3 receptors and induces trophic effects via activation of glial mGlu3 receptors.
  • 45718416.1 inhibition of GCPII increases NAAG and lowers glutamate, returning the system toward its basal state.
  • the synthesis first included conjugation of 2-PMPA to a cleavable PEG linker in presence of Azido-PEG-11-alcohol (EDC/DMAP/DMF for 24 hours at room temperature) to form 2-PMP-PEG-N3, as depicted in FIG.1.
  • the synthesis included conjugation of G4 hydroxylated polyamidoamine (PAMAM-OH) dendrimer (“D-OH”) to the previously synthesized 2-PMP-PEG-N3, by first conjugating a 56-hexynoic acid to D-OH in presence of EDC, DMAP, DMF for 24 hours at room temperature to form D-hexyne, then click chemistry-based conjugation of D-hexyne with 2-PMP-PEG-N3 in the presence of CuSO4.5H2O Na Ascorbate (DMF/THF/H2O for 6 hours at 50 ⁇ C in a microwave) to form D-2-PMPA (5), having a cleavable ester linkage , as depicted in FIG.2.
  • PAMAM-OH G4 hydroxylated polyamidoamine
  • D-OH dendrimer
  • mice were housed at a maximum of 5 per cage on a 12-hour dark/light cycle and provided food and water ad libitum. Mice were administered either 200 mg/kg D-2- PMPA or vehicle (HEPES buffer) control via intraperitoneal (IP) injection 5 days per week for 10 months.
  • CD11b+ macrophage isolation CD11b+ enriched macrophage and CD11b- cells in muscle from mice treated with either HEPES vehicle or G4-D-2-PMPA were isolated employing Miltenyi Biotec's magnetic-activated cell 45718416.1 sorting (MACS) system.
  • MCS Miltenyi Biotec's magnetic-activated cell 45718416.1 sorting
  • mice calf muscles were freshly harvested and diced into small pieces in DMEM buffer and underwent digestion with the skeletal muscle dissociation kit (Miltenyi Biotec) for 1.5 hours.
  • the digested homogenates were then filtered through 50- ⁇ m mesh-size cell strainers to collect mononuclear cells.
  • the collected mononuclear cells were magnetically labeled with anti- CD11b MicroBeads.
  • the cells were drained through the MACS separators with MACS magnetic stands, where the CD11b- cells were collected from the drained-through solution, and the CD11b+ cells were adhered to the columns and subsequently collected after relief from the magnetic field.
  • Enzyme activity measurements The GCPII enzyme activities in CD11b+ and CD11b- cells were measured after the CD11b+ macrophage isolation process. Briefly, isolated cells were carried out radiolabeled NAAG hydrolysis assays for about 3 hours at 37 °C using [3H]-NAAG (0.02 ⁇ M, 48.6 mCi/ ⁇ mol). Reactions were terminated with ice-cold sodium phosphate buffer (100 mM, pH 7) with 1 mM EDTA.96 well spin columns packed with anion exchange resin were used to separate the substrate and the reaction product. The reaction product [3H]-glutamate was eluted with 1 M formic acid and analyzed for radioactivity.
  • MRI Magnetic resonance imaging
  • mice were performed on a 9.4T MRI scanner. Animals were anesthetized for imaging using isoflurane with 3-5% at 0.6 L/min in an induction chamber for induction, and 1-2% at 0.6 L/min for maintenance during imaging.
  • MR imaging a physiological monitoring and gating system designed specifically to meet the physiological monitoring and gating requirements for anesthetized small animals such as mice and rats in the MR environment of very high magnetic fields will be used.
  • Subdermal Needle Electrodes (0.5”27G) were placed both in the sciatic nerve (stimulatory) 45718416.1 and adjacent muscle tendons (reference) to stimulate the gastrocnemius muscle. Electrical current was individually adjusted to produce maximal isometric force before isometric force testing. The force versus frequency relationship was determined with 500 ms trains of pulses between 1 and 150 Hz. Rotarod performance The rotarod performance was recorded monthly during the treatment. Briefly, on the day of the experiment, the animals were trained to stay on the bar at 5 rpm for 5 mins. They were then assessed on their ability to remain on a steady rotating bar for 5 mins at 15rpm over 3 separate trials, allowing the animals a 5-minute rest between trials.
  • CMAP Compound muscle action potential
  • a brief ( ⁇ 0.2 ms) electrical pulse was applied at the stimulating sites and the CMAP will be recorded using an Evidence 3102evo EMG system (Schreiber & T noirtechnik, Germany). Three recordings were taken at the maximal stimulation and supramaximal stimulation intensities. The highest amplitude and corresponding latency were recorded.
  • Single fiber electromyography (sfEMG) sfEMG was performed with mice anesthetized using 1-2% isoflurane. To record sfEMG, a recording sfEMG needle was placed in the gastrocnemius muscle while a stimulating electrode was placed near the sciatic nerve. Axons were stimulated at a rate of 10Hz with a stimulation intensity between 10-30 mA to avoid visible muscle contractions.
  • mice muscle showed increased GCPII activity specifically in CD11b + enriched macrophage cells (138.6 ⁇ 3.0 vs.336.6 ⁇ 52.9; p ⁇ 0.05) but not in CD11b- cells.
  • CMAP compound muscle action potential
  • Example 3 Dendrimer-2-PMPA (D-2PMPA) preferentially deliver 2-PMPA to muscle macrophages
  • D-2PMPA Dendrimer-2-PMPA
  • Aged mice were dosed with 10 ⁇ mg/kg Cy5-D-2PMPA, and the soleus and EDL muscles were harvested 24 ⁇ hours post-administration. Muscle sections were stained with CD68 (green) for macrophages and Cy5 (red) for D-2PMPA. The merged images clearly demonstrated that Cy5-D- 2PMPA preferentially accumulates in CD68+ macrophages.
  • D-2PMPA Dendrimer-2-PMPA
  • D2PMPA was administered three times per week (Monday, Wednesday, Friday) from 15 to 20 months of age.
  • CMAP compound muscle action potentials
  • rotarod performance was evaluated using a rotarod test with the Rotamex system (Columbus Instruments®, OH, US). Latency to fall off of the rotarod was measured monthly to index changes in motor coordination.
  • a grip strength test was performed using the AMETEK DFE Digital Force Gauge (AMETEK®, PA, US) to evaluate muscle performance. Grip strength performance was measured monthly and reported as a percentage of a baseline measurement taken before the treatment period. All data were analyzed using a two-way ANOVA followed by ⁇ idák’s post-hoc corrections for multiple comparisons and were reported.
  • mice treated with D-2PMPA exhibited significantly longer latencies to fall compared to vehicle-treated controls (105.8 ⁇ 3.4 s versus 84.7 ⁇ 4.4 s, p ⁇ 0.01) (FIG 12A).
  • both sexes showed improvement. Specifically, female mice showed latencies of 112.2 ⁇ 5.3 s versus 89.8 ⁇ 7.6 s (p ⁇ 0.05), and male mice exhibited latencies of 100.3 ⁇ 4.1 s versus 79.1 ⁇ 3.9 s (p ⁇ 0.01).
  • mice After 5 months of D-2PMPA treatment, aged mice maintained 97.5 ⁇ 1.4% of their maximum initial force, compared to 89.9 ⁇ 1.2% in the vehicle group (p ⁇ 0.001) (FIG 12B). Analysis by sex revealed improvements in both groups: female mice preserved 99.1 ⁇ 1.9% versus 90.5 ⁇ 1.8% (p ⁇ 0.01), and male mice preserved 96.1 ⁇ 2.1% versus 89.3 ⁇ 1.7% (p ⁇ 0.05).
  • D-2PMPA treatment preserved Muscle Volume and Force in aged mice Methods Calf muscle volume was monitored using a Bruker 9.4T MRI from 15 to 20 months of age.
  • CMAPs Compound Muscle Action Potentials
  • CMAP compound muscle action potentials

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Abstract

Inhibition of glutamate-carboxypeptidase-II in and around the neuromuscular junctions (NMJ) of aged subjects can reduce age related reduction in muscle mass and strength. Compositions and methods for treating sarcopenia have been developed. Hydroxylated dendrimers conjugated with glutamate-carboxypeptidase-II (GCPII) inhibitors are administered in an amount effective to reduce, delay or reverse one or more symptoms or markers of sarcopenia. The hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor is in a dosage unit effective to reduce glutamate and/or increase NAAG within and/or around muscle macrophage and neuromuscular junctions (NMJ) in the subject.

Description

DENDRIMER COMPOSITIONS FOR TARGETED DELIVERY OF GCPII INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of and priority to U.S. provisional application No. 63/563,705, filed March 11, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant AG078181 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION This invention is generally in the field of drug formulations to prevent or restore muscle loss, more specifically dendrimer GCPII drug-conjugates. BACKGROUND OF THE INVENTION Sarcopenia has been defined as an age related, involuntary loss of skeletal muscle mass and strength. Beginning as early as the 4th decade of life, evidence suggests that skeletal muscle mass and skeletal muscle strength decline in a linear fashion, with up to 50% of mass being lost by the 8th decade of life. Age-related loss of muscle mass and strength (sarcopenia) significantly impairs quality of life in the elderly yet lacks effective treatments. Sarcopenia has a multifactorial cause, with declines in activity and nutrition, disease states, inflammation, degeneration or deterioration in neuromuscular junctions, and aging related changes in mitochondria, apoptosis, and the angiotensin system recently found to be contributory. The primary symptoms of sarcopenia include low muscle function, low muscle strength, and low muscle mass. The loss of muscle mass is accompanied by the accumulation of fat in the muscles, which contributes to decreased muscle strength. As reported by Goates, et al. J Frailty Aging 2019;8(2):93-99 doi: 10.14283/jfa.2019.10, according to the National Institutes of Health, the total estimated cost of hospitalizations in individuals with sarcopenia was USD $40.4 billion with an average per person cost of USD $260. Currently the only treatments for sarcopenia include nutritional therapy and exercise therapy. An intervention combining nutritional therapy with a comprehensive exercise program that includes resistance training is more effective than a single intervention in improving sarcopenia, however there remains a significant need for effective therapeutic interventions that do not rely upon physical or nutritional activity. It is an object of the present invention to provide compositions for the effective treatment of sarcopenia. 45718416.1 It is also an object of the present invention to provide effective treatment regimens reducing or preventing one or more symptoms of sarcopenia. SUMMARY OF THE INVENTION It has been discovered that GCPII inhibitors can be covalently conjugated to hydroxylated dendrimers to produce therapeutic and/or prophylactic agents to reduce muscle function decline and/or decrease deterioration of neuromuscular junctions. Methods for treating or preventing one or more symptoms associated with sarcopenia in a subject in need thereof are provided. Typically, the methods include administering to a subject a pharmaceutically acceptable composition including hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor. This is formulated into a dosage form to provide an effective amount for administration to a human for reducing, delaying, inhibiting or reversing one or more symptoms associated with sarcopenia in the subject as compared to an untreated control subject. In one embodiment, the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to reduce glutamate and/or increase NAAG in muscle macrophage and/or around neuromuscular junctions (NMJ) in the subject. In some forms, the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is provided in an amount effective to reduce the amount of one or more markers of sarcopenia in the subject. Exemplary markers are selected from follistatin (FST), adiponectin, leptin, decrease interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). In some forms, the methods are effective to increase myostatin (MSTN), decrease follistatin (FST), increase irisin, increase brain-derived neurotrophic factor (BDNF), decrease adiponectin, decrease leptin, increase insulin-like growth factor-1 (IGF-1); increase dehydroepiandrosterone sulphate (DHEAS), decrease C-reactive protein (CRP); decrease interleukin-6 (IL-6), or decrease tumor necrosis factor α (TNF-α) in the subject, or combinations thereof. In some forms, the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to increase serum creatinine to serum cystatin C ratio in the subject. Exemplary symptoms of sarcopenia that can be treated or prevented by the conjugate include lack of strength, such as lack of hand-grip or limb strength, need for assistance in walking, difficulty rising from a chair, difficulty climbing stairs, increased number of falls, reduced muscle mass, reduced balance, and reduced muscular control. In some forms, the subject has a score of 4 or more on the SARC-F scale. Exemplary subjects are at least 40 years of age, 50 years of age, 60 years of age, or 70 years of age. In some forms, the subject does not have and/or has never been diagnosed as having one or more diseases or disorders selected from Alzheimer’s disease (AZ), Parkinson’s disease (PD) amyotrophic lateral sclerosis (ALS), dementia, Schizophrenia, multiple sclerosis, and depression. In other forms, the subject has, or is identified as being at risk of having, 45718416.1 one or more diseases or disorders selected from type II diabetes mellitus, albuminuria, sarcopenic obesity, sarcopenic arterial stiffness, and peripheral neuropathy. Methods for reducing, preventing or delating neuromuscular junction (NMJ) denervation or deterioration in a subject are also provided. Typically, the methods include administering to a human subject a pharmaceutically acceptable composition including hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor in an effective amount for reducing, delaying, inhibiting or reversing glutamate and/or increasing NAAG from muscle macrophage and/or around neuromuscular junctions (NMJ) in the subject. In some forms, the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor that is administered to the subject is effective to provide binding of the GCPII inhibitor to the GCPII enzyme on the surface of or inside target cells. Exemplary target cells include neuronal cells, glial cells, Schwann cells, and activated macrophages. Typically, the dendrimer includes a generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, or generation 8 dendrimer. In a preferred embodiment, the dendrimer is a poly(amidoamine) (PAMAM) dendrimer, preferably having greater than 40% or 50% free hydroxyl surface groups. In certain forms, the dendrimer is a generation 4 PAMAM dendrimer. In some forms, the dendrimer includes one or more surface-bound monosaccharides. Exemplary monosaccharides include glucose, galactose, glucosamine, galactose, mannose, and fructose. In some forms the dendrimer includes ten or more surface monosaccharide moieties. In some forms, the dendrimer is linked to the GCPII inhibitor via a spacer. In some forms, the spacer includes a cleavable linkage to the dendrimer. An exemplary cleavable linkage is selected from the group including ester, disulfide, phosphodiester, tri-glycyl peptide, and hydrazine linkages. In other forms, the spacer includes a non-cleavable linkage to the dendrimer. An exemplary non-cleavable linkage is selected from the group including amide, ether, and amino alkyl linkages. In certain forms, the spacer linking the dendrimer and the GCPII inhibitor includes a hydrocarbon such as an alkylene, a diethylene glycol moiety, and/or oligoethylene glycol chain. An exemplary spacer includes a triazole moiety. Exemplary GCPII inhibitors include 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA), ZJ-43, ZJ-11, ZJ-17, ZJ-38, VA-033, quisqualic acid, 2- [[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (GPI-5232), 2- (3-mercaptopropyl)pentanedioic acid (2-MPPA), 3-(2-carboxy-5-mercaptopentyl)benzoic acid, 3- (1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid, N- substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, phenylalkylphosphonamidates, 45718416.1 NAAG peptide analogs, and glutamate derivatives. In certain forms, the GCPII inhibitor includes 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA). In some forms, the composition provides sustained release of the GCPII-dendrimer conjugate to yield an effective amount in the subject for a period of 24 hours. Typically, the composition is formulated for systemic administration. In some forms the composition is formulated for enteral or parenteral administration. Exemplary compositions are formulated for oral, mucosal (intranasal, buccal, rectal, vaginal, sublingual, pulmonary), intramuscular, intravenous, or subcutaneous. For example, in some forms, the composition is in a form such as a hydrogel, nanoparticles or microparticles, suspensions, powders, tablets, capsules, or solutions. BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is a schematic depicting a synthetic route for producing an exemplary GCPII inhibitor conjugate with an enzyme sensitive ester linkage, showing conjugation of GCPII inhibitor, 2-PMPA (1), to a cleavable PEG linker in presence of Azido-PEG-11-alcohol (EDC/DMAP/DMF for 24 hours at room temperature) to form 2-PMP-PEG-N3(2). FIG 2 is a schematic showing conjugation of hydroxylated (OH) dendrimer (d) (D-OH; (3)) to the 2-PMP-PEG-N3 of FIG.1, by first conjugating a 56-hexynoic acid to (3) in presence of EDC, DMAP, DMF for 24 hours at room temperature to form D-hexyne (4), then click chemistry-based conjugation of (4) with (2) in the presence of CuSO4.5H2O Na Ascorbate (DMF/THF/H2O for 6 hours at 50 ˚C in a microwave) to form D-2-PMPA (5), having a cleavable ester linkage (indicated). FIGs 3A-3D are graphs of the comparative IC50s determined using human recombinant GCPII, showing %inhibition (-25%-125%) over concentration (Log M) for each of 2-PMPA alone (Fig.3A), 2-PMPA-PEG-Azide (Fig.3B), D-2-PMPA conjugate (Fig.3C), and D-OH alone (Fig. 3D), respectively. The structures of each respective molecule used in the assay, as well as the molecular weight are depicted at left of each graph, and the calculated IC50 value for each molecule is indicated under each curve, respectively. FIG 4 is a histogram of GCPII enzyme activity in mice muscle CD11b+ macrophage cells, showing GCPII Activity (0-600 fmol/mg/h) for each of young, old vehicle (controls) and Old D-2- PMPA, samples, respectively. Aged mice muscle showed increased GCPII activity specifically in CD11b+ enriched macrophage cells (138.6±3.0 vs.336.6±52.9; p<0.05), but not in CD11b- cells. Systemic D-2-PMPA therapy (20 mg/kg 2-PMPA equivalent; IP 3 × /week) completely inhibited the elevated GCPII activity (336.5±52.8 vs.21.8±9.2 fmol/mg/h; p<0.001). FIG 5 is a graph of mice muscle volume preservation after 5 months’ treatment, showing 20-month calf muscle volume relative to 15 month old (%) in each of groups treated with D- 2PMPA, and vehicle (control), respectively.5-months of D-2-PMPA therapy initiated with 15- 45718416.1 month-old mice led to significant preservation of calf muscle volume (95.0±0.8% vs.90.4±0.7%; p < 0.001). FIGs 6A-6B are graphs of mice muscle isometric force (Stim Frequency at 80 Hz), showing isometric force (0-400 mN) for each of groups treated with D-2-PMPA or vehicle (control), respectively, based on assays in female (Fig.6A) and male (Fig.6B) test animals, respectively. D- 2-PMPA enhanced isometric force (i.e., to 230.2±13.3 vs.184.0±9.0 mN, female p<0.05; and to 257.3±21.0 vs.189.7±19.3 mN, male p<0.05). FIG 7 is a graph of mice all limb grip strength preservation after 5 months’ treatment, showing 20-month all limb grip strength relative to 15 month old (%) in each of groups treated with D-2PMPA, and vehicle (control), respectively.5-months of D-2-PMPA therapy initiated with 15- month-old mice led to significant preservation of calf muscle volume (95.0±0.8% vs.90.4±0.7%; p < 0.001). D-2-PMPA improved grip strength (i.e., 96.1±1.6 vs.90.2±2.2 % of maximum p<0.001). FIGs 8A-8B are graphs of mice rotarod latency after 5 months’ treatment, showing rotarod latency (0-200 seconds) for each of groups treated with D-2-PMPA or vehicle (control), respectively, based on assays in female (Fig.8A) and male (Fig.8B) test animals, respectively. D-2- PMPA enhanced rotarod latency (i.e., from 119.0±6.6 vs.93.5±9.3 s, female p<0.05; and 104.5±5.2 vs.79.3±4.2 s, male p<0.001). FIGs 9A-9B are graphs depicting improved compound muscle action potential (CMAP) latency, showing CMAP latency (1.25-2.00 ms) (Fig.9A) and CMAP amplitude voltage (0-20 mV) (Fig.9B), respectively, for each of groups treated with D-2-PMPA or vehicle (control), respectively. D-2-PMPA improved compound muscle action potential (CMAP) latency (1.28±0.02 vs.1.38±0.04 ms; p< 0.05) and amplitude (15.2±0.5 vs.1.27±0.6 mV; p<0.01). FIG 10 is a graph of single fiber EMG (Jitter) of gastrocnemius (GTN) muscle after 5 months’ treatment, showing mean consecutive difference (2.5-12.5 µs) in each of groups treated with D-2PMPA, and vehicle (control), respectively. NMJ integrity was preserved with treatment as demonstrated by single fiber jitter (5.4±0.3 vs.7.5±0.5 µs; p<0.01). FIG 11 is a schematic showing experimental design for D-2PMPA treatment of mice from 15-20 months of age from baseline (15 month of age) to endpoint MRI (isometric force), with Motor function monitored monthly measurements of compound muscle action potentials (CMAP) grip strength and rotarod performance. FIGs 12A-12B are histograms showing rotarod latency to fall (s) over age (months) for each of vehicle (●) and D2PMPA (■), respectively (Fig.12A); and showing grip strength (% of initial) over age (months) for each of vehicle (●) and D2PMPA (■), respectively (Fig.12B). *P<0.05, 45718416.1 **P<0.01, ***P<0.001, ****P<0.0001, effect of treatment. ##P<0.01, ###P<0.001, ####P<0.0001, effect of time. FIGs 13A-13C are histograms showing calf muscle volume preservation (%) for each of vehicle and D2PMPA, respectively, in each of male (Fig.13A), female (Fig.13B), and combined (Fig.13C), groups, respectively. *P<0.05, ***P<0.001, ****P<0.0001. FIGs 13D-13F are histograms showing isometric force (mN) for each of vehicle and D2PMPA, respectively, in each of male (Fig.13D), female (Fig.13E), and combined (Fig.13F), groups, respectively. *P < 0.05. FIGs 14A-14D are histograms showing ankle stimulation CMAP amplitude (mV) (Fig.14A); Hip stimulation CMAP amplitude (mV) (Fig.14B); ankle stimulation CMAP latency (s) (Fig.14C); and Hip stimulation CMAP latency (s) (Fig.14D), respectively, over age (months) for each of vehicle (●) and D2PMPA (■), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, effect of treatment. #P < 0.05, ###P < 0.001, ####P < 0.0001, effect of time. DETAILED DESCRIPTION OF THE INVENTION I. Definitions The terms “active agent” or “biologically active agent” are used interchangeably to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, which may be prophylactic, therapeutic, or diagnostic. These may be a nucleic acid, a nucleic acid analog, a small molecule having a molecular weight less than 2 kD, more typically less than 1 kD, a peptidomimetic, a protein or peptide, carbohydrate or sugar, lipid, or a combination thereof. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of agents, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs. The term “therapeutic agent” refers to an agent that can be administered to treat one or more symptoms of a disease or disorder. The term “diagnostic agent” generally refers to an agent that can be administered to reveal, pinpoint, and define the localization of a pathological process. The diagnostic agents can label target cells that allow subsequent detection or imaging of these labeled target cells. “Analog” as relates to a given compound, refers to another compound that is structurally similar, functionally similar, or both, to the specified compound. Structural similarity can be determined using any criterion known in the art, such as the Tanimoto coefficient that provides a quantitative measure of similarity between two compounds based on their molecular descriptors. Preferably, the molecular descriptors are 2D properties such as fingerprints, topological indices, and maximum common substructures, or 3D properties such as overall shape, and molecular fields. Tanimoto coefficients range between zero and one, inclusive, for dissimilar and identical pairs of 45718416.1 molecules, respectively. A compound can be considered an analog of a specified compound, if it has a Tanimoto coefficient with the specified compound between 0.5 and 1.0, inclusive, preferably between 0.7 and 1.0, inclusive, most preferably between 0.85 and 1.0, inclusive. A compound is functionally similar to a specified compound, if it induces the same pharmacological effect, physiological effect, or both, as the specified compound. “Analog” can also refer to a modification including, but not limited to, hydrolysis, reduction, or oxidation products, of the compounds. Hydrolysis, reduction, and oxidation reactions are known in the art. The term “therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and/or onto dendrimers, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term “effective amount” refers to an amount of a therapeutic agent or prophylactic agent to reduce or diminish the symptoms of one or more diseases. The terms “inhibit” or “reduce” in the context of inhibition, mean to reduce, or decrease in activity and quantity. This can be a complete inhibition or reduction in activity or quantity, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, dendrimer compositions including one or more inhibitors may inhibit or reduce the activity and/or quantity of diseased neurons by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from the activity and/or quantity of the same cells in equivalent tissues of subjects that did not receive, or were not treated with, the dendrimer compositions. In some embodiments, the inhibition and reduction are compared at levels of mRNAs, proteins, cells, tissues, and organs. For example, an inhibition and reduction in the rate of neural loss, in the rate of decrease of muscle weight, or in the rate of decrease of muscle volume, as compared to an untreated control subject. The term “treating” or “preventing” means to ameliorate, reduce or otherwise stop a disease, disorder or condition from occurring or progressing in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by 45718416.1 administration of an analgesic agent even though such agent does not treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with sarcopenia are mitigated or eliminated, including, but are not limited to, reducing the level of anxiety, agitation, or restlessness, improving feelings of sadness, tearfulness, emptiness or hopelessness, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease. The phrase “pharmaceutically acceptable” or “biocompatible” refers to compositions, polymers, and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions, or vehicles, such as a liquid or solid filler, diluent, solvent, or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. The term “biodegradable” generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted in vivo. The degradation time is a function of composition and morphology. The term “dendrimer” includes, but is not limited to, a molecular architecture with an interior core, interior layers, or “generations” of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation. The term “functionalize” means to modify a compound or molecule in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by the introduction of a molecule that makes the molecule a strong nucleophile or strong electrophile. The term “targeting moiety” refers to a moiety that localizes to or away from a specific location. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The location may be a tissue, a particular cell type, a subcellular compartment, or a molecule such as a receptor. The term “prolonged residence time” refers to an increase in the time required for an agent to be cleared from a patient’s body, or organ or tissue of that patient. In certain embodiments, 45718416.1 “prolonged residence time” refers to an agent that is cleared with a half-life that is 10%, 20%, 50% or 75% longer than a standard of comparison such as a comparable agent without conjugation to a delivery vehicle such as a dendrimer. In certain embodiments, “prolonged residence time” refers to an agent that is cleared with a half-life of 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 target specific cell types. The terms “incorporated” and “encapsulated” refer to incorporating, formulating, or otherwise including an agent into and/or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The agent or other material can be incorporated into a dendrimer, by binding to one or more surface functional groups of such dendrimer (by covalent, ionic, or other binding interaction), by physical admixture, by enveloping the agent within the dendritic structure, and/or by encapsulating the agent inside the dendritic structure. As used herein, central nervous system (“CNS”) includes the brain and spinal cord. As used herein, peripheral nervous system (“PNS”) refers to the nerves other than in the brain and spinal cord. “Hydroxyl-terminated,” as relates to dendrimers, refers to dendrimers that have a hydroxyl group on their surface. These hydroxyl groups are not attached to the termini of the dendrimers via a sugar moiety (such as a saccharide moiety). “Sugar-terminated,” as relates to dendrimers, refers to dendrimers that contain a sugar moiety (such as a saccharide moiety) on their surface and not in their core. “Sugar-based,” as relates to dendrimers, refers to dendrimers that contain a sugar moiety (such as a saccharide moiety) in their core, or their core and on their surface. II. Compositions Compositions of dendrimers conjugated or complexed with one or more inhibitors of glutamate carboxypeptidase II (GCPII) have been developed. The compositions are particularly suited for treating and/or ameliorating one or more symptoms of age related reduction of muscle mass and strength. In exemplary embodiments, the dendrimers are glucose dendrimers or hydroxyl terminated dendrimers such as hydroxyl terminated PAMAM or sugar modified dendrimers, and the GCPII inhibitor is 2-phosphonomethyl-pentanedioic acid (2-PMPA). A. Glutamate carboxypeptidase II (GCPII) Inhibitor Active Agents Compositions including active agents that reduce, prevent or otherwise inhibit the biological functions of the Glutamate carboxypeptidase II (GCPII) enzyme are described. The GCPII inhibitor 45718416.1 is typically selectively specific to the target GCP enzymes. Typically, the GCPII inhibitors inhibit the biological function of GCPII in vivo. In some forms, the inhibitor of GCPII is a small molecule, a peptide, a lipid, a nucleic acid, a synthetic polymer, or combinations thereof. Typically, the GCPII inhibitor is a small molecule drug, for example, that binds to one or more active sites of biologically active GCPII and prevents, reduces or otherwise mediates the function of the GCPII relative to a GCPII in the absence of the inhibitor. Typically, the GCPII inhibitors and/or their derivatives bind to the GCPII enzyme on the surface of the target cell or inside the target cell. In some forms, the inhibitors of GCPII include a structure that is a glutarate moiety that binds to the C-terminal glutamate recognition site of GCPII, and or a zinc chelating group to coordinate the divalent zinc atoms at the enzyme’s active site, or both. 1. Glutamate carboxypeptidase II (GCPII) Glutamate carboxypeptidase II (GCPII), which is also known as prostate-specific membrane antigen (PSMA), N-acetylated-α-linked acidic dipeptidase (NAALADase), N-acetylaspartyl- glutamate (NAAG) peptidase, and folate hydrolase (FOLH1), is a type II extracellular membrane zinc metallopeptidase that catalyzes synaptically released N-acetylaspartylglutamate (NAAG) to form N-acetylaspartate (NAA) and glutamate in the nervous system. GCPII is an extracellular, glial enzyme with its active site in the extra-synaptic space, making it ideally positioned to control whether NAAG functions to block or drive glutamatergic transmission. Under basal conditions, GCPII activity appears low, permitting NAAG to function as an intact dipeptide. However, under conditions of high synaptic activity, NAAG release and its cleavage by GCPII is enhanced, serving to liberate glutamate that subsequently activates extra- synaptic glutamatergic receptors on surrounding neurons and glia. GCPII inhibitors appear to reverse this activated state, decreasing glutamate release and increasing NAAG, returning the system to its basal state. Glutamate is the primary excitatory neurotransmitter of the central nervous system (CNS), which has a crucial role in a complex communication network established between all residential brain cells. An excess release of glutamate leads to the activation of ionotropic and metabotropic receptors, resulting in the accumulation of toxic cytoplasmic Ca2+ and neuronal cell death, as observed in multiple neurological disorders like neuropathic pain, stroke, diabetic neuropathy, schizophrenia, addiction, multiple sclerosis, and traumatic brain injury. It has also been shown that GCPII is implicated in inflammatory bowel disease (IBD), as well as in the preclinical model of ovarian cancer. 45718416.1 A series of pioneering studies have demonstrated the ability of GCP-II inhibitors to be therapeutically beneficial in cases of glutamate-mediated neuronal damage from brain injuries and neurological. Under pathologic conditions with increased synaptic activity, elevated levels of glutamate and NAAG flood the synapse. NAAG that reaches the extra-synaptic space is rapidly cleaved by GCPII to liberate glutamate. The excess glutamate and NAAG activate both GluN2A- and GluN2B-rich NMDA receptors, increasing excitatory postsynaptic currents (EPSCs). These extra-synaptic NMDARs typically are enriched in GluN2B subunits, and are thought to constitute a major signaling pathway that triggers neuronal death. Blockade of GCPII under activated conditions prevents the breakdown of NAAG lowering overall glutamate levels. The resulting increased NAAG further decreases glutamate release through feedback inhibition via presynaptic mGlu3 receptors and induces trophic effects via activation of glial mGlu3 receptors. Overall, inhibition of GCPII increases NAAG and lowers glutamate, returning the system toward its basal state. Numerous studies have also shown that an increase in NAAG is neuroprotective against NMDA receptor mediated neurotoxicity without adverse side effects. Furthermore, GCP-II knockout mice exhibited a significantly smaller infarct volume than control littermates in a model of ischemic injury and displayed normal neurological function and behavior. 2. 2-Phosphonomethyl-pentanedioic acid (2-PMPA) In some forms, the GCPII inhibitor is 2-Phosphonomethyl-pentanedioic acid (2-PMPA). 2-PMPA is a phosphonate analogue of glutamate and is highly selective for GCPII with characteristics of low-molecular-weight and high aqueous solubility, having an IC50 value for GCPII of 0.3 nM. The pentanedioic acid portion of the inhibitor was designed to interact with the glutarate recognition site of GCPII while the phosphonate group was utilized to chelate to the active site zinc ions.2-PMPA was characterized as a competitive inhibitor with an IC50 value of 300 pM with exquisite selectivity having no activity at over 100 different transporter, enzymes and receptors, including several glutamate targets. A recently published study has reported a noninvasive method for delivering GCPII inhibitors to the brain via intranasal (i.n.) administration on rodents and nonhuman primates, where i.n. administration of 2-PMPA exhibited the highest level of brain penetration compared to intraperitoneal administration. 45718416.1 The structure of 2-PMPA is depicted in STRUCTURE A, below: STRUCTURE A: 2- . 3. Other GCPII Inhibitors In some forms, the inhibitor of GCPII is not 2-PMPA. Exemplary GCPII inhibitor molecules are described, for example, in Barinka et al., Curr Med Chem.2012; 19(6): 856–870. In some forms, the inhibitor of GCPII is one or more inhibitors including, but not limited to, a thiol- based inhibitor compound such as 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI-5693); 2-[[hydroxy[2,3,4,5,6 pentafluorophenyl)methyl]phosphinyl] methyl] pentanedoic acid (also known as GPI-5232); a 2-PMPA derivative/urea-based inhibitor compound such as ZJ-43, ZJ 11, ZJ 17, and ZJ 38; a 2-PMPA derivative compound such as 3-(2-carboxy-5- mercaptopentyl)benzoic acid, 3-(1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4- mercaptobutyl)thio]benzoic acid, N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acids; VA-033; phenylalkylphosphonamidates; non-hydrolyzable or conformationally restricted NAAG peptide analogs; glutamate derivatives and quisqualic acid. In some forms, the inhibitor of GCPII is 2-(3-mercaptopropyl)pentanedioic acid, also known as 2-MPPA, also known as GPI-5693, having an IC50 for the GCPII of 90 nM, and having a structure as set forth in STRUCTURE B, below. 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI-5693). 45718416.1 In some forms, the inhibitor of GCPII is 3-(2-carboxy-5-mercaptopentyl)benzoic acid, having an IC50 for the GCPII of 15 nM, and having a structure as set forth in STRUCTURE C, below. 3-(2-carboxy-5-mercaptopentyl)benzoic acid. In some forms, the inhibitor of GCPII is 3-(1-carboxy-4-mercaptobutoxy) benzoic acid, having an IC50 for the GCPII of 14 nM, and having a structure as set forth in STRUCTURE D, below. 3-(1-carboxy-4-mercaptobutoxy) benzoic acid. In some forms, the inhibitor of GCPII is 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid, having an IC50 for the CGPII of 32 nM, and having a structure as set forth in STRUCTURE E, below. 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid. 45718416.1 In some forms, the inhibitor of GCPII is 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, having an IC50 for the CGPII of 22 nM, and having a structure as set forth in STRUCTURE F, below. 3-(2- acid. In some forms, the inhibitor of GCPII is 2- [[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (also known as GPI-5232), having an IC50 for the GCPII of 82 nM, and having a structure as set forth in STRUCTURE G, below. 2-[[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (also known as GPI-5232). 45718416.1 In some forms, the inhibitor of GCPII is VA-033, having an IC50 for the GCPII of 12 nM, and having a structure as set forth in STRUCTURE H, below. In some forms, the inhibitor of GCPII is a prodrug of and/or derivative of 2-PMPA, such as ZJ-43, having a Ki for the GCPII of 0.8 nM, and having a structure as set forth in STRUCTURE J, below. ZJ-43. In some forms, the inhibitor of GCPII is a hydroxamate-based inhibitor having a core structure as set forth in STRUCTURE K, below. JHU 241. 45718416.1 B. Dendrimers Dendrimers are three-dimensional, hyperbranched, monodispersed, globular and polyvalent macromolecules including surface end groups (Tomalia, D. A., et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). The term “dendrimer” includes, but is not limited to, a molecular architecture with an interior core (“G0”) and layers (or “generations”) of repeating units which are attached to and extend from this interior core, each layer having one or more branching points, and an exterior surface of terminal groups attached to the outermost generation. In some embodiments, dendrimers have regular dendrimeric or “starburst” molecular structures. Generally, the dendrimers have a diameter between about 1 nm and about 60 nm, more preferably between about 1 nm and about 50 nm, between about 1 nm and about 40 nm, between about 1 nm and about 30 nm, 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 to about 2 nm. The preferred size of the dendrimers for crossing the blood brain barrier (“BBB”) is less than 5 nm, whereas those for not crossing the BBB and staying in the peripheral circulation are greater than 5 nm. In some embodiments, the dendrimers have a diameter effective to penetrate BBB and to be retained close to or within target neural and/or glial cells for delivery of the agents conjugated thereto. In some embodiments, the dendrimers have a diameter effective to penetrate a BBB and to be internalized into target neural and/or glial cells for delivery of the agents conjugated thereto, such as for example, neurons, oligodendrocytes, astrocytes, microglial, and neuroglial support cells. In some embodiments, the dendrimers have a diameter effective to penetrate a barrier interface, such as a blood nerve barrier (“BNB”), and to be internalized into neural and/or glial cells of the peripheral nervous system for delivery of the agents conjugated thereto such as for example, motoneurons, activated macrophage cells, Schwann cells, satellite cells, and neuroglial support cells. In some embodiments, the dendrimers have a diameter effective to be retained in the peripheral circulation for delivery of the agents conjugated thereto to target cells of the peripheral nervous system e.g., perisynaptic Schwann cells and glia, neuromuscular junctions (NMJ), and musculature. A major benefit of the use of dendrimer conjugates is the ability of the dendrimer to enhance the binding of the inhibitor of GCPII to its target receptor on target cells, for example, binding compounds to the GCPII on motoneurons and activated macrophage cells in the affected areas of the CNS, NMJ, and surrounding musculature. 45718416.1 In some embodiments, dendrimers have a molecular weight between about 500 Daltons and about 100,000 Daltons inclusive, between about 500 Daltons and about 50,000 Daltons inclusive, or between about 1,000 Daltons and about 20,000 Daltons inclusive. Dendrimer sizes of less than 30,000 Da are preferred for transport across the BBB, and sizes of greater than 50,000 Da are preferred for confinement to the periphery. In some embodiments, the dendrimers have 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 the prior layer of monomers via linkers such as polyethylene glycol chains. In some forms, the hypercore is dipentaerythritol and the monosaccharide branching unit is glucose-based branching unit, such as shown in Structures II-IV. In some forms, the dendrimers are made entirely of glucose building blocks. Exemplary dendrimers are G1 to G3 glucose dendrimers, such as G1, G2, and/or G3 glucose dendrimers. Suitable dendrimers scaffolds for use in the conjugates include, but are not limited to, poly(amidoamine), also known as PAMAM, or STARBURST™ dendrimers; polypropylamine (POPAM), polyethylenimine, polylysine, polyester, iptycene, aliphatic poly(ether), aromatic polyether dendrimers, dendrimer of a sugar (e.g., glucose, galactose, mannose, fructose, etc.), and copolymers thereof, such as a copolymer of a sugar and an alkylene glycol (e.g., a dendrimer formed by glucose and ethylene glycol building blocks). The dendrimers can have a plurality of surface functional groups, such as carboxylic, amine, hydroxyl, and/or acetamide. The terms “surface functional groups” and “terminal groups” are used interchangeably herein. In some embodiments, the dendrimers have surface hydroxyl groups. In some embodiments, one or more of these surface functional groups are further modified with other molecules, such as further modified with a sugar (e.g., glucose, galactose, mannose, fructose, etc.) and/or a polyalkylene glycol, for example, polyethylene glycol, and thus have sugar molecules and/or polyalkylene glycols as terminal moieties/molecules. Dendrimers can be any generation including, but not limited to, generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, generation 8, generation 9, or generation 10. In some preferred embodiments, the dendrimers are PAMAM dendrimers used as a platform and modified with functional groups for increased number of surface hydroxyl groups. Preferred PAMAM dendrimers include hydroxylated PAMAM dendrimers, particularly G3 to G6 hydroxyl-terminated PAMAM dendrimers, such as G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers. In some embodiments, the dendrimer-active agent conjugates can be confined to the peripheral circulation and specifically target a particular tissue region and/or cell type, e.g., 45718416.1 activated macrophages and Schwaan cells in the affected areas of the NMJ, and surrounding musculature, by using higher generation dendrimer (such as generation 4, 5, or 6 PAMAM dendrimer, generation 2, 3, or higher glucose-based dendrimers). Additionally, or alternatively, the dendrimer-active agent conjugates can be confined to the peripheral circulation by appropriate functionalization of the dendrimer (such as PEGylation). In some embodiments, the dendrimers can specifically target a particular tissue region and/or cell type of the central nervous system (CNS), the peripheral nervous system (PNS), and/or the periphery, especially activated macrophages and Schwann cells by using dendrimers of a certain generation, such as PAMAM dendrimers and/or glucose dendrimers of generation 2 (G2), G3, G4, and G5. 1. Monosaccharide-based Dendrimers In preferred embodiments, the branching units include monosaccharides. In some embodiments, the monosaccharide branching units are conjugated to the core or the prior layer of monomers via linkers such as polyethylene glycol chains. In some forms, the monosaccharide branching units are glucose-based branching units. In some embodiments, the branching units can include PEG and/or alkyl chain linkers between different dendrimer generations. For example, the glucose layers are connected via PEG linkers and triazole rings. In some embodiments, the branching units are the same for each generation of dendrimers generated from the core. Therefore, for example, the branching units are glucose-based branching units for generating generation 1 dendrimers, for generating generation 2 dendrimers, and for generating generation 3 dendrimers. In some embodiments, the dendrimers have a hypercore such as dipentaerythritol and one or more monosaccharide branching units. In some embodiments, the hypercore is dipentaerythritol and the monosaccharide branching unit is glucose-based branching unit. In further embodiments, spacer molecules can also be alkyl (CH2)n–hydrocarbon-like units. In some embodiments, dendrimers synthesized using glucose building blocks, with a surface made predominantly of glucose moieties, specifically targets cells including injured neurons, ganglion cells, and other neuronal cells in the brain, the eye, and/or in peripheral nervous system. In some embodiments, the glucose-based dendrimer selectively targets or is enriched inside target neural and/or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or enriches the surface of target neural and/or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched inside target neuronal cells and on the surface of the target neural and/or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched inside and/or on the surface of injured, diseased, and/or hyperactive neurons and/or glial cells. 45718416.1 In some cases, the dendrimers include an effective number of sugar molecules and terminal groups, for example, glucose and/or hydroxyl groups, for targeting to one or more neurons and/or glia of the CNS, PNS, and/or the eye. The terminal hydroxyl groups of these dendrimers may be part of terminal glucose molecules or extra hydroxyl groups that are not part of the glucose molecules, or a combination thereof. In some embodiments, all the terminal hydroxyl groups are part of the terminal glucose molecules. In some embodiments, the number of sugar molecules on the termination of dendrimer is determined by the generation number. In some embodiments, dendrimers are made of glucose and oligoethylene glycol building blocks. Exemplary glucose dendrimers are shown in Structures V and VII. Some exemplary glucose dendrimers include a generation 1 glucose dendrimer having 24 hydroxyl (-OH) end groups, a generation 2 glucose dendrimer having 96 hydroxyl (-OH) end groups, a generation 3 glucose dendrimer having 396 hydroxyl (-OH) end groups, and generation 4 glucose dendrimer having 1584 hydroxyl (-OH) end groups. For example, the glucose dendrimer is a generation 2 glucose-based dendrimer that has 24 glucose molecules at the periphery and 6 embedded glucose molecules in the backbone held together by PEG segments. Dendrimer compositions that can selectively accumulate inside neurons, particularly in the nucleus of injured and/or hyperactive neurons, referred to as “glucose dendrimers” also accumulate at a high level inside activated microglia. However, compared to hydroxyl dendrimers which primarily accumulate in microglia, these dendrimers primarily go to neurons. Glucose dendrimers are described in PCT/US23/17548 “Dendrimer Compositions for Targeted Delivery of Therapeutics to Neurons” by The Johns Hopkins University. Glucose dendrimers include (a) a central core, (b) one or more branching units, wherein the branching units are monosaccharide glucose-based branching units, optionally with a linker conjugated thereto; and optionally (c) one or more therapeutic, prophylactic and/or diagnostic agents. Generally, 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 hexa-propargylated derivative thereof. In some embodiments, the branching unit is conjugated to the central core via a linker such as a hydrocarbon or an oligoethylene glycol chain. In a preferred embodiment, the branching units are β-D- Glucopyranoside tetraethylene glycol azide having the structure of STRUCTURE L, or peracetylated derivatives thereof. 45718416.1 STRUCTURE L: β-D-Glucopyranoside tetraethylene glycol azide In some embodiments, the glucose dendrimer is a generation 1, generation 2, generation 3, generation 4, generation 5, or generation 6 dendrimer. In one embodiment, the dendrimer is a generation 1 dendrimer having the following structure: In some forms, the dendrimer is a generation 2 dendrimer having the following structure: 45718416.1
STRUCTURE N: Exemplary G2 Glucose dendrimer. In some embodiments, GCPII inhibitor agents, and/or diagnostic agents are encapsulated, associated, and/or conjugated in the dendrimer, at a concentration of between about 0.01% to about 30%, preferably about 1% to about 20%, more preferably about 5% to about 20% by weight. The dendrimers may also be conjugated to one or more diagnostic agents such as fluorescent dyes, near infra-red dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. In some embodiments, the dendrimer and the agent(s) are conjugated via one or more linkers or coupling agents such as one or more hydrocarbon or oligoethylene glycol chains. Exemplary linkages are disulfide, ester, ether, thioester, and amide linkages. 45718416.1 2. Core In some embodiments, dendrimers are prepared using methods in which the dendrimer is assembled from a multifunctional core, which is extended outward by a series of reactions. A multifunctional core moiety allows stepwise addition of branching units (i.e., generations) around the core. Exemplary chemical structures suitable as core moieties 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-hexachloro-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 core, nitrilotrimethanol, ethylene diamine, propane-1,3-diamine, butane-1,4-diamine, 2,2',2''- nitrilotris(ethan-1-ol), alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, Cucurbituril, benzene-1,2,3,4,5,6-hexathiol, monosaccharide, disaccharides, trisaccharides, oligosaccharides, or azide- , alkyne-modified moieties thereof. In some embodiments, the core moiety is chitosan. Thus, azide-modified chitosan, or alkyne-modified chitosan are suitable for conjugating to branching units using click chemistry. In a preferred embodiment, the central core is dipentaerythritol or a hexa- propargylated derivative thereof. In some embodiments, the core moiety is ethylenediamine, or tetra(ethylene oxide). In some embodiments, the core moiety is dipentaerythritol. Exemplary chemical structures suitable for use as core moieties are shown in Table 1 below. Table 1. Structural representation of various building blocks (cores, branching units, surface functional groups, monomers) for the synthesis of dendrimers. Building Blocks Structure 45718416.1 Building Blocks Structure 2-(aminomethyl)-2- 45718416.1 Building Blocks Structure tetrakis(3-(trichlorosilyl) 45718416.1 Building Blocks Structure 1,3,5- 45718416.1 Building Blocks Structure 45718416.1 Building Blocks Structure gamma cyclodextrin 3. Branching Units Exemplary chemical structures suitable as branching units include monosaccharides. In some embodiments, the monosaccharide branching units are conjugated to the core or the prior layer of monomers via linkers such as polyethylene glycol chains. In some forms, the monosaccharide branching units are glucose-based branching units. Exemplary glucose-based branching units are shown in Structures II-IV. These are spacer molecules, so can also be alkyl (CH2)n – hydrocarbon-like units. The branching units are the PEG or alkyl chain linkers between different dendrimer generations, for example, the glucose layers are connected via PEG linkers and triazole rings. 45718416.1 In some forms, the branching units are the same for each generation of dendrimers generated from the core. Therefore, in one embodiment, the branching units are glucose-based branching units for generating generation 1 dendrimers as shown in Structures V-VII. In some embodiments, the branching units are hyper-monomers i.e., ABn building blocks. Exemplary hyper-monomers include AB4, AB5, AB6, AB7, AB8 building blocks. Hyper-monomer strategy drastically increases the number of available end groups. An exemplary AB4 hypermonomer is peracetylated β-D-Glucopyranoside tetraethylene glycol azide as shown in Structure III. The chemical structures listed in Table 1, are also suitable as building blocks to form the branching units of the dendrimer. For example, the branching units of the dendrimers are formed by 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-hexachloro-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 core, nitrilotrimethanol, ethylene diamine, propane-1,3-diamine, butane-1,4-diamine, 2,2',2''-nitrilotris(ethan-1-ol), alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, Cucurbituril, benzene-1,2,3,4,5,6-hexathiol, monosaccharide, disaccharides, trisaccharides, oligosaccharides, or azide- , alkyne-modified moieties thereof, or a combination thereof. Other examples of chemical structures that are suitable for forming the branching units of the dendrimers include, but are not limited to, sugar moieties, such as glucose, galactose, mannose, and fructose, and alkylene glycol, such as ethylene glycol, and combinations thereof. In some embodiments, the branching unit is chitosan. Thus, azide- modified chitosan, or alkyne-modified chitosan are suitable for conjugating to the core moiety or additional same or different branching units using click chemistry. In some embodiments, the branching unit is methyl acrylate or ethylenediamine, or a combination thereof. In some embodiments, the branching unit is polyethylene glycerol linear or branched. In some embodiments, the branching unit is a copolymer of an alkylene glycol (such as ethylene glycol) and a sugar moiety, such as glucose, galactose, mannose, and/or fructose. 45718416.1 4. Surface Functional Groups Surface functional groups/molecules of the dendrimers are not limited to a primary amine end group, a hydroxyl end group, a carboxylic acid end group, an acetamide end group, a sugar molecule, an oligo- or poly-alkylene glycol, and/or a thiol end group. In some embodiments, the desired terminal functional groups can be added via one of the conjugation methods for the core and branching unit. In some embodiments, the surface functional groups are hydroxyl groups, for example those of PAMAM dendrimers, of generation 2 OEG dendrimer as shown in Structure I, or of the terminal glucose of dendrimers prepared with glucose-based branching units as shown in Structures V and VII. In some embodiments, desired surface functional groups can be modified or added via one of the conjugation methods for the core and branching unit. Exemplary surface functional groups include hydroxyl end groups, amine end groups, carboxylic acid end groups, acetamide end group, and thiol end groups, and combinations thereof. In some embodiments, the dendrimers can specifically target a particular tissue region and/or cell type, such as the cells and tissues of the central nervous system (CNS), the peripheral nervous system (PNS). In some embodiments, the dendrimers specifically target neurons and/or glia of the CNS. In some embodiments, the dendrimers specifically target neurons and/or glia of the PNS. In some embodiments, the dendrimers specifically target non-neural and/or non-glial cells such as activated macrophage cells in and around neuromuscular junctions. In some embodiments, the glucose dendrimers are those of generation 1 (G1), G2, G3, G4, and G5. In some embodiments, the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more neurons and/or glia of the CNS, the PNS, and/or the eye. In some embodiments, the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more non-neural and/or non-glial cells such as activated macrophage cells in and around neuromuscular junctions. In some embodiments, dendrimers are made of glucose and oligoethylene glycol building blocks. Exemplary generation 1 glucose dendrimer is shown in Structure VI, and generation 2 glucose dendrimers is shown in Structure VIII. In some embodiments, the dendrimers have a plurality of surface functional groups, such as hydroxyl (-OH) groups, amine groups, acetamide groups, and/or carboxyl groups on the periphery of the dendrimers (also referred to herein as surface functional groups or peripheral functional groups). In some embodiments, the surface density of such peripheral functional groups is at least 1 group/nm2 (number of the surface functional groups/surface area in nm2). For example, in some embodiments, the surface density of the surface functional groups, such as hydroxyl groups, is more 45718416.1 than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm2, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm2. In some embodiments, the volumetric density of surface functional groups, such as hydroxyl groups, is between about 1 and about 50 groups/nm3, between about 5 and about 30 groups/nm3, or between about 10 and about 20 groups/nm3. In further embodiments, the surface density of the surface functional groups, such as hydroxyl groups, is between about 1 and about 50, preferably 5-20 group/nm2 (number of surface functional groups/surface area in nm2), while each surface functional moiety has a molecular weight of between about 100 Da and about 10 kDa, preferably between about 100 Da and 1000 Da. In some embodiments, the amount of the surface functional groups, such as any one of those described above, e.g., hydroxyl groups, of the dendrimer is at least 30%, at least 40%, at least 50%, more than 40%, more than 50%, or in a range from more than 30% to 100%. %. In preferred embodiments, the amount of surface hydroxyl groups of the dendrimer is preferably more than 35%. In some embodiments, one or more of the surface functional groups, such as any one of those described above, on the periphery of the dendrimers are further modified by conjugating with one or more carbohydrate molecules and/or more or more polyalkylene glycols, such as polyethylene glycols. In these embodiments, the surface density of the terminal carbohydrate moieties/molecules and/or polyalkylene glycols can have any of the ranges described above for hydroxyl groups. Hydroxyl-terminated PAMAM dendrimers, PAMAM dendrimer modified on the surface with sugar moieties (with >10% of surface groups modified by sugars, especially by glucose, and glucose dendrimers (where the dendrimers are made of glucose building blocks are preferred). For delivery to the brain, constructs with a total molecular weight of <30,000 Da are preferred. For confinement primarily to the peripheral circulation, constructs with a total molecular weight of >50,000 Da are preferred. When dendrimers are formed of, or include sugar moieties/molecules at termination, for example, glucose, the terminal hydroxyl groups of these dendrimers may be part of the terminal sugar moieties/molecules or extra hydroxyl groups that are not part of the sugar moieties/molecules, or a combination thereof. In some embodiments, all of the terminal hydroxyl groups are part of the terminal sugar moieties/molecules. a. Hydroxyl-terminated Dendrimers In some embodiments, the dendrimers include a plurality of hydroxyl groups. Some exemplary high-density hydroxyl groups-containing dendrimers include commercially available polyester dendritic polymer such as hyperbranched 2,2-Bis(hydroxyl- methyl)propionic acid polyester polymer (for example, hyperbranched bis-MPA polyester-64- hydroxyl, generation 4), dendritic polyglycerols. In some embodiments, the hydroxyl terminated 45718416.1 dendrimers include hydroxyl-terminated PAMAM dendrimers, particularly G3 to G6 hydroxyl- terminated PAMAM dendrimers, such as G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers. In some embodiments, the high-density hydroxyl groups-containing dendrimers are oligo ethylene glycol (OEG)-like dendrimers. For example, a generation 2 OEG dendrimer (D2-OH-60) as shown in Structure I can be synthesized using highly efficient, robust and atom economical chemical reactions such as Cu (I) catalyzed alkyne–azide click and photo catalyzed thiol-ene click chemistry. Highly dense polyol dendrimer at very low generation in minimum reaction steps can be achieved by using an orthogonal hypermonomer and hypercore strategy, for example as described in WO2019094952. In some embodiments, the dendrimer backbone has non-cleavable polyether bonds throughout the structure to avoid the disintegration of dendrimer in vivo and to allow the elimination of such dendrimers as a single entity from the body (non-biodegradable). dendrimer. 45718416.1 In some embodiments, the dendrimers have a plurality of hydroxyl (-OH) groups on the periphery of the dendrimers. In some embodiments, the surface density of hydroxyl (-OH) groups is at least 1 OH group/nm2 (number of surface hydroxyl groups/surface area in nm2). For example, in some embodiments, the surface density of hydroxyl groups, per nm2, is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm2, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm2. In some embodiments, the volumetric density of hydroxyl groups is between about 1 and about 50 groups/nm3, between about 5 and about 30 groups/nm3, or between about 10 and about 20 groups/nm3. In further embodiments, the surface density of hydroxyl (-OH) groups is between about 1 and about 50, or between 5 and 20 OH group/nm2 (number of surface hydroxyl groups/surface area in nm2) while having a molecular weight of between about 100 Da and about 1000 Da. In some embodiments, the amount of the surface hydroxyl groups of the dendrimer is preferably greater than 35%, at least 40%, at least 50%, more than 40%, more than 50%, or in a range from more than 40% to 100. In some embodiments, the dendrimers may have a fraction of the hydroxyl groups exposed on the outer surface, with the others in the interior core of the dendrimers. In some embodiments, the dendrimer specifically targets a particular tissue region and/or cell types following administration into the body. In some embodiments, the dendrimer specifically targets a particular tissue region and/or cell type without a targeting moiety. In some embodiments, the dendrimers include an effective number of hydroxyl groups for targeting CNS cells and/or PNS cells, such as microglial, astrocytes, and/or neurons associated with a disease, disorder, or injury of the central nervous system or the peripheral nervous system. In some embodiments, the dendrimer specifically targets a particular tissue region and/or cell type without a targeting moiety and the active agent conjugated thereto bind directly to a receptor on the surface and/or interior of target neural and/or glial cells, such as activated macrophage cells in and around neuromuscular junctions. Unmodified PAMAM dendrimers with hydroxyl end groups do not enrich in the neurons of brain and/or retinal ganglion cells (RGCs) in the eye as much as glucose dendrimers. The glucose dendrimers with terminal glucose monosaccharide and a high density of hydroxyl functional groups effectively target the neurons in a generation dependent manner. Generation 2 (G2), and G3 and G4 should be efficacious. G5 and above are more difficult to use. In preferred embodiments, the dendrimers include an effective number of terminal glucose and/or hydroxyl groups for targeting to one or more motoneurons. The hydroxyl groups on the dendrimer surface are part of glucose molecules. There are no extra hydroxyls in addition to the glucose molecules on the surface. The number of sugar molecules on the surface is determined by the generation number. All generations are expected to target neurons. 45718416.1 Some exemplary glucose dendrimers include a generation 1 glucose dendrimer having 24 hydroxyl (-OH) end groups, a generation 2 glucose dendrimer having 96 hydroxyl (-OH) end groups, a generation 3 glucose dendrimer having 396 hydroxyl (-OH) end groups, and generation 4 glucose dendrimer having 1584 hydroxyl (-OH) end groups. In some forms, the glucose dendrimer is a generation 2 glucose based dendrimer that has 24 glucose molecules at the periphery and 6 embedded glucose molecules in the backbone held together by PEG segments. b. Dendrimers Modified with Carbohydrates In some embodiments, the dendrimers contain one or more carbohydrate molecules at the termination. These terminal carbohydrate molecules can be prepared by conjugating one or more surface functional groups of a dendrimer, such as amine groups, carboxyl groups, or hydroxyl groups, with one or more carbohydrate molecules. In some forms, the dendrimers, prior to carbohydrate conjugation, are hydroxyl-terminated dendrimers such as hydroxyl-terminated PAMAM dendrimers and one or more of the hydroxyl groups are conjugated with one or more carbohydrate molecules. In some embodiments, hydroxyl-terminated dendrimers modified with surface glucose molecules selectively target activated macrophage cells in and around neuromuscular junctions in vitro and in vivo; and/or selectively accumulate on the surface and/or within these targets, such that the active agent(s) conjugated thereto bind to one or more receptors on/in the target activated macrophage cells in and around neuromuscular junctions. In some embodiments, hydroxyl- terminated dendrimers modified with surface glucose molecules selectively target immune system cells, such as activated macrophage cells in and around neuromuscular junctions in vitro and in vivo; and/or selectively accumulate on the surface and/or within these targets, such that the active agent(s) conjugated thereto bind to one or more receptors on/in the target activated macrophage cells in and around neuromuscular junctions. In some embodiments, the carbohydrate moieties used to modify one or more surface functional groups of the dendrimers are monosaccharides. Exemplary monosaccharides suitable for modifying the dendrimers include glucose, glucosamine, galactose, mannose, fructose, dehydroascorbic acid, urate, myo-inositol. In some embodiments, the dendrimers are conjugated to glucose and thus contain glucose as terminal moieties/molecules. In some embodiments, hydroxyl- terminated dendrimers are modified with one or more glucose moieties to the dendrimer (“D-Glu”). In some embodiments, the dendrimers are conjugated to galactose. In some embodiments, the dendrimers are conjugated to mannose. In some embodiments, the dendrimers are conjugated to fructose. In some embodiments, the dendrimers are conjugated to one or more monosaccharides other than glucose, such as galactose, mannose, and/or fructose. For example, the carbohydrate 45718416.1 moieties are oligosaccharides which terminate in one or more monosaccharides including glucose, glucosamine, mannose, fructose, thus exposing these sugar moieties on the surface for binding. In preferred embodiments, the glucose or hydroxyl-terminated PAMAM dendrimers, or carbohydrate-functionalized dendrimers, are conjugated to one or more active agents that have affinity to and are suitable for binding directly or indirectly, to GCPII at the surface of, or within target cells. In some embodiments, the dendrimers are conjugated to one or more carbohydrates moieties that have affinity to and are suitable for binding directly or indirectly to one or more of AMPA receptors, NMDA receptors, EGFR1 receptors, EGFR2 receptors, histamine (H1) receptors, GABA receptors, and trace amine-associated receptor 1 (TAAR1). Evidence indicates these are internalized once bound to GCPII on the surface of cells In some embodiments, the dendrimers, with or without carbohydrate moieties, are conjugated to one or more active agents that have affinity to and are suitable for transport via one or more of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, and GLUT14. These are all the transporters that the glucose dendrimers could take to be transported into neurons and may be glia in a receptor- mediated way. In further embodiments, the dendrimers are conjugated to one or more glucose and/or glucosamine moieties. In some embodiments, the dendrimers contain carbohydrate moieties which enable transport of the active agent to target cells/receptors, wherein the activity at the target cell or receptor is driven by the active agent. In these embodiments, the carbohydrates and glucose moieties enable better drug targeting to cells and/or receptors of interest. For example, in some embodiments, the dendrimers are conjugated to one or more glucose and/or glucosamine moieties. In other embodiments, the dendrimers are conjugated to one or more oligosaccharides terminating in glucose and/or glucosamine moieties, i.e., glucose and/or glucosamine moieties are exposed on the surface of the dendrimer conjugates suitable for binding to one or more of the GLUTs, 5HT receptors, NE receptors, DA receptors and/or transporters. In some embodiments, the dendrimers have a plurality of carbohydrate moieties/molecules such as monosaccharides, e.g., glucose, on the periphery of the dendrimers. In some embodiments, the surface density of carbohydrate molecules such as monosaccharides, e.g., glucose, is at least 1 carbohydrate molecule/nm2 (number of surface carbohydrate groups/surface area in nm2). In some embodiments, the surface density of carbohydrate molecules, per nm2, is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups/nm2, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups/nm2. For example, surface density of carbohydrate molecules, per nm2, is more than 10. In some embodiments, the volumetric density of surface carbohydrate molecules is between about 1 45718416.1 and about 50 groups/nm3, between about 5 and about 30 groups/nm3, or between about 10 and about 20 groups/nm3. In further embodiments, the surface density of carbohydrate molecules is between about 1 and about 50, between about 5 and about 20, per nm2 (number of surface carbohydrate molecules/surface area in nm2) while each carbohydrate moiety having a molecular weight of between about 100 Da and about 1000 Da. In these embodiments, i.e., one or more surface functional groups of the dendrimer are modified to introduce one or more sugar moieties/molecules at termination, the terminal hydroxyl groups may be part of the terminal sugar moieties/molecules or extra hydroxyl groups that are not modified with sugar moieties/molecules and thus are not part of the sugar moieties/molecules, or a combination thereof. In some embodiments, carbohydrate molecules such as monosaccharides, e.g., glucose, are present in an amount by weight that is between about 1% and 40% of the total weight of the glycosylated dendrimer, for example, between about 2% and 20%, between about 5% and 15%, or between 9 % and 12 % of the total weight of the glycosylated dendrimer. For example, in some embodiments, the carbohydrate moieties are present in an amount that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the glycosylated dendrimer following conjugation. In some embodiments, conjugation of carbohydrate molecules through one or more surface functional groups occurs via about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimers prior to the conjugation. In other embodiments, the conjugation of carbohydrate molecules occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40% of total available surface functional groups of the dendrimers prior to the conjugation. c. Dendrimers Modified with Polyalkylene Glycol (“PEG”) In some embodiments, the dendrimers contain one or more polyalkylene glycols at the termination. These terminal polyalkylene glycols can be prepared by conjugating one or more of surface functional groups of the dendrimers, such as hydroxyl groups, with a polyalkylene glycol, such as PEG. In some embodiments, the dendrimers, prior to conjugation, are hydroxyl-terminated dendrimers such as hydroxyl-terminated PAMAM dendrimers and at least a portion of the surface hydroxyl groups are conjugated with PEG. In some embodiments, the dendrimers have a plurality of polyalkylene glycols such as PEG on the periphery of the dendrimers. In some embodiments, the surface density of polyalkylene glycols such as PEG, is at least 1 polyalkylene glycol/nm2 (number of surface polyalkylene glycol/surface area in nm2). In some embodiments, the surface density of polyalkylene glycols, per nm2, is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 polyalkylene glycol/nm2, such as at least 10, 15, 20, 45718416.1 25, 30, 35, 40, 45, 50, or more than 50 polyalkylene glycol/nm2. For example, surface density of polyalkylene glycols, per nm2, is more than 10. In some embodiments, the volumetric density of surface polyalkylene glycols is between about 1 and about 50 groups/nm3, between about 5 and about 30 groups/nm3, or between about 10 and about 20 groups/nm3. In further embodiments, the surface density of polyalkylene glycols such as PEG is between about 1 and about 50, between about 5 and about 20, per nm2 (number of surface polyalkylene glycols/surface area in nm2) while having a molecular weight of between about 100 Da and about 1000 Da. In some embodiments, the polyalkylene glycol molecules such as PEG can be present in an amount by weight that is between about 1% and 40% of the total weight of the pegylated dendrimer, for example, between about 2% and 20%, between about 5% and 15%, or between 9 % and 12 % of the total weight of the pegylated dendrimer. For example, in some embodiments, the polyalkylene glycol molecules, such as PEG, are present in an amount that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the pegylated dendrimer following conjugation. In some embodiments, conjugation of polyalkylene glycol molecules such as PEG through one or more surface functional groups of the dendrimer occurs via about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimers prior to the conjugation. In other embodiments, the conjugation of polyalkylene glycol molecules such as PEG occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40% of total available surface functional groups of the dendrimers prior to the conjugation. C. Coupling Agents and Spacers Dendrimer-GCPII inhibitor agent conjugates can be formed from one or more active agents covalently conjugated or non-covalently attached to a dendrimer. In preferred embodiments, the one or more active agents are covalently conjugated to the dendrimer. Optionally, the one or more active agents are conjugated to the dendrimer via one or more spacers. The term “spacer” includes chemical moieties and functional groups used for linking an active agent to the dendrimer. The spacer can be either a single chemical entity or two or more chemical entities linked together. The spacer can include any small chemical entity, peptide or polymers having sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone, carbonate, etc. In some embodiments, the spacer via which the active agent is conjugated to the dendrimer contains different linkages such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazides, ether, and amide linkages. The spacer between a dendrimer and an active agent can be designed to provide a releasable or non-releasable form of the dendrimer conjugate in vivo. In some 45718416.1 embodiments, the conjugation between active agent and dendrimer is via an appropriate spacer that contains an ester bond between the active agent and the dendrimer. In some embodiments, one or more spacers between a dendrimer and active agents can provide desired and effective release kinetics in vivo. These spacers may contain cleavable linkages (e.g., ester, disulfide, phosphodiester, triglycyl peptide, and hydrazine) or non-cleavable linkages (e.g., amide, ether, and amino alkyl). The conjugation between active agents and dendrimers can be performed using reaction known in the art, such as click chemistry, acid-amine coupling, Steglich esterification, etc. In some embodiments, the conjugation between active agent and dendrimer is via a spacer that contains disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, ether, or amide linkages, or a combination thereof. In some embodiments, the conjugation between active agent and dendrimer is via an appropriate spacer that contains an ester linkage or an amide linkage between the agent and the dendrimer depending on the desired release kinetics of the agent. The spacer can be chosen from among a class of compounds terminating in sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone and carbonate group. The spacer can include thiopyridine terminated compounds such as dithiodipyridine, N-Succinimidyl 3-(2-pyridyldithio)- propionate (SPDP), Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or Sulfo-LC-SPDP. The spacer can also include peptides wherein the peptides are linear or cyclic, having sulfhydryl groups 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). The spacer can be a mercapto acid derivative such as 3 mercapto propionic acid, mercapto acetic acid, 4 mercapto butyric acid, thiolan-2-one, 6 mercaptohexanoic acid, 5 mercapto valeric acid and other mercapto derivatives such as 2 mercaptoethanol and 2 mercaptoethylamine. The spacer can be thiosalicylic acid and its derivatives, (4- succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridithio]propionyl hydrazide, The spacer can have maleimide terminations wherein the spacer includes polymer or small chemical entity such as bis-maleimido diethylene glycol and bis-maleimido triethylene glycol, Bis- Maleimidoethane, bismaleimidohexane. The spacer can include vinylsulfone such as 1,6-Hexane- bis-vinylsulfone. The spacer can include thioglycosides such as thioglucose. The spacer can be reduced proteins such as bovine serum albumin and human serum albumin, any thiol terminated compound capable of forming disulfide bonds. The spacer can include polyethylene glycol having maleimide, succinimidyl, and/or thiol terminations. D. Dendrimer-Agent Conjugates or Complexes Conjugate molecules including a dendrimer coupled and/or complexed with one or more GCPII inhibitor(s) are provided. Dendrimer-active agent conjugates can be formed of GCPII 45718416.1 inhibitor agents covalently conjugated or non-covalently attached to a dendrimer, a dendritic polymer, or a hyperbranched polymer. Methods for conjugation of one or more active agents to a dendrimer are known, such as those described in U.S. Published 2011/0034422, 2012/0003155, and 2013/0136697. In general, conjugation to the dendrimer may further improve safety and efficacy of these agents. For example, dendrimer conjugation may change specific receptor activity and/or modify biodistribution. For example, use of higher generation dendrimers and/or dendrimers with molecular weights greater than 24 kDa can confine these agents to the peripheral nervous system in order to preclude their effects elsewhere, such as in the brain. In some embodiments, one or more GCPII inhibitor agents are covalently conjugated to one or more terminal groups of the dendrimer such as terminal hydroxyl groups. In some embodiments, dendrimer conjugates include one or more GCPII inhibitor agents conjugated to the dendrimer via one or more spacers. The spacer between a dendrimer and an active agent can be designed to provide a releasable or non-releasable form of the dendrimer conjugate in vivo. For example, the spacer can be cleavable or contain a chemical linkage that is cleavable, for example, by exposure to the intracellular compartments of target neural and/or glial cells or upon binding to the receptor on the surface or in the interior of the target neural and/or glial cells in vivo. Examples of cleavable linkages that can be used in a spacer of the dendrimer-GCPII inhibitor agent conjugates include, esterase sensitive ester bond, glutathione sensitive disulfide bond, phosphatase-sensitive phosphodiester bond, oligopeptide such as triglycyl peptide linker capable of lysosomal release, acid cleavable hydrazine linkage etc. In some embodiments, the spacer between a dendrimer and active agents can provide desired and effective release kinetics in vivo. In some embodiments, the spacer between the dendrimer and the active agent can be non-cleavable or contain a chemical linkage that is non-cleavable, such as amide, ether, and amino alkyl linkages. Generally, the spacer between the dendrimer and active agent has a length sufficient for the active agent conjugated thereto to reach and bind to the target receptor on the surface and/or inside of the target cell. For example, the spacer between the dendrimer and active agent has a length in a range from 50 Da to 2,000 Da, depending on the release kinetics desired, and the receptor binding flexibility desired. The length of the spacer can vary, depending on the location of the target receptor (for example, on the cell surface, in the cytoplasm of the cell, or in an intercellular compartment of the cell) and/or density of the receptor when located on the cell surface. The dendrimer can be a generation 2, generation 3, generation 4, generation 5, generation 6, and up to generation 10. In some embodiments, the dendrimer is conjugated to one or more active 45718416.1 agents via spacers containing cleavable (ester, disulfide, phosphodiester, triglycyl peptide, and hydrazine) or non-cleavable (amide, ether, and amino alkyl) linkages. The density of active agents covalently conjugated to or non-covalently attached to the dendrimer can be adjusted based on the specific GCPII inhibitor agent being delivered, the target receptors, the target neural and/or glial cells, the location of the target neural and/or glial cells, etc. For example, a plurality of active agents conjugated to the dendrimer are on the periphery of the dendrimer and the surface density of the active agent is at least 1 active agent/nm2 (number of active agent conjugated/surface area in nm2). For example, in some embodiments, the surface density of active agent per nm2 is more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH/nm2, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH/nm2. In some embodiments, the volumetric density of active agent is between about 1 and about 50 groups/nm3, between about 5 and about 30 groups/nm3, or between about 10 and about 20 groups/nm3. Typically, the dendrimer-active agent conjugates have a hydrodynamic volume in the nanometer range. For example, in some embodiments, the glucose dendrimer-active agent conjugates including one or more GCPII inhibitor agents conjugated to the dendrimer have a diameter of about 2 nm to about 100 nm, or more than 100 nm, up to 500 nm, depending upon the generation of dendrimer, the chemical composition and amount of active agent conjugated thereto. In some embodiments, a dendrimer-active agent conjugate including one or more GCPII inhibitor agents conjugated to the dendrimer has a diameter effective to penetrate nervous-system tissues and to retain on the surface and/or in target neural and/or glial cells for a period of time sufficient for the active agent to bind to the targeted receptors on the surface and /or in the target neural and/or glial cells. In some embodiments, a dendrimer-active agent conjugate including one or more GCPII inhibitor agents conjugated to the dendrimer has a diameter effective to remain in the peripheral circulation and to retain on the surface and/or in target neural and/or glial cells for a period of time sufficient for the active agent to bind to the targeted receptors on the surface and/or in the target neural and/or glial cells such as for example, neural and/or glial cells of the gastrointestinal system. The dendrimer-active agent conjugates can be neural, have a positive charge or a negative charge. In some embodiments, the dendrimer-GCPII inhibitor agent conjugates are neutral. The presence of GCPII inhibitor agents can affect the surface charge of the dendrimer conjugates. In some embodiments, the surface charge of the dendrimer conjugated to GCPII inhibitor agents is between -100 mV and 100 mV, between -50 mV and 50 mV, between -25 mV and 25 mV, between -20 mV and 20 mV, between -10 mV and 10 mV, between -10 mV and 5 mV, between -5 mV and 5 mV, or between -2 mV and 2 mV. The range above is inclusive of all values from -100 mV to 100 45718416.1 mV. In preferred embodiments, the surface charge of the dendrimer-GCPII inhibitor agent conjugates is neutral or near-neutral, i.e., from about -10 mV to about 10 mV, inclusive. An exemplary dendrimer-GCPII inhibitor agent conjugate is represented by Formula (I). The dendrimer of the exemplary conjugate contains surface hydroxyl groups, wherein one or more of the surface hydroxyl groups are conjugated to one or more active agents via one or more spacers as shown in Formula (I), below: wherein D can be a or 2 to generation 10 dendrimer, such as any one of those described above, for example, PAMAM (such as hydroxyl- terminated PAMAM dendrimer) or a glucose-based dendrimer; each occurrence of L can be any suitable chemical moiety, preferably containing a triazole moiety; Y can be a bond or a linkage selected from secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonate (-O-C(O)-O-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinols (- CHOH-, -CROH-), disulfide groups, phosphodiester ), hydrazino group, hydrazones, hydrazides, ester (-C(O)-O-), ether (- triglycyl peptide), wherein R is an alkyl group, an aryl group, or a heterocyclic group; each occurrence of X can be a GCPII inhibitor agent, wherein a functional group of X (such as an amino group including primary amino, secondary amino, or tertiary amino group; a carboxylic group; or a hydroxyl group) forms a portion of linkage Y; n can be an integer from 1 to 100; and m can be an integer from 16 to 4096. The dendrimer can be PAMAM (such as hydroxyl-terminated PAMAM) or a glucose dendrimer, which is 100% hydroxyl. m and n depend on the size of the dendrimer D, n should be such that the weight percent of the drug in the total conjugate is 5-20%. This range is also appropriate for binding and internalization. The oxygen atom shown in Formula (I) is from the surface functional group of the dendrimer, such as a surface hydroxyl group, where the surface hydroxyl group may or may not be part of a terminal sugar moiety/molecule (e.g., glucose). Although not illustrated in Formula (I), one 45718416.1 or more hydroxyl groups of the dendrimer that are not conjugated to active agents may be modified with one or more carbohydrates and/or polyalkylene glycols, such as PEG. When administered to a subject in need thereof, the GCPII inhibitor agent (“X” of Formula (I)) can bind to a target receptor on the surface of the target cell or inside the target cell. In some embodiments, when the GCPII inhibitor agent X binds to the target receptor, the agent X remains conjugated to the dendrimer. In these embodiments, following binding, the agent X may be released from the dendrimer or remain conjugated to the dendrimer as an intact dendrimer-active agent conjugate. In some embodiments, the GCPII inhibitor agent X is released from the dendrimer at close proximity to the target receptor and then binds to the target receptor. In some embodiments, each occurrence of L can be represented by -A’-L1-B’-L2-, wherein A’ can be a carbonyl (-C(O)-) or a bond (including single, double, and triple bonds, for example a single bond); B’ can be a bond (including single, double, and triple bonds, for example a single bond), an amide, an ester, an ether, a thiol, a dithiol, an aryl, a heteroaryl, a polyaryl, a heteropolyaryl, or a heterocyclic; and L1 and L2 can be independently a bond, an alkylene, a heteroalkylene, an aryl, an aralkyl, an ether, a polyether, a thiol, a dithiol, a thiolether, a polythioether, an oligopeptide, a polypeptide, an oligo(alkylene glycol), or a polyalkylene glycol, or L1 and L2 can be independently composed of a combination of these groups, such as a combination of alkylene and polyether, a combination of alkylene and thiol or dithiol, a combination of alkylene and oligopeptide, a combination of alkylene, polyether, and thiol or dithiol, or a combination of polyether and thiol or dithiol. In some forms, L1-B’-L2- together form a chemical moiety selected from an -alkylene-triazole-di(alkylene glycol)-, a -di(alkylene glycol)-triazole-alkylene-, -alkylene- triazole-oligo(alkylene glycol)-, an -oligo(alkylene glycol)-triazole-alkylene-, an -alkylene-triazole- poly(alkylene glycol)-, -poly(alkylene glycol)-triazole-alkylene-, an -alkylene-triazole-ether-, an - alkylene-triazole-alkylene-, an -alkylene-amide-alkylene-, and combinations thereof. In some embodiments, B’ can be a bond (including single, double, and triple bonds, for example a single bond), an amide group, or a heterocyclic group, such as a triazole group. In some embodiments, L1 can be a bond; an alkylene, such as a C1-C10 alkylene, a C1-C8 alkylene, a C1-C6 alkylene, a C1-C5 alkylene, a C1-C4 alkylene, or a C1-C3 alkylene; or an oligo- or poly-(alkylene glycol), such where p is an integer from 1 to 20, from 1 to 18, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or 1 or 2. 45718416.1 In some embodiments, L2 can be a bond; an alkylene, such as a C1-C10 alkylene, a C1-C8 alkylene, a C1-C6 alkylene, a C1-C5 alkylene, a C1-C4 alkylene, or a C1-C3 alkylene; an oligo- or poly-(alkylene glycol), such where p is an integer from 1 to 20, from 1 to 18, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 4, or or an oligo- or poly- peptide, such as a triglycyl peptide; a thiol; or a dithiol; or L2 is composed of a combination of two or more of alkylene, oligo- or poly-(alkylene glycol), oligo- or poly-peptide, thiols, and dithiols. For example, L2 is represented by , to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2, such as 0, 1, or 2; and G’ is a thiol, a dithiol, an oligo- peptide such as a triglycyl peptide, or a poly-peptide. In some embodiments, Y is a linkage that is minimally cleavable in vivo. In some embodiments, Y is a linkage that is cleavable in vivo. In some embodiments, Y is an amide (- CONH-), an ester (-C(O)-O-), an ether (-O-), a phosphodiester, or a disulfide group. In some embodiments, L and Y are both a single bond, and D is directly conjugated to X (an active agent or analog thereof) via an ether linkage. In some embodiments, D is a generation 2 PAMAM dendrimer, a generation 3 PAMAM dendrimer, a generation 4 PAMAM dendrimer, a generation 5 PAMAM dendrimer, a generation 6 PAMAM dendrimer, a generation 1 glucose dendrimer, a generation 2 glucose dendrimer, a generation 3 glucose dendrimer, a generation 4 glucose dendrimer, a generation 5 glucose dendrimer, or a generation 6 glucose dendrimer. Typically, the GCPII inhibitors and/or their derivatives bind to a target receptor on the surface of the target cell or inside the target cell. In some forms, when GCPII inhibitors and/or their derivatives bind to the target receptor, the agent remains conjugated to the dendrimer. In these embodiments, following binding, the agent may be released from the dendrimer or remain conjugated to the dendrimer as an intact dendrimer-active agent conjugate. In some embodiments, the GCPII inhibitors and/or their derivatives are released from the dendrimer at close proximity to the target receptor and then binds to the target receptor on the target neural and/or glial cell. 45718416.1 III. Methods of Making Dendrimer Conjugates Methods of synthesizing dendrimers and making dendrimer-GCPII inhibitor conjugate nanoparticles are also described. A. Methods of Making Dendrimers Dendrimers can be prepared via a variety of chemical reaction steps. Dendrimers are usually synthesized according to methods allowing controlling their structure at every stage of construction. The dendritic structures are mostly synthesized by two main different approaches: divergent or convergent. In some embodiments, dendrimers are prepared using divergent methods, in which the dendrimer is assembled from a multifunctional core, which is extended outward by a series of reactions, commonly a Michael reaction. The strategy involves the coupling of monomeric molecules that possesses reactive and protective groups with the multifunctional core moiety, which leads to stepwise addition of generations around the core followed by removal of protecting groups. For example, PAMAM-NH2 dendrimers are first synthesized by coupling N-(2-aminoethyl) acryl amide monomers to an ammonia core. In other embodiments, dendrimers are prepared using convergent methods, in which dendrimers are built from small molecules that end up at the surface of the sphere, and reactions proceed inward, building inward, and are eventually attached to a core. Many other synthetic pathways exist for the preparation of dendrimers, such as the orthogonal approach, accelerated approaches, the Double-stage convergent method or the hypercore approach, the hypermonomer method or the branched monomer approach, the Double exponential method; the Orthogonal coupling method or the two-step approach, the two monomers approach, AB2–CD2 approach. In some embodiments, the core of the dendrimer, one or more branching units, one or more spacers, and/or one or more surface functional groups can be modified to allow conjugation to further functional groups (branching units, spacers, surface functional groups, etc.), monomers, and/or agents via click chemistry, employing one or more Copper-Assisted Azide-Alkyne Cycloaddition (CuAAC), Diels-Alder reaction, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules.2015 May 20;20(5):9263-94). In some embodiments, pre-made dendrons are clicked onto high-density hydroxyl polymers. ‘Click chemistry’ involves, for example, the coupling of two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface functional group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or equivalent thereof) on the surface of the first moiety and an azide moiety (e.g., present on a triazine composition or equivalent thereof), or any active end group such as, for 45718416.1 example, a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc. on the second moiety. In some embodiments, one or more hydroxyl groups on the surface of the dendrimer (hydroxyl-terminated PAMAM dendrimer, or glucose dendrimer) are modified to contain an alkyl group and a drug is modified to contain an azide group. Alternatively, one or more hydroxyl groups on the surface of the dendrimer (hydroxyl-terminated PAMAM dendrimer, or glucose dendrimer) are modified to contain an azide group and a drug is modified to contain an alkyne group. The azide and alkyne are then reacted via a 1,3-dipolor cycloaddition reaction to form a triazole moiety. In some embodiments, dendrimer synthesis relies upon one or more reactions such as thiol- ene click reactions, thiol-yne click reactions, CuAAC, Diels-Alder click reactions, azide-alkyne click reactions, Michael Addition, epoxy opening, esterification, silane chemistry, and a combination thereof. In some embodiments, methods involve one or more protection and deprotection steps of the function groups (e.g., hydroxyl groups) on the central core, branching units, and/or therapeutic, prophylactic or diagnostic agents to facilitate addition of branching units to generate desired dendrimer molecules, or addition of therapeutic, prophylactic or diagnostic agents to generate desired dendrimer conjugates. In the case of hydroxyl groups, they may be protected by formation of an ether, an ester, or an acetal. Other exemplary protection groups include Boc and Fmoc. Any existing dendritic platforms can be used to make dendrimers of desired functionalities, i.e., with a high-density of surface hydroxyl groups by conjugating high-hydroxyl containing moieties such as 1-thio-glycerol or pentaerythritol. Exemplary dendritic platforms such as polyamidoamine (PAMAM), poly (propylene imine) (PPI), poly-L-lysine, melamine, poly (etherhydroxylamine) (PEHAM), poly (esteramine) (PEA) and polyglycerol can be synthesized and explored. Dendrimers also can be prepared by combining two or more dendrons. Dendrons are wedge- shaped sections of dendrimers with reactive focal point functional groups. Many dendron scaffolds are commercially available. They come in 1, 2, 3, 4, 5, and 6th generations with, respectively, 2, 4, 8, 16, 32, and 64 reactive groups. In certain embodiments, one type of agents is linked to one type of dendron and a different type of agent is linked to another type of dendron. The two dendrons are then connected to form a dendrimer. The two dendrons can be linked via click chemistry i.e., a 1,3- dipolar cycloaddition reaction between an azide moiety on one dendron and alkyne moiety on another to form a triazole linker. 45718416.1 Exemplary methods of making dendrimers are described in detail in International Patent Publication Nos. WO2009/046446, WO2015168347, WO2016025745, WO2016025741, WO2019094952, and U.S. Patent No.8,889,101. 1. Methods of Making Glucose Dendrimers The glucose-based dendrimers are assembled from a multifunctional core, which is extended outward by a series of reactions. The strategy involves the coupling of monomeric molecules that possess reactive and protective groups with the multifunctional core moiety, which leads to stepwise addition of generations around the core followed by removal of protecting groups. In some embodiments, glucose dendrimers are synthesized by coupling AB4 peracetylated β-D glucose-PEG4-azide monomers to hexapropargylated core. In preferred embodiments, the hypercore is prepared from dipentaerythritol, for example by performing propargylation of dipentaerythritol to achieve the hexa-propargylated core. An exemplary scheme for preparing such a glucose dendrimer is shown by Scheme I. In some embodiments, the branching units are hypermonomers i.e., ABn building blocks. Exemplary hypermonomers include AB3, AB4, AB5, AB6, AB7, AB8 building blocks. Hypermonomer strategy drastically increases the number of available end groups. An exemplary hypermonomer is AB4 orthogonal hypermonomer including one azide functional group and four allyl groups prepared from dipentaerythritol with five allyl groups reacted with monotosylated triethylene glycol azide. In some embodiments, the branching unit is polyethylene glycerol linear or branched e.g., as shown by Formula III. Other monomers include disaccharides and oligosaccharides, as well as saccharides such as fructose, lactose, and sucrose. 45718416.1 a. Synthesis of AB4 building block Some exemplary synthesis methods of hypermonomer AB4 are described below. In some embodiments, the hypermonomer AB4 is based on glucose molecules. In preferred embodiments, the hypermonomer AB4 is conjugated to a polyethylene glycerol, for example, tetraethylene glycol (PEG4). In one embodiment, the hypermonomer AB4 is peracetylated β-D-Glucopyranoside tetraethylene glycol azide. In some embodiments, the synthesis of glucose-Oac-TEG-Ots involves the following steps: a solution of peracetylated β-D-glucopyranoside (10g, 25.6mmol) was dissolved in 50mL of anhydrous dichloromethane (DCM) followed by addition of 2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.2g, 17.9mmol) and the reaction mixture was cooled to 0°C. Boron trifluoride diethyl etherate (2.5 eq.) was added and the reaction was allowed to come to room temperature. The reaction was monitored with the help of TLC and quenched after 5hrs by the addition of saturated sodium bicarbonate solution at 0°C. After 10 minutes of stirring, DCM (300mL) was added and the organic layer was washed with saturated sodium bicarbonate solution 3 times 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 / hexanes (70:30) mixture as eluents. The desired compound was achieved in 60% yield. Structure of glucose-Oac (“acetoxy”)- triethylene glycol (“TEG”)-tosylate (“Ots”) is shown below: In some embodiments, the synthesis of glucose-Oac-TEG-N3 involves the following steps: a solution of glucose-Oac-TEG-Ots (6g, 8.8mmoles) is dissolved in 40 mL of anhydrous DMF followed by the addition of sodium azide (2eq) and the reaction mixture is heated to 50 oC for overnight. Upon completion, the reaction mixture is filtered and DMF is evaporated. Once dried, the crude reaction mixture is passed through combiflash using ethyl acetate:hexane (70:30) as eluent. Structure of glucose-Oac-TEG-N3 is shown below: 45718416.1 In some embodiments, the synthesis of glucose-OH-TEG-N3 involves the following steps: the peracetylated β-D-Glucopyranoside tetraethylene glycol azide is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH around 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with Amberlist IR- 120+ around 6-7. The reaction mixture is separated by filtration and the solvent removed by rotary evaporation. Structure of glucose-OH-TEG-N3 is shown below. b. Synthesis of Glucose Dendrimers In some embodiments, glucose dendrimers are synthesized by coupling AB4 peracetylated β-D glucose-PEG4-azide monomers to hexapropargylated core. In preferred embodiments, the hexapropargylated core is linked to AB4 β-D-glucose-PEG4-azide building block (2) via click reaction to obtain generation 1 dendrimer. In some embodiments, generation one dendrimer D1-Glu6-Oac24 is prepared according to the following: Hexapropargylated compound (0.5g, 1mmoles) and an azido derivative ((4.1g, 7.4mmoles) 1.2 eq. per acetylene) are suspended in a 1:1 mixture of DMF and water in a 20mL microwave vial equipped with a magnetic stir bar. CuSO4·5H2O (5mol%/acetylene, 75mg) and sodium ascorbate (5mol%/acetylene, 60mg) dissolved in the minimum amount of water 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 dialysis containing EDTA. The EDTA is further removed by extensive water dialysis. The product is lyophilized to obtain D1-Glu6-Oac24. Structure of D1-Glu6-Oac24 is shown below. 45718416.1
Structure V: D1-Glu6-Oac24 In some embodiments, generation one dendrimer D1-Glu6-OH24 is prepared according to the following: the peracetylated generation 1 glucose dendrimer (1g, 0.26mmoles) is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH to around 8.5-9. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with AMBERLIST® IR-120+ around 6-7. The reaction mixture is separated by filtration and the solvent removed by rotary evaporation, followed by water dialysis. Structure of generation one glucose dendrimer, D1-Glu6-OH24, is shown below. 45718416.1
Structure VI: D1-Glu6-OH24 In some embodiments, generation one glucose dendrimer D1-Glu6-OH24 is propargylated to provide D1-Acetylene24 according to the following: 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) is slowly added in portions at 0°C to the solution with stirring. The solution is stirred for an additional 15 minutes at 0°C. This is followed by the addition of propargyl bromide (3.85 mL, 34.608 mmol, 80% w/w solution in toluene) at 0°C and the stirring is continued at room temperature for another 6h. The reaction mixture is quenched with ice and water, filtered, and dialyzed against DMF, followed by the water dialysis to afford D1- acetylene24. Structure of D1-acetylene24 is shown below. 45718416.1
Structure VII: D1-acetylene24 In some embodiments, generation one dendrimer D1-acetylene24 is further reacted with AB4 β-D-glucose-PEG4-azide to provide generation 2 dendrimer with 24 glucose molecules containing 96 surface hydroxyl groups. An exemplary generation two dendrimer D2-Glu24-Oac96 is prepared according to the following: D1-acetylene dendrimer24 (0.5g, 0.13 mmoles) and glucose-Oac-TEG-azide (2.2g, 4mmoles) 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 CuSO4·5H2O (5mol%/acetylene, 5mg) and sodium ascorbate (5mol%/acetylene, 10mg) dissolved in the minimum amount of water 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 dialysis containing EDTA. The EDTA is further removed by extensive water dialysis. The product is lyophilized to obtain D2-Glu24-Oac96. In some embodiments, generation two dendrimer D2-Glu24-OH96 is prepared according to the following: the peracetylated generation 2 glucose dendrimer D2-Glu24-OH96 is dissolved in anhydrous methanol and sodium methoxide is added to adjust the pH around 8.5-9.0. The reaction is stirred overnight at room temperature, then diluted with methanol and pH is adjusted with 45718416.1 AMBERLIST® IR-120+ around 6-7. The reaction mixture is filtered to remove the resin and the filtrate is evaporated by rotary evaporation followed by water dialysis to obtain the product as off- white solid. Structure of generation two glucose dendrimer, D2-Glu24-OH96, is shown below. In some embodiments, generation two 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 generation two dendrimer D2-Glu24-OH96 is propargylated according to the following: D2-Glu24- OH96 (5b) (200 mg, 0.016 mmol) is dissolved in anhydrous dimethylformamide (DMF, 10 mL) by sonication. To this stirring solution, sodium hydride [60% dispersion in mineral oil] (22 mg, 0.934 mmol) is slowly added in portions at 0°C. The solution is additionally stirred for 15 minutes at 0°C. This is followed by the addition of propargyl bromide (18.0 µL, 80% w/w solution in toluene) at 0°C and the stirring is continued at room temperature for another 6h. The solvent is evaporated 45718416.1 using V10 evaporator system and the crude product is purified by passing through PD10 SEPHADEX® G25 M column. The aqueous solution is lyophilized to afford the product as off- white solid. In some embodiments, one or more fluorescent dyes such as infrared fluorescent Cy5 dyes are conjugated to generation two dendrimer D2-Glu24-OH96. In one embodiment, Cy5-D2-Glu24- OH96 (compound 7 of FIG.1B) is prepared according to the following: 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 25mL round bottom flask equipped with a magnetic stir bar. To this, CuSO4·5H2O (5mol%/acetylene, 0.3 mg) and sodium ascorbate (10 mol%/acetylene, 0.5 mg) dissolved in the minimum amount of water are added. The reaction is stirred at room temperature for 24 h. Upon completion, the DMF is evaporated using V10 and the purification is performed using PD10 Sephadex G25 M column. The aqueous solution is lyophilized to afford the product as blue solid. 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 out of total 96 available hydroxyl groups of the exemplary generation 2 dendrimer with 24 glucose molecules containing 96 surface hydroxyl groups. B. Methods of Making Dendrimer-Agent Conjugates Methods for conjugating agents with dendrimers are generally known in the art, for example, as described by Menjoge, et al. "Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications." Drug discovery today 15.5-6 (2010): 171-185; US 2011/0034422, US 2012/0003155, and US 2013/0136697. In some embodiments, one or more GCPII inhibitor agents are covalently attached to the dendrimers. In some embodiments, the agents are attached to the dendrimer via a spacer that is designed to be non-cleavable in vivo. In some embodiments, the agents are attached to the dendrimer via a spacer that is designed to be cleaved in vivo. For example, the spacer can be designed to be cleaved hydrolytically, enzymatically, or combinations thereof, so as to provide for the sustained release of the GCPII inhibitor agents in vivo. In some embodiments, both the chemical structure of the spacer and its point of attachment to the GCPII inhibitor agent, can be selected so that cleavage of the spacer releases either an agent, or a suitable prodrug thereof. The chemical structure of the spacer can also be selected in view of the desired release rate of the agents. In some embodiments, the conjugation between the GCPII inhibitor agent and dendrimer is via one or more of disulfide, ester, ether, phosphodiester, triglycyl peptide, hydrazine, amide, or amino alkyl linkages. In some embodiments, the conjugation between the GCPII inhibitor agent and dendrimer is via an appropriate spacer that provides an ester bond or an amide bond between the 45718416.1 agent and the dendrimer depending on the desired release kinetics of the agent. In some cases, an ester or disulfide bond is introduced for releasable form of GCPII inhibitor agents. In other cases, an amide or amino alkyl bond is introduced for non-releasable form of GCPII inhibitor agents. Spacers generally contain one or more organic functional groups. Examples of suitable organic functional groups contained in the spacers include secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonate (-O-C(O)-O-), ureas (-NHCONH-; - NRCONH-; -NHCONR-, -NRCONR-), carbinols (-CHOH-, -CROH-), disulfide groups, hydrazones, hydrazides, ethers (-O-), and esters (-COO-, –CH2O2C-, CHRO2C-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. In general, the identity of the one or more organic functional groups within the spacer is chosen in view of the desired release rate of the GCPII inhibitor agents. In addition, the one or more organic functional groups can be selected to facilitate the covalent conjugation of the agents to the dendrimers. In some embodiments, the conjugation between the GCPII inhibitor agent and dendrimer is via an appropriate spacer that provides a disulfide bridge between the agent and the dendrimer. In some embodiments, the dendrimer-active agent conjugates are capable of rapid release of the agent in vivo by thiol exchange reactions, under the reduced conditions found in body. In certain embodiments, the spacer contains one or more of the organic functional groups described above in combination with a linking group. The linking group can be composed of any assembly of atoms, including oligomeric and polymeric chains; for example, the total number of atoms in the linking group is between 3 and 200 atoms, between 3 and 150 atoms, between 3 and 100 atoms, or between 3 and 50 atoms. Examples of suitable linking groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Variation of the linking group provides additional control over the release of the GCPII inhibitor agents in vivo. In embodiments where the spacer includes a linking group, one or more organic functional groups will generally be used to connect the linking group to both the GCPII inhibitor agent and the dendrimers. Reactions and strategies useful for the covalent conjugation of agents 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, U.S.A. and Menjoge, et al "Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications." Drug discovery today 15.5-6 (2010): 171-185. 45718416.1 Appropriate methods for the covalent conjugation of a given agent can be selected in view of the linking moiety desired, as well as the structure of the agents and dendrimers as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds. The amount of active agent in the dendrimer-active agent conjugates (drug loading) depends on many factors, including the choice of active agent, dendrimer structure and size, and tissues to be treated. In some embodiments, the one or more GCPII inhibitor agents are conjugated to the dendrimer at a concentration between about 0.01% and about 45%, inclusive; between about 0.1% and about 30%, inclusive; between about 0.1% and about 20%, inclusive; between about 0.1% and about 10%, inclusive; between about 1% and about 10%, inclusive; between about 1% and about 5%, inclusive; between about 3% and about 20% by weight, inclusive; or between about 3% and about 10% by weight, inclusive. However, specific drug loading for any given active agent, dendrimer, and site of target can be determined by routine methods, such as those described herein. In some embodiments, the conjugation of GCPII inhibitor agents/spacers occurs via about 1%, 2%, 3%, 4%, or 5% of the total available surface functional groups, such as hydroxyl groups, of the dendrimers prior to the conjugation. In other embodiments, the conjugation of agents/spacers occurs on 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% total available surface functional groups of the dendrimers prior to the conjugation with active agents. In some embodiments, dendrimer-GCPII inhibitor agent conjugates retain an effective amount of surface functional groups for targeting to activated macrophage cells in and around motoneuron/muscle junctions, whilst conjugated to an effective amount of GCPII inhibitor agents to treat, or prevent age-related muscular decline. In some embodiments, dendrimer-GCPII inhibitor agent conjugates retain an effective amount of GCPII inhibitor agents for targeting to target cells and binding to target receptors on the surface or in the interior of the target cells. More specific methods for preparing exemplary dendrimer-GCPII inhibitor agent conjugates are described in the Examples below. IV. Pharmaceutical Formulations Pharmaceutical compositions including dendrimer-GCPII inhibitor agent conjugates may be formulated in a conventional manner using one or more physiologically acceptable carriers, optionally including excipients which facilitate processing of the GCPII inhibitor compounds into preparations which can be used pharmaceutically. Although it may be possible to formulation, for mucosal (intranasal, buccal, sublingual, vaginal, rectal or pulmonary), compositions are typically administered orally or by injection (intravenous, subcutaneous, intraperitoneal, intramuscular, or 45718416.1 intrathecal administration).epresentative excipients include aqueous buffers, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS) and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. Generally, pharmaceutically acceptable salts of the actives can be prepared by reaction of the free acid or base forms of an agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p.704. Examples of ophthalmic drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac. The compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The phrase “dosage unit form” refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total single administration of the compositions will be decided by the attending physician within the scope of sound medical judgment. The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs, or extrapolated from human data. Therapeutic efficacy and toxicity of conjugates can be determined by standard pharmaceutical procedures in experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and is expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use. The compositions of dendrimer-GCPII inhibitor agent conjugates can be formulated together with one or more additional active agents, for example, agents that are known or contemplated to exert some beneficial effect to a subject. 45718416.1 V. Methods of Use The compositions can be administered to treat or alleviate one or more symptoms of reduction in muscle mass and/or strength, for example, associated with sarcopenia. The compositions are typically administered in an amount effective to provide a therapeutic effect, but insufficient to provide an adverse effect. In preferred embodiments, the dendrimer compositions selectively target specific cells and specific receptors on the cells to address a variety of diseases, disorders and conditions. The methods include administering to a subject in need, the compositions in an amount effective to increase binding of the GCPII enzyme inhibitor compound to GCPII on the surface of target cells or at specific receptors in specific cells, particularly activated immune cells, such as activated macrophages in the muscles, and Schwaan cells at the neuromuscular junctions (NMJ), and surrounding synapses. A. Methods of Treatment It has been established that inhibition of glutamate-carboxypeptidase-II in and around the neuromuscular junctions (NMJ) of aged subjects can increase muscle mass and thereby strength. Typicallythe hydroxylated dendrimers conjugated with glutamate-carboxypeptidase-II (GCPII) inhibitors are administered to a subject in an amount effective to reduce, delay or reverse one or more symptoms or markers of sarcopenia in the subject. In exemplary forms, an amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is administered in an amount effective to reduce glutamate and/or cytoplasmic Ca2+ concentrations within and/or around neuromuscular junctions (NMJ) in the subject and thereby prevent or reduce neuronal cell damage and death in the subject. In some forms, the amount of dendrimers conjugated with GCPII inhibitors is effective to bind to GCPII at the surface of target cells, including neuronal cells, glial cells, Schwann cells, and activated macrophages. The compositions can be administered to prevent, treat, and/or manage the symptoms of ofther disorders, diseases, and conditions in additiona to age-related decrease in muscle mass, age- related decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and related disorders. In some forms, when the dendrimer is complexed to one or more GCPII inhibitors, the compositions can be administered to reduce, prevent, delay or reverse one or more neurological disorders such as a decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), neuromuscular transmission defects, and related disorders in a subject in need thereof. In some forms, the dendrimer-GCPII inhibitor conjugates may be administered to a subject prophylactically, e.g., to prevent, delay or otherwise mediate the onset or rate of age-related decrease in effective 45718416.1 neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and related disorders. 1. Site-Specific Targeting to Target Cells The compositions and methods are designed to circumvent existing challenges in selective drug delivery to specific target cells and/or tissues, motoneurons, perisynaptic Schwann cells, and activated macrophage cells within and around neural synapses and neuromuscular junctions (NMJ) and are administered peripherally. Glutamate-carboxypeptidase-II (GCPII) inhibitors conjugated to hydroxylated dendrimers can be selectively delivered to specific cell types in vivo to reduce, prevent or delay neuromuscular junction (NMJ) denervation in a subject. Typically the hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor is administered in an effective amount to provide binding of the GCPII inhibitor to one or more receptors on the surface of or inside target cells such as neuronal cells, glial cells, Schwann cells, and activated macrophages. Typically, the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is administered in an amount effective for reducing, delaying, inhibiting or reversing glutamate and/or cytoplasmic Ca2+ concentrations within and/or around neuromuscular junctions (NMJ) in the subject. Macrophage influx specifically in the muscle tissue of a test animal model for neurodegenerative disease was found to destabilize the perisynaptic Schwann cells, which are important for NMJ health. In contrast, reducing macrophage activation was shown to prolong the NMJ integrity, indicating that activated macrophages can be detrimental to perisynaptic Schwann cells (Van Dyke, et al., Experimental neurology.2016; (277) pp.275–282). One mechanism for infiltrating macrophages to exacerbate the axonal withdrawal is through the upregulation of GCPII activity and increased glutamate production. While acetylcholine (Ach) is the canonical neurotransmitter at the neuromuscular synapse, it has been demonstrated that glutamate also plays an importantrole during neuromuscular synapse development. Activated macrophages may, in part, elicit neurotoxic effects through enhanced release of glutamate. PAMAM-OH and PEG-OL dendrimers may target microglia/macrophages ), and glucose dendrimers that target both neurons and microglia. The role of upregulated GCPII activity on infiltrating macrophages is likely where activated macrophages have detrimental effects on Schwann cells and axonal integrity such as Charcot-Marie-Tooth disease or even the general aging process of axonal withdrawal. GCPII inhibition may be altering the immune response and affecting disease progression by reducing harmful immune cell activation. Chronic immune cell activation in the muscle can be pathological and immune cells are known to modulate the extracellular matrix, which is a key 45718416.1 component of nerve regeneration and repair. Therefore, administering dendrimer-GCPII inhibitor conjugates to selectively target activated macrophages at or near the NMJ may prevent, reduce or reverse the effects of increased/deleterious GCPII receptor activity at or near motoneurons and the NMJ. In some forms, the dendrimer-GCPII inhibitor conjugates increase drug bioavailability in and around motoneurons and neuromuscular junctions by one or more of the following: (i) increasing drug solubility, (ii) facilitating target engagement i.e., increasing site-specific binding, (iii) improving drug pharmacokinetics, and (iv) targeting to activated immune cells at an near the neuromuscular junctions. For example, in some forms, the methods permit selective delivery of compounds to the neuromuscular junctions, thereby increasing the potential of the compositions to be used to selectively treat neuromuscular impairment and related diseases and disorders. B. Conditions and Diseases to be Treated In some forms, the compositions are used to prevent, treat, and/or manage symptoms of sarcopenia, including age-related decrease in muscle mass, age-related decrease in effective neuromuscular function (e.g., decrease in maximal muscular force/ strength), age-related increase in neuromuscular transmission defects, and sarcopenic obesity and arterial stiffness. 1. Sarcopenia In some forms, the methods reduce, prevent, delay or reverse one or more symptoms of sarcopenia in a subject in need thereof. Sarcopenia has been defined as a generalized disease that causes decreased muscle mass and muscle function; it is a chronic, progressive skeletal muscle disease characterized by low muscle strength and quantity or quality, leading to reduced physical performance and increased physical frailty. Sarcopenia significantly worsens prognosis of, and is related to a higher risk of polypharmacy, impaired quality of life, falls and fractures, loss of physical independence, and in can lead directly to death. a. Symptoms of Sarcopenia In some forms, the methods are used to treat or prevent one or more symptoms of sarcopenia in a subject. The symptoms/signs associated with sarcopenia include dynapenia (low muscle strength), reduced multisensory integration (balance), reduced muscle mass, increased physical frailty, such as falls and difficulties in lifting and carrying load (e.g., 4.5 kg or more), moving, getting up from a chair/bed, or going up a flight of stairs. In some forms, symptoms of sarcopenia are determined using one or more of a maximal handgrip strength test (upper limbs), total body mass and/or body mass index (BMI; e.g., calculated by the ratio between body mass and height (meters) squared (body mass/height2), and based on 45718416.1 responses to self-assessment of strength, walking aids, difficulty getting up from a chair, difficulty climbing stairs, and falls (e.g., based on the SARC-F score determined from a 5-item questionnaire, described below); or ability in the Senior Fitness Test (SFT; to evaluate physical function in healthy elderly people including six tests: the 30-s Chair Stand Test (CST), the 30-s arm curl test (ACT), the chair sit and reach test, the back-scratch test (BST), the 8-foot up-and-go test (FUG), and the 6- min walk test (6MWT)). Therefore, in some forms, an amount of a dendrimer conjugated to one or more GCPII inhibitors is administered in an amount effective to treat one or more symptoms of sarcopenia such as lack of strength, need for assistance in walking, difficulty rising from a chair, difficulty climbing stairs, increased number of falls, reduced muscle mass, reduced balance and reduced muscular control in a subject in need thereof. For example, in some forms, the conjugates are effective to increase strength, reduce the need for assistance in walking, reduce difficulty rising from a chair, reduce difficulty climbing stairs, reduce the number of falls, increase muscle mass, increase balance and/or increase muscular control in a subject relative to an untreated control subject. In some forms, the methods are effective to slow or stop the progression of sarcopenia in a subject, relative to an untreated control subject. For example, in some forms, the methods are effective to reduce the rate of loss of strength, reduce the rate of loss of muscle mass, reduce the rate or extent of loss of balance and/or reduce the rate of loss of muscular control in a subject as compared to an untreated control subject. In some forms, the methods increase the speed of walking, increase the speed of muscular contractions, increase vigor, increase energy and/or reduce body fat, increase lifespan and/or decrease mortality in a subject identified as having sarcopenia. i. SARC-F Test Score In some forms, symptoms of sarcopenia are determined using the SARC-F test. An exemplary scoring system for a SARC-F test is as follows: Q1: Strength: How much difficulty do you have in lifting and carrying 10 pounds? A1: None =0; Some =1; A lot or unable =2. Q2: Assistance in walking: How much difficulty do you have walking across a room? A2: None =0; Some =1; A lot, use aids, or unable =2. Q3: Rise from a chair: How much difficulty do you have transferring from a chair or bed? A3: None =0; Some =1; A lot or unable without help =2. Q4: Climb stairs: How much difficulty do you have climbing aflight of 10 stairs? 45718416.1 A4: None =0; Some =1; A lot or unable =2. Q5: Falls: How many times have you fallen in the past year? A5: None =0; 1ess than 3 falls=1; 4 or more falls =2. Therefore, in some forms, a subject has a SARC-F score of between 1 and 10, inclusive before administration of the dendrimer-GCPII inhibitor conjugates. In some forms, the subject has a SARC-F score of 2, 3, 4, 5, 6 , 7, 8, 9 or 10 before administration of the dendrimer-GCPII inhibitor conjugates. Data suggests that a SARC-F score of ≥4 best predicts the need for further, more comprehensive evaluation. In some forms, the methods administer to a subject an amount of the dendrimer-GCPII inhibitor conjugates effective to reduce the SARC-F score of the subject by 1 or more points, such as by 2, 3, 4 ,56, 7, 8, 9 or 10 points, relative to an untreated control. For example, the dendrimer-GCPII inhibitor conjugates reduce the SARC-F score of a subject from 4 or more to less than 4. b. Biomarkers of Sarcopenia Thedendrimer-GCPII inhibitor conjugates may be effective to alter the presence of one or more biomarkers of sarcopenia in the subject. The pathogenesis of sarcopenia includes various complex molecular mechanisms and is not yet fully understood, however several biomarkers that are associated with sarcopenia are known in the art, for example, as described in Gugliucci, et al., J Clin Med.2024 Feb; 13(4): 1107, which is incorporated herein by reference in its entirety. Generally, biomarkers related to sarcopenia can be divided into markers evaluating musculoskeletal status (biomarkers specific to muscle mass, markers of the neuromuscular junction, or myokines), and markers assuming causal factors (adipokines, hormones, and inflammatory markers). Exemplary biomarkers associated with assessment of sarcopenia include: myostatin (MSTN, also known as GDF-8; decreased in Sarcopenia); follistatin (FST; increased in Sarcopenia); irisin (decreased in Sarcopenia); brain-derived neurotrophic factor (BDNF; decreased in Sarcopenia); procollagen type III N-terminal peptide (PIIINP; P3NP – varies depending upon other factors, but typically changed in sarcopenia); sarcopenia index (serum creatinine to serum cystatin C ratio, decreased in Sarcopenia); adiponectin (increased in Sarcopenia); leptin (increased in Sarcopenia); insulin-like growth factor-1 (IGF-1; decreased in Sarcopenia); dehydroepiandrosterone sulphate (DHEAS; decreased in Sarcopenia); C-reactive protein (CRP; increased in Sarcopenia); interleukin-6 (IL-6; increased in Sarcopenia), and tumor necrosis factor α (TNF-α; increased in Sarcopenia). 45718416.1 i. Myostatin The one or more dendrimer-GCPII inhibitor conjugates may be administered in an amount effective to increase the relative amount of myostatin (MSTN, also known as GDF-8) in a subject. It may be that myostatin muscle protein turnover decreases during the progression/development of sarcopenia. Methods of measuring myostatin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. Therefore, in some forms, the methods administer one or more dendrimer-GCPII inhibitor conjugates to a subject in an amount effective to increase myostatin muscle protein turnover in a subject identified as having sarcopenia, as compared to an untreated control. ii. Follistatin The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of follistatin (FST) in a subject. It may be that follistatin muscle protein turnover is acutely increased with exercise during the progression/development of sarcopenia. Methods of measuring follistatin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. Therefore, in some forms, the methods administer one or more dendrimer-GCPII inhibitor conjugates to a subject in an amount effective to decrease follistatin muscle protein in a subject identified as having sarcopenia, as compared to an untreated control. iii. Irisin Theone or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of irisin in a subject. It may be that irisin muscle protein turnover is decreased during the progression/development of sarcopenia. Methods of measuring irisin levels and muscle mass in a subject are known in the art, including LC-MS/MS measurement methods. iv. Brain-derived neurotrophic factor (BDNF) and/or Glial cell line-derived neurotrophic factor (GDNF) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a human subject in an amount effective to increase the relative amount of brain-derived neurotrophic factor (BDNF) and/or glial cell line-derived neurotrophic factor (GDNF) in a subject. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are markers associated with the neuromuscular junction and neuroinflammation. These are neurotrophins released from both neurons and muscles and play an important role in muscle development and metabolism and the regulation of synapse function. It may be that BDNF and/or GDNF is decreased due to remodeling of the neuromuscular junctions (NMJ) during the progression/development of 45718416.1 sarcopenia. Methods of measuring BDNF and/or GDNF serum levels in a subject are known in the art, including immunoassay measurement methods. v. Procollagen type III N-terminal peptide (PIIINP; P3NP) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to alter the relative amount of procollagen type III N-terminal peptide (PIIINP; P3NP) in a subject. PIIINP is produced during type III collagen synthesis, and PIIINP levels increase with higher BMI and age. Its concentrations seem to be related to aging, body composition, and physical performance. One study showed an association between higher PIIINP levels and lower lean body mass in postmenopausal women. It may be that the amount of PIIINP changes due to muscle collagen turnover during the progression/development of sarcopenia. Methods of measuring PIIINP in a subject are known in the art. vi. Sarcopenia index (serum creatinine to serum cystatin C ratio) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative Sarcopenia index (SI; serum creatinine to serum cystatin C ratio) in a subject. SI is a formula for evaluating muscle mass; Serum creatinine concentrations reflect muscle protein turnover but are also determined by renal function. That is why, in this formula, creatinine level is related to another marker of renal function—cystatin C. SI has been correlated with CT skeletal muscle cross-sectional surface area, calf circumference, BMI, handgrip strength, and gait speed. The SI is an algorithm developed to reflect muscle mass, reclassified as the marker of muscle turnover subclass; SI = serum creatinine (mg/dL)/serum cystatin C (mg/L)) × 100 It may be that the SI is decreased due to reduction in muscle mass during the progression/development of sarcopenia. Methods of measuring SI in a subject are known in the art. vii. Adiponectin The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of adiponectin in a subject. Adiponectin could play a role in a compensatory mechanism for mitigating sarcopenia resulting from chronic inflammation and oxidative stress. It may be that the anti-inflammatory factor adiponectin is increased due to increased muscle-fat crosstalk during the progression/development of sarcopenia. Methods of measuring adiponectin serum levels in a subject are known in the art, including immunoassay measurement methods. 45718416.1 viii. Leptin The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of leptin in a subject. Leptin is an adipokine that, among other things, improves immune response and induces lipid catabolism; leptin serum levels are positively correlated with muscle and fat mass and negatively correlated with muscle strength—increased leptin levels are linked to a higher risk of dynapenia (low muscle strength). It may be that the adipokine factor leptin is increased due to increased fat deposition during the progression/development of sarcopenia. Methods of measuring leptin serum levels in a subject are known in the art, including immunoassay measurement methods. ix. Insulin-like growth factor-1 (IGF-1) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of insulin-like growth factor-1 (IGF-1) in a subject. Age-related decline in IGF-1 levels is considered a possible factor contributing to the development of sarcopenia and it may be that serum levels of IGF-1 are decreased due to age- related anabolic metabolism during the progression/development of sarcopenia. Methods of measuring IGF-1 serum levels in a subject are known in the art, including immunoassay measurement methods. x. Dehydroepiandrosterone sulphate (DHEAS) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to increase the relative amount of dehydroepiandrosterone sulphate (DHEAS) in a subject. Its role in sarcopenia has long been studied, and the age-related decrease in its levels is perceived as a causal factor for muscle loss; DHEAS concentrations correlate positively with older patients’ skeletal muscle mass and strength and seem to be decreased in sarcopenic individuals. It may be that levels of serum DHEAS are decreased due to age-related anabolic metabolism during the progression/development of sarcopenia. Methods of measuring DHEAS serum levels in a subject are known in the art, including immunoassay measurement methods. xi. C-reactive protein (CRP ) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of C-reactive protein (CRP) in a subject. One of the crucial mechanisms that may be related to sarcopenia pathogenesis is chronic inflammation. Inflammatory cytokines have been linked to muscle wasting, promoting protein catabolism and suppressing muscle tissue synthesis. It may be that the C-reactive protein (CRP) is increased due to increased chronic inflammation during the progression/development of sarcopenia. Methods of 45718416.1 measuring C-reactive protein (CRP) levels in a subject are known in the art, including immunoassay measurement methods. xii. Interleukin-6 (IL-6) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of Interleukin-6 (IL-6) in a subject. It may be that IL-6 is increased due to increased low-grade chronic inflammation associated with reduced muscle mass during the progression/development of sarcopenia. Methods of measuring C-reactive protein IL-6 levels in a subject are known in the art, including immunoassay measurement methods. xiii. Tumor necrosis factor α (TNF-α) The one or more dendrimer-GCPII inhibitor conjugates may be administered to a subject in an amount effective to decrease the relative amount of tumor necrosis factor α (TNF-α) in a subject. It may be that tumor necrosis factor α (TNF-α) is increased due to increased low-grade chronic inflammation associated with reduced muscle mass during the progression/development of sarcopenia. Methods of measuring C-reactive protein TNF-α levels in a subject are known in the art, including immunoassay measurement methods. C. Subjects Sarcopenia is an age-related disease and affects approximately 10% to 27% of the population aged ≥60 years in the USA. The frequency of sarcopenia in nursing homes is higher than in community-dwelling older adults and is estimated at 51% in men and 31% in women. Typically, a subject has one or more symptoms of sarcopenia, or is identified as being at risk of developing sarcopenia. Systems and methods for the diagnosis of sarcopenia are known in the art, and the method can include selecting a subject having or at risk of having sarcopenia based on any one or more of the known systems or methods for diagnosing sarcopenia known in the art. Exemplary methods for diagnosis of sarcopenia include methods proposed by the European Working Group on Sarcopenia in Older People (EWGSOP), methods proposed by the Asia Working Group for Sarcopenia (AWGS), methods proposed by the International Working Group on Sarcopenia (IWGS), methods proposed by Society for Sarcopenia Cachexia and Wasting Disorders (SCWD), and methods proposed by the Foundation for the National Institutes of Health (FNIH) Sarcopenia Project. In an exemplary form, identification of sarcopenia in a subject is based on one or more procedures including Screening assessment using the SARC-F scale; measuring hand grip strength (or performing a Chair Stand Test) to diagnose low muscle strength; estimating muscle quantity with dual-energy X-ray absorptiometry (DXA); bioelectrical impedance analysis (BIA); magnetic resonance imaging (MRI); and computed tomography (CT) of one or more regions of the body of 45718416.1 the subject. In some forms, the methods include one or more additional physical performance tests, such as the Timed-Up-and-Go test (TUG), Short Physical Performance Battery (SPPB), gait speed measurement, and a defined distance Walk Test, such as a 400 meter walk test. In some forms, these methods identify a subject as having sarcopenia. The subject may have one or more additional or underlying medical indications in addition to one or more symptoms of sarcopenia. For example, in some forms, a subject has type II diabetes mellitus, albuminuria, sarcopenic obesity, sarcopenic arterial stiffness and or peripheral neuropathy. In some forms, the subject does not have an underlying indication and is otherwise a healthy subject. In some forms, the subject does not have and/or has never had been diagnosed with a neurological disease. For example, in some forms, the subject has not had or been diagnosed as having Alzheimer’s disease (AZ), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS). In some forms, the subject has not had or has never been diagnosed as having dementia, depression, or mental illness. Because of several shared pathways between the two diseases, sarcopenia is also a risk factor for developing type 2 diabetes mellitus (T2DM) in older patients. Therefore, in some forms, the subject has or is identified as being at risk of having type 2 diabetes mellitus (T2DM). In Exemplary forms, the subject has a score of 4 or more on the SARC-F scale, such as a score of 5, 6, 7, 8, 9, or 10 on the SARC-F scale. Exemplary subjects are at least 40 years of age, or at least 50 years of age, or at least 60 years of age, or at least 70 years of age. In exemplary forms, the subject does not have and/or has never been diagnosed as having one or more diseases or disorders selected from Alzheimer’s disease (AZ), Parkinson’s disease (PD) amyotrophic lateral sclerosis (ALS), dementia, Schizophrenia, multiple sclerosis, and depression. D. Dosage and Effective Amounts Dosage and dosing regimens are dependent on the severity and location of the disorder and/or methods of administration, as well as the specific agent being delivered. This can be determined by those skilled in the art. Typically, an effective amount of dendrimer complexes including a combination of a dendrimer with one or more therapeutic GCPII inhibitor active agents are administered to an individual in need thereof. The dendrimers may also include a targeting agent, but as demonstrated by the examples, these are not required for delivery to activated macrophage cells associated with the nervous system, neuromuscular junctions and muscles surrounding synapses. Typically, the dosage of dendrimer complexes include an effective amount of one or more GCPII inhibitors attached or conjugated to a dendrimer, which are capable of preferentially releasing the drug at the target receptor. The GCPII inhibitor agent can be either covalently attached or intra-molecularly 45718416.1 dispersed or encapsulated. The amount of dendrimer complexes administered to the subject is selected to deliver an effective amount to reduce, prevent, or otherwise alleviate one or more clinical or molecular symptoms of the disease or disorder to be treated, such as sarcopenia, as compared to a control, for example, a subject treated with the active agent without dendrimer. In some embodiments, dosages are expressed in mg/kg, particularly when the expressed as an in vivo dosage of composition(s) of dendrimer-GCPII inhibitor conjugates. As noted above, the preferred route of administration is oral or subcutaneous injection Typically, doses are in the range from microgram/kg up to about 100 mg/kg of body weight of the recipient. Exemplary dosages are, for example 0.01 mg/kg to about 1,000 mg/kg, or 0.5 mg/kg to about 1,000 mg/kg, or 1 mg/kg to about 1,000 mg/kg, or about 10 mg/kg to about 500 mg/kg, or about 20 mg/kg to about 500 mg/kg per dose, or 20 mg/kg to about 100 mg/kg per dose, or 25 mg/kg to about 75 mg/kg per dose, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg/kg per dose. Efficacy has been shown at 20 mg/kg administered orally. Preferably, the compositions of dendrimer-GCPII inhibitor agents do not target or otherwise impact non-activated immune cells, or cells that not within or associated with impacted tissues/motoneurons, or do so at a reduced level compared to activated immune cells and motoneurons associated with a disease or disorder such as a sarcopenia. In this way, by-products and other side effects associated with the compositions are reduced. Therefore, in preferred embodiments, dendrimer compositions are administered in an amount that leads to an improvement, or enhancement, function in an individual with a disease or disorder, such as sarcopenia or an associated disorder. The actual effective amounts of the composition can vary according to factors including the specific agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder. Generally, for intravenous injection or infusion, the dosage will be lower than for oral administration. In general, the timing and frequency of administration will be adjusted to balance the efficacy of a given treatment or diagnostic schedule with the side effects of the given delivery system. In some embodiments, dosages are administered daily, biweekly, weekly, every two weeks or less frequently in an amount to provide a therapeutically effective increase in the blood level of the therapeutic agent. Where the administration is by other than an oral route, the compositions may be delivered over a period of more than one hour, e.g., 3-10 hours, to produce a therapeutically effective dose within a 24-hour period. Alternatively, the compositions can be formulated for controlled release, wherein the composition is administered as a single dose that is repeated on a regimen of once a week, or less frequently. It will be understood by those of ordinary skill that a 45718416.1 dosing regimen can be any length of time sufficient to treat the disorder in the subject. In some embodiments, the regimen includes one or more cycles of a round of therapy followed by a drug holiday (e.g., no drug). The drug holiday 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. Any of the therapeutic effects of the described compositions including one or more dendrimer-GCPII inhibitor agents can be compared to a suitable control. In some embodiments, a control includes an equivalent amount of GCPII inhibitor agents delivered alone, or bound to dendrimers without a similar generation, molecular weight, and/or surface group density (e.g., hydroxyl groups). VI. Kits The compositions can be packaged in kit. The kit can include a single dose or a plurality of doses of a composition including one or more GCPII inhibitors associated with or conjugated to a dendrimer (e.g., one or more hydroxyl-terminated PAMAM dendrimers or glucose dendrimers as described in the Examples), and instructions for administering the compositions. Specifically, the instructions direct that an effective amount of the dendrimer composition be administered to an individual with 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. The present invention will be further understood by reference to the following non-limiting examples. Example 1: Synthesis of Hydroxyl-Polyamidoamine (PAMAM-OH) dendrimer 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA) conjugates NAAG is synthesized pre-synaptically by NAAG synthetase and packaged into vesicles by sialin. After release, intact NAAG interacts with mGlu3 and NMDA receptors and/or is hydrolyzed by glial GCPII to release glutamate outside the synaptic cleft. Under “basal”/non-pathological conditions, GCPII activity and synaptic NAAG and glutamate concentrations are relatively low. NAAG modulates synaptic activity by activating presynaptic mGlu3 receptors and postsynaptic GluN2A-rich NMDA receptors. Further, NAAG that reaches the extra-synaptic space inhibits GluN2B-rich NMDA receptor-mediated EPSCs and stimulates glial mGlu3 receptors which induces trophic effects. Under pathologic conditions with increased synaptic activity, elevated levels of glutamate and NAAG flood the synapse. NAAG that reaches the extra-synaptic space is rapidly cleaved by GCPII to liberate glutamate. The excess glutamate and NAAG activate both GluN2A- and GluN2B-rich NMDA receptors, increasing EPSCs. Blockade of GCPII under activated conditions prevents the breakdown of NAAG lowering overall glutamate levels. The resulting increased NAAG further decreases glutamate release through feedback inhibition via presynaptic mGlu3 receptors and induces trophic effects via activation of glial mGlu3 receptors. Overall, 45718416.1 inhibition of GCPII increases NAAG and lowers glutamate, returning the system toward its basal state. Methods The synthesis of 4th generation hydroxyl PAMAM dendrimers to 2-(Phosphonomethyl)- pentanedioic Acid (2-PMPA) conjugates was achieved using attachment of a cleavable azido- polyethylene glycol (11) alcohol linker to 2-PMPA, followed by conjugation of 2-PMPA on the surface of hydroxyl PAMAM dendrimer, to form a Dendrimer-2-PMPA conjugate (D-2-PMPA) having a cleavable ester linkage. Briefly, the synthesis first included conjugation of 2-PMPA to a cleavable PEG linker in presence of Azido-PEG-11-alcohol (EDC/DMAP/DMF for 24 hours at room temperature) to form 2-PMP-PEG-N3, as depicted in FIG.1. Secondly, the synthesis included conjugation of G4 hydroxylated polyamidoamine (PAMAM-OH) dendrimer (“D-OH”) to the previously synthesized 2-PMP-PEG-N3, by first conjugating a 56-hexynoic acid to D-OH in presence of EDC, DMAP, DMF for 24 hours at room temperature to form D-hexyne, then click chemistry-based conjugation of D-hexyne with 2-PMP-PEG-N3 in the presence of CuSO4.5H2O Na Ascorbate (DMF/THF/H2O for 6 hours at 50 ˚C in a microwave) to form D-2-PMPA (5), having a cleavable ester linkage , as depicted in FIG.2. Results The stepwise synthesis depicted in the schematic of FIGS.1-2 produced a hydroxylated PAMAM dendrimer-2-PMPA (“D-2-PMPA”) conjugate via click chemistry with an enzyme sensitive ester linkage. Example 2: Validation of Hydroxyl-Polyamidoamine (PAMAM-OH) dendrimer 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA) conjugates for treating Sarcopenia Materials and Methods Animals C57BL/6 mice were obtained from the National Institute on Aging (NIA) and housed under specific pathogen-free conditions at Johns Hopkins University. All procedures were approved by the relevant Institutional Animal Care and Use Committees. A combination of male and female C57BL/6 mice were used. Mice were housed at a maximum of 5 per cage on a 12-hour dark/light cycle and provided food and water ad libitum. Mice were administered either 200 mg/kg D-2- PMPA or vehicle (HEPES buffer) control via intraperitoneal (IP) injection 5 days per week for 10 months. CD11b+ macrophage isolation CD11b+ enriched macrophage and CD11b- cells in muscle from mice treated with either HEPES vehicle or G4-D-2-PMPA were isolated employing Miltenyi Biotec's magnetic-activated cell 45718416.1 sorting (MACS) system. Briefly, the young or aged mice calf muscles were freshly harvested and diced into small pieces in DMEM buffer and underwent digestion with the skeletal muscle dissociation kit (Miltenyi Biotec) for 1.5 hours. The digested homogenates were then filtered through 50-µm mesh-size cell strainers to collect mononuclear cells. Following the removal of debris and red blood cells, the collected mononuclear cells were magnetically labeled with anti- CD11b MicroBeads. After 1.5 hours of magnetic labeling, the cells were drained through the MACS separators with MACS magnetic stands, where the CD11b- cells were collected from the drained-through solution, and the CD11b+ cells were adhered to the columns and subsequently collected after relief from the magnetic field. Enzyme activity measurements The GCPII enzyme activities in CD11b+ and CD11b- cells were measured after the CD11b+ macrophage isolation process. Briefly, isolated cells were carried out radiolabeled NAAG hydrolysis assays for about 3 hours at 37 °C using [3H]-NAAG (0.02 µM, 48.6 mCi/µmol). Reactions were terminated with ice-cold sodium phosphate buffer (100 mM, pH 7) with 1 mM EDTA.96 well spin columns packed with anion exchange resin were used to separate the substrate and the reaction product. The reaction product [3H]-glutamate was eluted with 1 M formic acid and analyzed for radioactivity. Finally, total protein was quantified per the manufacturer’s instructions using the Bio-Rad DC Protein Assay kit, and GCPII activity data were calculated as fmoles of NAAG hydrolysis per mg protein per hour (fmol/mg/h). Noninvasive magnetic resonance imaging (MRI) MRI of mice were performed on a 9.4T MRI scanner. Animals were anesthetized for imaging using isoflurane with 3-5% at 0.6 L/min in an induction chamber for induction, and 1-2% at 0.6 L/min for maintenance during imaging. During MR imaging a physiological monitoring and gating system designed specifically to meet the physiological monitoring and gating requirements for anesthetized small animals such as mice and rats in the MR environment of very high magnetic fields will be used. Once the scan is complete, the animal will be placed on a warm blanket until it can move around and is fully recovered. MRI data were further analyzed with Matlab to obtain the total volume of mice calf muscle. Contractility A nerve-evoked contractile function evaluation of gastrocnemius muscles was performed with an Aurora Scientific Incorporated High-Power Bi-Phase Current Stimulator (701B) and Dual- Mode Lever System (305C-LR-FP). The assessment included anesthetizing the mice and placing them in a supine position with the knee position fixed and the foot secured to the footplate (300C- FP). Subdermal Needle Electrodes (0.5”27G) were placed both in the sciatic nerve (stimulatory) 45718416.1 and adjacent muscle tendons (reference) to stimulate the gastrocnemius muscle. Electrical current was individually adjusted to produce maximal isometric force before isometric force testing. The force versus frequency relationship was determined with 500 ms trains of pulses between 1 and 150 Hz. Rotarod performance The rotarod performance was recorded monthly during the treatment. Briefly, on the day of the experiment, the animals were trained to stay on the bar at 5 rpm for 5 mins. They were then assessed on their ability to remain on a steady rotating bar for 5 mins at 15rpm over 3 separate trials, allowing the animals a 5-minute rest between trials. The animals were also tested on an accelerating rotarod that began at 4 rpm and steadily increased up to 40 rpm over 5 mins for 3 separate trials, allowing the animals a 5-minute rest between trials. The time it took for the animals to fall from the bar was recorded and an average of the 3 trials was used for assessments. Compound muscle action potential (CMAP) CMAP Recordings were measured monthly during the treatment. Briefly, mice were anesthetized with 1-2% isoflurane, and stimulating electrodes were placed along the sciatic nerve at the sciatic notch and ankle. Recording electrodes were placed in the muscles of the footpad. A brief (<0.2 ms) electrical pulse was applied at the stimulating sites and the CMAP will be recorded using an Evidence 3102evo EMG system (Schreiber & Tholen Medizintechnik, Germany). Three recordings were taken at the maximal stimulation and supramaximal stimulation intensities. The highest amplitude and corresponding latency were recorded. Single fiber electromyography (sfEMG) sfEMG was performed with mice anesthetized using 1-2% isoflurane. To record sfEMG, a recording sfEMG needle was placed in the gastrocnemius muscle while a stimulating electrode was placed near the sciatic nerve. Axons were stimulated at a rate of 10Hz with a stimulation intensity between 10-30 mA to avoid visible muscle contractions. The mean consecutive difference in the single fiber action potentials was subsequently analyzed. Results The D-2-PMPA conjugate inhibited GCPII with and IC50= 3.50±0.05 nM using human recombinant GCPII, as demonstrated in FIGs 3A-3D. Aged mice muscle showed increased GCPII activity specifically in CD11b+ enriched macrophage cells (138.6±3.0 vs.336.6±52.9; p<0.05) but not in CD11b- cells. Systemic D-2-PMPA therapy (20 mg/kg 2-PMPA equivalent; IP 3 × /week) completely inhibited the elevated GCPII activity (336.5±52.8 vs.21.8±9.2 fmol/mg/h; p<0.001), as demonstrated in FIG 4. 45718416.1 5-months of D-2-PMPA therapy initiated with 15-month-old mice led to significant preservation of calf muscle volume (95.0±0.8% vs.90.4±0.7%; p < 0.001), as demonstrated by the relative percentage of mice calf muscle preserved after 5 months of treatment of D-2-PMPA or vehicle (depicted in FIG 5), as well as enhanced isometric force (230.2±13.3 vs.184.0±9.0 mN, female p<0.05; 257.3±21.0 vs.189.7±19.3 mN, male p<0.05), as demonstrated in female and male mouse calf muscle after 5-months treatment of D-2-PMPA or vehicle (see FIGs 6A-6B), as well as improved grip strength (96.1±1.6 vs.90.2±2.2 % of maximum p<0.001), as demonstrated by the relative percentage of mice all limb grip strength preserved after 5-months treatment of D-2-PMPA or vehicle (depicted in FIG 7), and increased rotarod latency (119.0±6.6 vs.93.5±9.3 s, female p<0.05; 104.5±5.2 vs.79.3±4.2 s, male p<0.001) as demonstrated in female and male mice after 5- months treatment of D-2-PMPA or vehicle (depicted in FIGs 8A-8B). Age-related decline of nerve signal conductivity to muscles was also delayed by treatment with D-2-PMPA, as observed by improved compound muscle action potential (CMAP) latency (1.28±0.02 vs.1.38±0.04 ms; p< 0.05) and amplitude (15.2±0.5 vs.1.27±0.6 mV; p<0.01), as demonstrated in mice hind paw compound muscle action potential (CMAP) latency and amplitude with ankle site stimulation after 5-month treatment of D-2-PMPA or vehicle (as depicted in FIGs 9A-9B). Further, NMJ integrity was preserved by D-2-PMPA treatment, as demonstrated by single fiber jitter (5.4±0.3 vs.7.5±0.5 µs; p<0.01) in mice gastrocnemius muscle single-fiber EMG measured after 5-month treatment of D-2-PMPA or vehicle in FIG 10. Together, the data support that excessive GCPII activity in muscle macrophages is associated with age-related muscle atrophy, which is improved by GCPII inhibition. This study highlights GCPII inhibition in macrophages as a new therapeutic approach to delay sarcopenia. Example 3: Dendrimer-2-PMPA (D-2PMPA) preferentially deliver 2-PMPA to muscle macrophages Methods Aged mice were dosed with 10^mg/kg Cy5-D-2PMPA, and the soleus and EDL muscles were harvested 24^hours post-administration. Muscle sections were stained with CD68 (green) for macrophages and Cy5 (red) for D-2PMPA. The merged images clearly demonstrated that Cy5-D- 2PMPA preferentially accumulates in CD68+ macrophages. Results Using Cy5-labeled D-2PMPA (Cy5-D-2PMPA), it was demonstrated that the dendrimer nanoparticle preferentially targets and delivers 2-PMPA to macrophages within both fast and slow 45718416.1 muscle tissue of aged (24-month-old) mice. This targeted delivery was sustained for at least 24 hours. Example 4: Dendrimer-2-PMPA (D-2PMPA) treatment enhances motor function in aged mice Methods D2PMPA was administered three times per week (Monday, Wednesday, Friday) from 15 to 20 months of age. The experimental design included: 15-month-old mice (n=15M and 15F) underwent 5 months of IP treatment with D-2PMPA (20 mg/kg) or empty dendrimer vehicle. Motor function was monitored via monthly measurements of compound muscle action potentials (CMAP) and assessments of grip strength and rotarod performance (See FIG 11). Motor performance was evaluated using a rotarod test with the Rotamex system (Columbus Instruments®, OH, US). Latency to fall off of the rotarod was measured monthly to index changes in motor coordination. A grip strength test was performed using the AMETEK DFE Digital Force Gauge (AMETEK®, PA, US) to evaluate muscle performance. Grip strength performance was measured monthly and reported as a percentage of a baseline measurement taken before the treatment period. All data were analyzed using a two-way ANOVA followed by Šidák’s post-hoc corrections for multiple comparisons and were reported. Results At 20 months, mice treated with D-2PMPA exhibited significantly longer latencies to fall compared to vehicle-treated controls (105.8 ± 3.4 s versus 84.7 ± 4.4 s, p < 0.01) (FIG 12A). When the mice were analyzed by sex, both sexes showed improvement. Specifically, female mice showed latencies of 112.2±5.3 s versus 89.8±7.6 s (p < 0.05), and male mice exhibited latencies of 100.3±4.1 s versus 79.1±3.9 s (p < 0.01). After 5 months of D-2PMPA treatment, aged mice maintained 97.5 ± 1.4% of their maximum initial force, compared to 89.9 ± 1.2% in the vehicle group (p < 0.001) (FIG 12B). Analysis by sex revealed improvements in both groups: female mice preserved 99.1 ± 1.9% versus 90.5 ± 1.8% (p < 0.01), and male mice preserved 96.1 ± 2.1% versus 89.3 ± 1.7% (p < 0.05). Example 5: D-2PMPA treatment preserved Muscle Volume and Force in aged mice Methods Calf muscle volume was monitored using a Bruker 9.4T MRI from 15 to 20 months of age. To determine whether D-2PMPA treatment protects against calf muscle volume loss in aged mice, an MRI setup was used to measure calf muscle volume, and a reconstructed 3D MRI image of the left and right calf muscles was used for volume quantification. (n=18–20 Data were presented as mean ± SEM and analyzed with unpaired t-test. 45718416.1 The gastrocnemius muscle isometric force was measured in vivo using the 3-in-1 whole animal system for mice (1300A, Aurora Scientific Corp). An in vivo setup was used to measure isometric torque in the gastrocnemius muscle as traces of isometric contraction in the gastrocnemius muscle at stimulation frequencies ranging from 10 Hz to 150 Hz. Data were presented as mean ± SEM and analyzed with unpaired t-test. Results In the vehicle-treated group, total calf muscle volume declined by 9.6% between 15 and 20 months, consistent with age-related muscle atrophy. In contrast, D-2-PMPA treatment significantly preserved muscle volume, with treated mice retaining 95.0±0.8% of baseline muscle mass compared to 90.4±0.7% in controls (p < 0.001). When the mice were analyzed by sex, both sexes showed improvement. In females, the preservation percentage was 94.8±4.2% versus 90.7±3.4%, p < 0.05, and in males, 95.2±1.9% versus 90.1±2.5%, p < 0.05. The percentage of baseline calf muscle volume maintained after the 5-month treatment period in male mice (n=9–10) (FIG 13A), female mice (n=9–10) (FIG 13B), and combined male and female cohorts (FIG 13C). respectively. These findings indicate that D-2-PMPA mitigates age- related muscle waste. After 5 months of D-2PMPA treatment, the isometric force of the gastrocnemius muscle was significantly preserved relative to controls (244.7± 12.9 mN versus 187.2 ± 11.3 mN, p<0.01). When the mice were analyzed by sex, both sexes showed improvement. Isometric force measured at an 80-Hz stimulation frequency, shown separately for male (n=8–9) (FIG 13D), female (n=7) (FIG 13E) , and combined male and female cohorts (n=16) (FIG 13F). In females, the force was 230.2 ± 13.3 mN versus 184.0 ± 9.0 mN, p < 0.05, and in males, 257.3 ± 21.0 mN versus 189.7 ± 19.3 mN, p < 0.05). These data demonstrated that D-2PMPA treatment enhances gastrocnemius muscle contractility in aged mice. Example 6: D-2PMPA chronic treatment preserved neuromuscular conduction Methods Compound Muscle Action Potentials (CMAPs) were recorded and analyzed using Neuro- MEP software (Neurosoft) to assess neuromuscular conduction. Amplitude of compound muscle action potentials (CMAP) was recorded following stimulation at the ankle and hip. Latency between stimulus onset and the initiation of the CMAP trace in the ankle and hip was analyzed by treatment condition (n=x). Data were presented as mean ± SEM and analyzed using two-way ANOVA followed by Šidák’s post hoc correction for multiple comparisons. Results Chronic treatment with D-2PMPA significantly delayed the age-related decline in nerve signal conductivity, as demonstrated by an improved CMAP latency (1.28 ± 0.02 ms versus 1.38 ± 45718416.1 0.04 ms; p < 0.05) and enhanced amplitude (15.2 ± 0.5 mV versus 12.7 ± 0.6 mV; p < 0.01). When the mice were analyzed by sex, female mice showed a significant improvement in amplitude (15.5 ± 3.5 versus 12.0 ± 3.0 mV, p < 0.01) while male didn’t (14.9 ± 1.9 mV versus 13.5 ± 3.9 mV, p=0.21). Amplitude of compound muscle action potentials (CMAP) recorded following stimulation at the ankle (FIG.14A) and hip (FIG.14B), and latency between stimulus onset and the initiation of the CMAP trace in the ankle (FIG.14C) and hip (FIG.14D) were analyzed by treatment condition (n=x). These results indicate that D-2PMPA treatment effectively maintains neuromuscular function by preserving both nerve conduction and NMJ integrity. 45718416.1

Claims

We claim: 1. A dosage composition comprising a hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor in an effective amount for reducing, delaying, inhibiting or reversing one or more symptoms or markers associated with muscle loss or motor neuron function.
2. The dosage composition of claim 1, wherein the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to reduce glutamate and/or increase NAAG within and/or around muscle macrophages and neuromuscular junctions (NMJ) in the subject.
3. The dosage composition of claim 1 or 2, wherein the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to reduce the amount of one or more biomarkers of sarcopenia in the subject, such as markers selected from the group consisting of follistatin (FST), adiponectin, leptin, decrease interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α).
4. The dosage composition of any one of claims 1-3, wherein the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is effective to increase myostatin (MSTN), decrease follistatin (FST), increase irisin, increase brain-derived neurotrophic factor (BDNF), decrease adiponectin, decrease leptin, increase serum creatinine to serum cystatin C ratio, increase insulin-like growth factor-1 (IGF-1); increase dehydroepiandrosterone sulphate (DHEAS), decrease C-reactive protein (CRP); decrease interleukin-6 (IL-6), and/or decrease tumor necrosis factor α (TNF-α) in the subject.
5. A method of reducing, preventing, or delaying neuromuscular junction (NMJ) denervation in a subject, comprising administering to a subject a pharmaceutically acceptable composition comprising a hydroxylated dendrimer conjugated to a glutamate-carboxypeptidase-II (GCPII) inhibitor in an effective amount for reducing, delaying, inhibiting or reversing glutamate and/or increasing NAAG within and/or around muscle macrophages and neuromuscular junctions (NMJ) in the subject.
6. The method of claim 5, wherein the amount of hydroxylated dendrimer conjugated to a GCPII inhibitor is administered in an amount effective for binding of the GCPII inhibitor to one or more receptors on the surface of or inside target cells.
7. The method of claim 6, wherein the target cells are selected from the group consisting of neuronal cells, glial cells, Schwann cells, and activated macrophages.
8. The dosage composition of any one of claims 1-4 or method of any one of claims 5- 7, wherein the dendrimer comprises a generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, or generation 8 dendrimer. 45718416.1
9. The dosage composition of claim 8, wherein the dendrimer comprises a glucose dendrimer.
10. The dosage composition of claim 8, wherein the dendrimer comprises a hydroxylated poly(amidoamine) (OH-PAMAM) dendrimer, optionally wherein the OH-PAMAM dendrimer comprises greater than 40% or 50% free hydroxyl surface groups.
11. The dosage composition of claim 10, wherein the dendrimer comprises a generation 4 PAMAM dendrimer.
12. The dosage composition of claim 8 wherein the dendrimer comprises one or more surface-bound monosaccharides selected from the group consisting of glucose, galactose, glucosamine, galactose, mannose, and fructose, preferably ten, or more than ten surface monosaccharide moieties.
13. The dosage composition of claim 8, wherein the dendrimer is linked to the GCPII inhibitor via a spacer, optionally wherein the spacer comprises a cleavable linkage to the dendrimer such as a cleavable linkage selected from the group consisting of ester, disulfide, phosphodiester, tri-glycyl peptide, and hydrazine linkages, or a non-cleavable linkage such as a amide, ether, or amino alkyl linkage.
14. The dosage composition of claim 13, wherein the spacer linking the dendrimer and the GCPII inhibitor comprises a hydrocarbon such as an alkylene, a diethylene glycol moiety, and/or oligoethylene glycol chain or a triazole moiety.
15. The dosage composition of claim 8 wherein the GCPII inhibitor is selected from the group consisting of 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA), ZJ-43, ZJ-11, ZJ-17, ZJ- 38, VA-033, quisqualic acid, 2-[[hydroxy[2,3,4,5,6pentafluorophenyl)methyl] phosphinyl]methyl] pentanedoic acid (GPI-5232), 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), 3-(2-carboxy-5- mercaptopentyl)benzoic acid, 3-(1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4- mercaptobutyl)thio]benzoic acid, N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, phenylalkylphosphonamidates, NAAG peptide analogs, and glutamate derivatives.
16. The dosage composition of claim 15, wherein the GCPII inhibitor comprises 2- (Phosphonomethyl)-pentanedioic Acid (2-PMPA).
17. A composition comprising a hydroxylated dendrimer conjugated to a glutamate- carboxypeptidase-II (GCPII) inhibitor, wherein the dendrimer comprises a generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, or generation 8 dendrimer. 45718416.1
18. The composition of claim 17, wherein the dendrimer comprises a hydroxylated poly(amidoamine) (PAMAM) dendrimer, preferably having greater than 40% or 50% free hydroxyl surface group, preferably wherein the dendrimer comprises a generation 4 PAMAM dendrimer.
19. The composition of claim 17, wherein the dendrimer comprises a glucose dendrimer, preferably a generation 1, generation 2, generation 3 or generation 4 glucose dendrimer.
20. The composition of any one of claims 17-19, wherein the GCPII inhibitor is selected from the group consisting of 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA), ZJ-43, ZJ-11, ZJ- 17, ZJ-38, VA-033, quisqualic acid, 2- [[hydroxy[2,3,4,5,6pentafluorophenyl)methyl]phosphinyl]methyl] pentanedoic acid (GPI-5232), 2- (3-mercaptopropyl)pentanedioic acid (2-MPPA), 3-(2-carboxy-5-mercaptopentyl)benzoic acid, 3- (1-carboxy-4-mercaptobutoxy) benzoic acid, 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid, N- substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid, phenylalkylphosphonamidates, NAAG peptide analogs, and glutamate derivatives thereof.
21. The composition of claim 18 comprising a generation 4 hydroxylated PAMAM dendrimer conjugated with 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA), wherein the 2-PMPA is conjugated to the dendrimer via a cleavable ester linker. 45718416.1
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