WO2011053618A2 - Hydroxyl-terminated dendrimers - Google Patents

Hydroxyl-terminated dendrimers Download PDF

Info

Publication number
WO2011053618A2
WO2011053618A2 PCT/US2010/054202 US2010054202W WO2011053618A2 WO 2011053618 A2 WO2011053618 A2 WO 2011053618A2 US 2010054202 W US2010054202 W US 2010054202W WO 2011053618 A2 WO2011053618 A2 WO 2011053618A2
Authority
WO
WIPO (PCT)
Prior art keywords
dendrimer
agent
agents
embodiments
acid
Prior art date
Application number
PCT/US2010/054202
Other languages
French (fr)
Other versions
WO2011053618A3 (en
Inventor
James R. Baker, Jr.
Yuehua Zhang
Original Assignee
The Regents Of The University Of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US25675909P priority Critical
Priority to US61/256,759 priority
Application filed by The Regents Of The University Of Michigan filed Critical The Regents Of The University Of Michigan
Publication of WO2011053618A2 publication Critical patent/WO2011053618A2/en
Publication of WO2011053618A3 publication Critical patent/WO2011053618A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002Dendritic macromolecules
    • C08G83/003Dendrimers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/56Medicinal 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
    • A61K47/59Medicinal 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
    • A61K47/595Polyamides, e.g. nylon
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002Dendritic macromolecules
    • C08G83/003Dendrimers
    • C08G83/004After treatment of dendrimers

Abstract

The present invention relates to dendrimer synthesis, dendrimer compositions, and related methods of use. Specifically, the present invention relates to hydroxyl-terminated PAMAM dendrimers bearing, e.g., multiple terminal hydroxyl groups and/or terminal oligo (ethylene glycol) groups. In some embodiments, the hydroxyl-terminated PAMAM dendrimers are further conjugated with functional ligands (e.g., therapeutic agents, pro-drugs, targeting agents, trigger agents, imaging agents).

Description

HYDROXYL- TERMINATED DENDRIMERS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to pending U.S. Provisional Patent Application No. 61/256,759, field October 30, 2009, the contents of which are incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract number R01

CA119409 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to dendrimer synthesis, dendrimer compositions, and related methods of use. Specifically, the present invention relates to hydroxyl-terminated PAMAM dendrimers bearing, e.g., multiple terminal hydroxyl groups and/or terminal oligo (ethylene glycol) groups. In some embodiments, the hydroxyl-terminated PAMAM dendrimers are further conjugated with functional ligands (e.g., therapeutic agents, pro-drugs, targeting agents, trigger agents, imaging agents).

BACKGROUND OF THE INVENTION

Cancer remains the number two cause of mortality in the United States, resulting in over 500,000 deaths per year. Despite advances in detection and treatment, cancer mortality remains high. New compositions and methods for the imaging and treatment (e.g., therapeutic) of cancer may help to reduce the rate of mortality associated with cancer.

Severe, chronic pain is observed a variety of subjects. For example, there exist large numbers of individuals with severe pain associated with arthritis, autoimmune disease, injury, cancer, and a host of other conditions.

Additionally, inflammatory diseases such as rheumatoid arthritis, chronic

inflammatory bowel disease, pelvic inflammatory disease, and autoimmune diseases involving inflammation contribute greatly to increased mortality, morbidity, and disability. Rheumatoid arthritis, for example, affects 1% of the human population worldwide. In addition to causing considerable pain and disability, rheumatoid arthritis is associated with a 5 to 10-year reduction in lifespan on average.

There exists a need for compositions, methods and systems for delivering agents (e.g. diagnostic and/or therapeutic (e.g., cancer therapeutics, pain relief agents, inflammatory disease agents) to subjects that provide effective therapy (e.g., disease treatment, symptom relief, etc.) with reduced or eliminated side effects, even when administered in high doses. Functionalized dendrimers, such as PAMAM dendrimers conjugated to functional ligands relevant to cancer therapy and/or pain alleviation, have been developed for such purposes. However, multi-step conjugation strategies used to attach different functional groups to the surfaces of nanoparticles (e.g., dendrimers, PAMAM dendrimer branches) introduce higher polydispersity and require multiple processing steps, thereby complicating synthesis and increasing cost of manufacture. In addition, increased polydispersity of functionalized dendrimer products can negatively affect properties such as therapeutic potency,

pharmacokinetics, or effectiveness for multivalent targeting.

Improved methods of synthesis of dendrimers resulting in decreased polydispersity are needed. In particular, compositions and methods that facilitate one-step chemistry (e.g., "one-pot" synthesis reactions) for use in synthesis of functionalized dendrimers are needed.

SUMMARY OF THE INVENTION

The present invention relates to dendrimer synthesis and methods of use.

Specifically, the present invention relates to hydroxyl-terminated PAMAM dendrimers bearing, e.g., multiple hydroxyl groups or an oligo (ethylene glycol) group, and which are compatible with further conjugation with functional ligands (e.g., therapeutic agents, prodrugs, trigger agents, targeting agents, imaging agents).

Experiments conducted during the course of developing some embodiments of the present invention determined that PAMAM dendrimers bearing multiple hydroxyl groups

(HO-PAMAM dendrimers) at dendrimer terminal branches provide significant improvements over existing PAMAM dendrimers. For example, benefits of hydroxyl terminated dendrimers as compared to non-hydroxyl-terminated dendrimers include but are not limited to simpler methods of synthesis, lower toxicity, lower cost of manufacture, lack of need to cap terminal groups before or after conjugation with functional ligands, compatibility with one-step (e.g., "one-pot") synthesis reactions for further conjugation with functional ligands, lack of formation of undesired cross-linked by-products during synthesis of HO-PAMAM dendrimers, improved water solubility, enhanced reproducibility of synthesis methods, and ease of large-scale synthesis. It was determined that such hydroxyl terminated PAMAM dendrimers could be synthesized by, e.g., 1) reaction of ester-terminated dendrimer (e.g., ester-terminated

PAMAM dendrimer of N and half generation (e.g., G.n.5 where N is an integer)) and an amino alcohol to result in the formation of an amide bond; and/or 2) reaction of amino- terminated dendrimer (e.g., amino-terminated dendrimer of generation N (where N is an integer)) with a saccharide acid or saccharide lactone to result in the formation of an amide bond. Experiments conducted during the development of some embodiments of the present invention further demonstrated that dendrimers of lower generation (e.g., generation 2, generation 3, generation 4) and half-generation dendrimers (e.g., generation 2.5, generation 3.5, generation 4.5) could be used for synthesis of HO-PAMAM dendrimers.

In addition, such hydroxyl terminated dendrimers may be further functionalized through conjugation with one or more functional ligands (e.g., a therapeutic agent, a prodrug, a trigger agent, a targeting agent, an imaging agent). Conjugation with one or more functional ligands is not limited to a particular manner of conjugation. In some embodiments, conjugation of hydroxyl terminated dendrimers (e.g., HO-PAMAM dendrimers) with one or more functional ligands occurs via a one-step (e.g, "one pot") reaction (e.g., as shown in Example 1). In some embodiments, such one-step conjugation is facilitated, for example, by ester coupling agents (e.g., 2-chloro-l-methylpyridinium iodide and 4-(dimethylamino) pyridine) (see, e.g., U.S. Patent App. No. 61/226,993, herein incorporated by reference in its entirety).

The present invention is not limited to utilizing a particular type or form of dendrimer. Indeed, examples of dendrimers finding use in the present invention include, but are not limited to, a polyamideamine (PAMAM) dendrimer, a Baker-Huang PAMAM dendrimer (see, e.g., U.S. Provisional Patent Application No. 61/251,244, herein incorporated by reference in its entirety), a polypropylamine (POP AM) dendrimer, and a PAMAM-POPAM dendrimer. The type of dendrimer used is not limited by the generation number of the dendrimer. Dendrimer molecules may be generation 0, generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, or higher than generation 7. In some embodiments, half-generation dendrimers may be used. In certain embodiments, a generation 5 amine-terminated PAMAM dendrimer is used. In certain embodiments, a generation 5 alkyne-terminated PAMAM dendrimer is used. In some embodiments, the dendrimer is at least partially acetylated.

Dendrimers are not limited by their method of synthesis. The dendrimer may be synthesized by divergent synthesis methods or convergent synthesis methods. In certain embodiments of the present invention, dendrimer molecules may be modified. Modifications may include but are not limited to the addition of amine-blocking groups (e.g., acetyl groups), ligands, functional groups, conjugates, and/or linkers not originally present on the dendrimer. Modification may be partial or complete. In some embodiments, all of the termini of the dendrimer molecules are modified. In some embodiments, not all of the dendrimer molecules are modified. In preferred embodiments, methods and systems of the present invention permit identification and isolation of subpopulations of dendrimers with known numbers of ligand attachments (e.g., conjugations) per dendrimer molecule, thereby yielding samples or subpopulations of dendrimer compositions with high structural uniformity.

The present invention is not limited to particular ligand types (e.g., functional groups)

(e.g., for conjugation with dendrimers). Examples of ligand types (e.g., functional groups) include but are not limited to therapeutic agents, targeting agents, trigger agents, and imaging agents. In some embodiments, the ligand is an alkyne ligand that includes an alkyne. In some embodiments, the ligand is an azide ligand that includes N3. In some embodiments, the ligand includes an aromatic group. Methods, systems, and compositions of the present invention are not limited by the number of different ligand types used. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of ligands attached to a dendrimer molecule.

In some embodiments, conjugation between a ligand and a functional group or between functional groups is accomplished through use of a 1,3-dipolar cycloaddition reaction ("click chemistry"). 'Click chemistry' involves, for example, the coupling of two different moieties (e.g., a therapeutic agent and a functional group) (e.g., a first functional group and a second functional group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or equivalent thereof) on the surface of the first moeity and an azide moiety (or equivalent thereof) (or any active end group such as, for example, a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.) on the second moiety. 'Click' chemistry is an attractive coupling method because, for example, it can be performed with a wide variety of solvent conditions including aqueous environments. For example, the stable triazole ring that results from coupling the alkyne with the azide is frequently achieved at quantitative yields and is considered to be biologically inert (see, e.g., Rostovtsev, V. V.; et al, Angewandte Chemie-International Edition 2002, 41, (14), 2596; Wu, P.; et al, Angewandte Chemie-International Edition 2004, 43, (30), 3928-3932; each herein incorporated by reference in their entireties). The present invention is not limited to particular functional groups (e.g., for conjugation with dendrimers). Examples of functional groups include but are not limited to therapeutic agents, targeting agents, trigger agents, and imaging agents.

In some embodiments, the functional group(s) is attached with the dendrimer via a linker. The present invention is not limited to a particular type or kind of linker. In some embodiments, the linker comprises a spacer comprising between 1 and 8 straight or branched carbon chains. In some embodiments, the straight or branched carbon chains are

unsubstituted. In some embodiments, the straight or branched carbon chains are substituted with alky Is.

Examples of therapeutic agents include, but are not limited to, a chemotherapeutic agent, an anti-oncogenic agent, an anti-angiogenic agent, a tumor suppressor agent, an antimicrobial agent, an expression construct comprising a nucleic acid encoding a therapeutic protein, a pain relief agent, a pain relief agent antagonist, an agent designed to treat an inflammatory disorder, an agent designed to treat an autoimmune disorder, an agent designed to treat inflammatory bowel disease, and an agent designed to treat inflammatory pelvic disease. In some embodiments, the agent designed to treat an inflammatory disorder includes, but is not limited to, an antirheumatic drug, a biologicals agent, a nonsteroidal antiinflammatory drug, an analgesic, an immunomodulator, a glucocorticoid, a TNF-a inhibitor, an IL-1 inhibitor, and a metalloprotease inhibitor. In some embodiments, the antirheumatic drug includes, but is not limited to, leflunomide, methotrexate, sulfasalazine, and

hydroxychloroquine. Examples of biologicals agents include, but are not limited to, rituximab, fmfliximab, etanercept, adalimumab, and golimumab. In some embodiments, the nonsteroidal anti-inflammatory drug includes, but is not limited to, ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, and diclofenac. In some embodiments, the analgesic includes, but is not limited to, acetaminophen, and tramadol. In some embodiments, the immunomodulator includes but is not limited to anakinra, and abatacept. In some

embodiments, the glucocorticoid includes, but is not limited to, prednisone, and

methylprednisone. In some embodiments, the TNF-a inhibitor includes but is not limited to adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some

embodiments, the autoimmune disorder and/or inflammatory disorder includes, but is not limited to, arthritis, psoriasis, lupus erythematosus, Crohn's disease, and sarcoidosis. In some embodiments, examples of arthritis include, but are not limited to, osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudo-gout, juvenile idiopathic arthritis, psoriatic arthritis, Still's disease, and ankylosing spondylitis. Ligands suitable for use in certain method embodiments of the present invention are not limited to a particular type or kind of targeting agent. In some embodiments, the targeting agent is configured to target the composition to cancer cells. In some embodiments, the targeting agent comprises folic acid. In some embodiments, the targeting agent binds a receptor selected from the group consisting of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR. In some embodiments, the targeting agent comprises an antibody that binds to a polypeptide selected from the group consisting of p53, Mucl, a mutated version of p53 that is present in breast cancer, HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, and MSA breast carcinoma glycoprotein. In some embodiments, the targeting agent comprises an antibody selected from the group consisting of human carcinoma antigen, TP1 and TP3 antigens from osteocarcinoma cells, Thomsen- Friedenreich (TF) antigen from adenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigen from cystadenocarcinoma, DF3 antigen from human breast carcinoma, and p97 antigen of human melanoma, carcinoma or orosomucoid-related antigen. In some embodiments, the targeting agent is configured to permit the composition to cross the blood brain barrier. In some embodiments, the targeting agent is transferrin. In some embodiments, the targeting agent is configured to permit the composition to bind with a neuron within the central nervous system. In some embodiments, the targeting agent is a synthetic tetanus toxin fragment. In some embodiments, the synthetic tetanus toxin fragment comprises an amino acid peptide fragment. In some embodiments, the amino acid peptide fragment is HLNILSTLWKYR.

In some embodiments, the ligand comprises a trigger agent. The present invention is not limited to particular type or kind of trigger agent. In some embodiments, the trigger agent is configured to have a function such as, for example, a) a delayed release of a functional group from the dendrimer, b) a constitutive release of the therapeutic agent from the dendrimer, c) a release of a functional group from the dendrimer under conditions of acidosis, d) a release of a functional group from a dendrimer under conditions of hypoxia, and e) a release of the therapeutic agent from a dendrimer in the presence of a brain enzyme.

Examples of trigger agents include, but are not limited to, an ester bond, an amide bond, an ether bond, an indoquinone, a nitroheterocyle, and a nitroimidazole.

Ligands suitable for use in certain method embodiments of the present invention are not limited to a particular type or kind of imaging agent. In some embodiments, the imaging agent comprises fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and/or cis- parinaric acid. In some embodiments, the imaging agent comprises 3-azido-coumarine. In certain embodiments, the present invention provides a composition comprising a dendrimer, wherein one or more terminal branches of said dendrimer comprises two or more OH groups. In some embodiments, the dendrimer is a PAMAM dendrimer. In some embodiments, the PAMAM dendrimer has a generation between 0 and 3. In some embodiments, the one or more terminal branches of the dendrimer compring two or more OH groups is represented by the following formula:

Figure imgf000008_0001
wherein X is a moiety such as an amino alcohol comprising two or more hydroxyl groups, saccharide lactone comprising three or more hydroxyl groups, and a saccharide acid comprising three or more hydroxyl groups. In some embodiments, the amino alcohol is represented by the following formula:
Figure imgf000008_0002

wherein m is an integer between 2 and 6, wherein R is a chemical group comprising two or more carbon molecules (e.g., 2 carbon molecules, 5 carbon molecules, 10 carbon molecules, 15 carbon molecules, 25 carbon molecules, 30 carbon molecules, 50 carbon molecules). In some embodiments, the chemical group comprising two or more carbon molecules is an alkyl group comprising two or more carbon molecules. In some embodiments, the amino alcohol is a type such as glucamine or isomers thereof, 1 -amino- 1-deoxy-D-manitol or isomers thereof, glucosamine or isomers thereof, l-amino-2,5-anhydro-l-deoxy-D-mannitol or isomers thereof, tri(hydroxyl)aminomethane or isomers thereof, 3-amino-l,2-propanediol or isomers thereof, and amino-oligo(ethylene glycol). In some embodiments, the saccharide lactone comprising three or more hydroxyl groups and the saccharide acid comprising three or more hydroxyl groups is represented by the following formula:

Figure imgf000008_0003

wherein m is an integer equal to or higher than 3, and wherein R is a chemical group comprising two or more carbon molecules (e.g., 2 carbon molecules, 5 carbon molecules, 10 carbon molecules, 15 carbon molecules, 25 carbon molecules, 30 carbon molecules, 50 carbon molecules). In some embodiments, m is between 3 and 10. In some embodiments, the saccharide lactone is a type such as D-(+)-gluconic acid δ-lactone, D-gulonic acid γ- lactone, L-gulonic acid γ-lactone, D-(+)-glucuronic acid γ-lactone, α,β-glucooctanoic γ- lactone, and D-glucoheptono-l,4-lactone. In some embodiments, the saccharide acid is a type such as D-glucuronic acid, D-gluconic acid, and lactobionic acid. In some

embodiments, the dendrimer is conjugated with one or more functional ligands such as a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent. In some embodiments, conjugation of the one or more functional ligands with the dendrimer is represented by the following formula: X (Functional Ligand)y

, wherein y is at least 1. In some embodiments, y is between 1 and 10. In some embodiments, the therapeutic agent is a type such as chemotherapeutic agents, anti-oncogenic agents, anti-angiogenic agents, tumor suppressor agents, anti- microbial agents, expression constructs comprising a nucleic acid encoding a therapeutic protein, pain relief agents, pain relief agent antagonists, agents designed to treat arthritis, agents designed to treat inflammatory bowel disease, agents designed to treat an autoimmune disease, and agents designed to treat inflammatory pelvic disease. In some embodiments, the chemotherapeutic agent is methotrexate. In some embodiments, the targeting agent is a type such as an agent binding a receptor selected from the group consisting of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR; an antibody that binds to a polypeptide selected from the group consisting of p53, Mucl, a mutated version of p53 that is present in breast cancer, HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, and MSA breast carcinoma glycoprotein; an antibody selected from the group consisting of human carcinoma antigen, TP1 and TP3 antigens from osteocarcinoma cells, Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigen from

cystadenocarcinoma, DF3 antigen from human breast carcinoma, and p97 antigen of human melanoma, carcinoma or orosomucoid-related antigen; transferrin; and a synthetic tetanus toxin fragment. In some embodiments, the targeting agent is folic acid. In some

embodiments, the trigger agent is configured for a function such as permitting a delayed release of a functional group from the dendrimer, permitting a constitutive release of the therapeutic agent from the dendrimer, permitting a release of a functional group from the dendrimer under conditions of acidosis, permitting a release of a functional group from a dendrimer under conditions of hypoxia, and permitting a release of the therapeutic agent from a dendrimer in the presence of a brain enzyme. In some embodiments, the imaging agent is an agent such as fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and cis- parinaric acid. In some embodiments, the dendrimer further comprises nanomaterials such asgold nanoparticles, iron oxide nanoparticles, polymers, silica, albumin, quantum dots, and carbon nanotubes.

In certain embodiments, the present invention provides a method of synthesizing a dendrimer comprising at least one terminal branch comprising two or more hydroxyl groups, comprising: a) providing a dendrimer comprising at least one ester-terminated terminal branch; b) providing an amino alcohol comprising two or more hydroxyl groups; c) reacting the ester-terminated terminal branch of the dendrimer with the amino alcohol resulting in the formation of an amide bond, wherein the resulting dendrimer comprises at least one terminal branch comprising two or more hydroxyl groups. In some embodiments, the dendrimer is a PAMAM dendrimer. In some embodiments, the amino alcohol is a type such as glucamine or isomers thereof, 1 -amino- 1-deoxy-D-manitol or isomers thereof, glucosamine or isomers thereof, l-amino-2,5-anhydro-l-deoxy-D-mannitol or isomers thereof,

tri(hydroxyl)aminomethane or isomers thereof, 3-amino-l,2-propanediol or isomers thereof, and amino-oligo(ethylene glycol). In some embodiments, the ester-terminated dendrimer is of generation N and one half, wherein N is an integer. In some embodiments, the value of N does not exceed 5.

In certain embodiments, the present invention provides a method of synthesizing a dendrimer comprising at least one terminal branch comprising two or more hydroxyl groups, comprising: a) providing an NH2-terminated dendrimer; b) providing a reactant such as a saccharide lactone and a saccharide acid; and c) reacting said NH2-terminated dendrimer with the saccharide lactone or the saccharide acid resulting in the formation of an amide bond. In some embodiments, the dendrimer is a PAMAM dendrimer. In some embodiments, the saccharide lactone is a type such as D-(+)-gluconic acid δ-lactone, D-gulonic acid γ-lactone, L-gulonic acid γ-lactone, D-(+)-glucuronic acid γ-lactone, α,β-glucooctanoic γ-lactone, and D-glucoheptono-l,4-lactone. In some embodiment, the saccharide acid is a type such as D- glucuronic acid, D-gluconic acid, and lactobionic acid.

In some embodiments, the methods of the present invention further comprise conjugation with a functional ligand such as a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent. In some embodiments, the therapeutic agent is an agent such as chemotherapeutic agents, anti-oncogenic agents, anti-angiogenic agents, tumor suppressor agents, anti-microbial agents, expression constructs comprising a nucleic acid encoding a therapeutic protein, pain relief agents, pain relief agent antagonists, agents designed to treat arthritis, agents designed to treat inflammatory bowel disease, agents designed to treat an autoimmune disease, and agents designed to treat inflammatory pelvic disease. In some embodiments, the chemotherapeutic agent is methotrexate. In some embodiments, the targeting agent is a type such as an agent binding a receptor selected from the group consisting of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR; an antibody that binds to a polypeptide selected from the group consisting of p53, Mucl, a mutated version of p53 that is present in breast cancer, HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, and MSA breast carcinoma glycoprotein; an antibody selected from the group consisting of human carcinoma antigen, TPl and TP3 antigens from osteocarcinoma cells, Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigen from cystadenocarcinoma, DF3 antigen from human breast carcinoma, and p97 antigen of human melanoma, carcinoma or orosomucoid- related antigen; transferrin; and a synthetic tetanus toxin fragment. In some embodiments, the targeting agent is folic acid. In some embodiments, the trigger agent is configured for a function such as permitting a delayed release of a functional group from the dendrimer, permitting a constitutive release of the therapeutic agent from the dendrimer, permitting a release of a functional group from the dendrimer under conditions of acidosis, permitting a release of a functional group from a dendrimer under conditions of hypoxia, and permitting a release of the therapeutic agent from a dendrimer in the presence of a brain enzyme. In some embodiments, the imaging agent is an agent such as fluorescein isothiocyanate (FITC), 6- TAMARA, acridine orange, and cis-parinaric acid.

In certain embodiments, the present invention provides methods for treating a disorder selected from the group consisting of any type of cancer or cancer-related disorder (e.g., tumor, a neoplasm, a lymphoma, or a leukemia), a neoplastic disease, osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudo-gout, juvenile idiopathic arthritis, psoriatic arthritis, Still's disease, and ankylosing spondylitis, comprising administering to a subject suffering from the disorder a dendrimer generated with the methods of the present invention. In some embodiments, the dendrimer is co-administered with an additional agent(s) so as to enhance such a treatment.

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein. DESCRIPTION OF THE DRAWINGS

Figure 1 shows synthesis of hydroxyl-terminated PAMAM dendrimer via reaction of ester-terminated PAMAM dendrimer (generation N.5) and amino-alcohol.

Figure 2 shows synthesis of glucamine-terminated G.3 PAMAM dendrimer and conjugate of folic acid, methotrexate, and glucamine-terminated G.3 PAMAM dendrimer.

Figure 3 shows synthesis of saccharide-terminated PAMAM dendrimer via the reaction of amino-terminated PAMAM dendrimer and saccharide acid or saccharide lactone.

Figure 4 shows synthesis of conjugate of methotrexate and saccharide-terminated G.3 PAMAM dendrimer.

Figure 5 shows synthesis of conjugate of folic acid, methotrexate, and saccharide- terminated G.3 PAMAM dendrimer.

Figure 6 shows 1H NMR spectra of G.3 PAMAM dendrimer (G.3-NH2), saccharide- terminated G.3 PAMAM dendrimer (G.3-(OH)6), conjugate of methotrexate and saccharide- terminated G.3 PAMAM dendrimer (G.3-MTX5.2), and conjugate of folic acid,

methotrexate, and saccharide-terminated G.3 PAMAM dendrimer (G.3-FA1.7-MTX4.3).

Figure 7 shows MALDI-TOF spectra of G.3 PAMAM dendrimer (G.3-NH2), saccharide-terminated G.3 PAMAM dendrimer (G.3-(OH)e), conjugate of methotrexate and saccharide-terminated G.3 PAMAM dendrimer (G.3-MTXs.2), and conjugate of folic acid, methotrexate, and saccharide-terminated G.3 PAMAM dendrimer (G.3-FA1.7-MTX4.3).

Figure 8 shows HPLC chromatograms of G.3-MTX5.2 and G.3-FA1.7-MTX4.3. 1H

NMR was used to confirm the surface modification of the PAMAM dendrimer.

Figure 9 shows competition of binding of "G5-FI-FA-MTX" conjugate by the newly synthesized G3-FA. Into KB cells plated in 24 well-plates were added G5-FI-FA-MTX (100 nM) and different concentrations of the G3-FA simultaneously. Samples were incubated for 1 h at 37°C. The cells were rinsed and the bound fluorescence was determined by flow cytometry. Note that IC50 for binding by the G3-FA is -25 nM at 100 nM of the G5-FI-FA- MTX, indicating higher affinity of the G3-FA vs. G5-FI-FA-MTX.

Figure 10 shows treatment of KB cells with 6 μΜ each of free FA, G3-FA or G3- MTX for 20 h to down-regulate the FAR. Cells were rinsed to remove these agents, and the dose-depending binding of "G5-FI-FA-MTX" (1 h incubation) was determined by flow cytometry. Note that free FA and G3-FA completely down-regulated the FAR whereas the low FAR-affinity-binding G3-MTX only partially down-regulated the FAR. These results confirm that the G3-FA and G3-MTX internalize into KB cells through the FAR. Figure 1 1 shows cytotoxicity data for the G3-MTX conjugates. KB cells plated in 96- well plates were incubated with different concentrations of the indicated conjugates for 4 days and the cytotoxicity was determined by XTT assay. The results show that all conjugates were cytotoxic, except the control dendrimer G3-OH. The number given after each ligands show the numbers of the ligands present per dendrimer molecule. The number in the parentheses indicates the lot number of the synthesized conjugates.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "subject suspected of having cancer" refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass) or is being screened for a cancer (e.g., during a routine physical). A subject suspected of having cancer may also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer. However, a "subject suspected of having cancer" encompasses an individual who has received a preliminary diagnosis (e.g., a CT scan showing a mass) but for whom a confirmatory test (e.g., biopsy and/or histology) has not been done or for whom the stage of cancer is not known. The term further includes people who once had cancer (e.g., an individual in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and is sometimes found to not have cancer.

As used herein, the term "subject diagnosed with a cancer" refers to a subject who has been tested and found to have cancerous cells. The cancer may be diagnosed using any suitable method, including but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present invention.

As used herein, the term "initial diagnosis" refers to a test result of initial cancer diagnosis that reveals the presence or absence of cancerous cells (e.g., using a biopsy and histology).

As used herein, the term "identifying the risk of said tumor metastasizing" refers to the relative risk (e.g., the percent chance or a relative score) of a tumor metastasizing. As used herein, the term "identifying the risk of said tumor recurring" refers to the relative risk (e.g. , the percent chance or a relative score) of a tumor recurring in the same organ as the original tumor.

As used herein, the term "subject at risk for cancer" refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental expose, and previous incidents of cancer, preexisting non-cancer diseases, and lifestyle.

As used herein, the term "characterizing cancer in subject" refers to the identification of one or more properties of a cancer sample in a subject, including but not limited to, the presence of benign, pre-cancerous or cancerous tissue and the stage of the cancer.

As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body and where the cancer has spread (e.g., within the same organ or region of the body or to another organ).

As used herein, the term "providing a prognosis" refers to providing information regarding the impact of the presence of cancer on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis).

As used herein, the term "non-human animals" refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

As used herein, the term "drug" is meant to include any molecule, molecular complex or substance administered to an organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical and prophylactic applications. The term "drug" is further meant to include any such molecule, molecular complex or substance that is chemically modified and/or operatively attached to a biologic or biocompatible structure.

As used herein, the term "purified" or "to purify" or "compositional purity" refers to the removal of components (e.g., contaminants) from a sample or the level of components (e.g., contaminants) within a sample. For example, unreacted moieties, degradation products, excess reactants, or byproducts are removed from a sample following a synthesis reaction or preparative method.

The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using screening methods known in the art.

As used herein, the term "nanodevice" or "nanodevices" refer, generally, to compositions comprising dendrimers of the present invention. As such, a nanodevice may refer to a composition comprising a dendrimer of the present invention that may contain one or more ligands, linkers, and/or functional groups (e.g., a therapeutic agent, a targeting agent, a trigger agent, an imaging agent) conjugated to the dendrimer.

As used herein, the term "degradable linkage," when used in reference to a polymer refers to a conjugate that comprises a physiologically cleavable linkage (e.g., a linkage that can be hydrolyzed (e.g., in vivo) or otherwise reversed (e.g., via enzymatic cleavage). Such physiologically cleavable linkages include, but are not limited to, ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal linkages (See, e.g., U.S.

Pat. No. 6,838,076, herein incorporated by reference in its entirety). Similarly, the conjugate may comprise a cleavable linkage present in the linkage between the dendrimer and functional group, or, may comprise a cleavable linkage present in the polymer itself (See, e.g., U.S. Pat. App. Nos. 20050158273 and 20050181449, each of which is herein

incorporated by reference in its entirety).

A "physiologically cleavable" or "hydrolysable" or "degradable" bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydro lyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

As used herein, the term "NAALADase inhibitor" refers to any one of a multitude of inhibitors for the neuropeptidase NAALADase (N-acetylated-alpha linked acidic

dipeptidase). Such inhibitors of NAALADase have been well characterizied. For example, an inhibitor can be selected from the group comprising, but not limited to, those found in U.S. Pat. No. 6,011,021, herein incorporated by reference in its entirety.

A "hydro lyrically stable" linkage or bond refers to a chemical bond (e.g., typically a covalent bond) that is substantially stable in water (i.e., does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time).

Examples of hydro lyrically stable linkages include, but are not limited to, carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like.

As used herein, the term "click chemistry" refers to chemistry tailored to generate substances quickly and reliably by joining small modular units together (see, e.g., Kolb et al. (2001) Angewandte Chemie Intl. Ed. 40:2004-2011; Evans (2007) Australian J. Chem.

60:384-395; Carlmark et al. (2009) Chem. Soc. Rev. 38:352-362; each herein incorporated by reference in its entirety).

As used herein, an "ester coupling agent" refers to a reagent that can facilitate the formation of an ester bond between two reactants. The present invention is not limited to any particular coupling agent or agents. Examples of coupling agents include but are not limited to 2-chloro-l-methylpyridium iodide and 4-(dimethylamino) pyridine, or

dicyclohexylcarbodiimide and 4-(dimethylamino) pyridine or diethyl azodicarboxylate and triphenylphosphine or other carbodiimide coupling agent and 4-(dimethylamino)pyridine.

As used herein, the term "glycidolate" refers to the addition of a 2,3-dihydroxylpropyl group to a reagent using glycidol as a reactant. In some embodiments, the reagent to which the 2,3-dihydroxylpropyl groups are added is a dendrimer. In some embodiments, the dendrimer is a PAMAM dendrimer. Glycidolation may be used generally to add terminal hydroxyl functional groups to a reagent.

As used herein, the term "ligand" refers to any moiety covalently attached (e.g., conjugated) to a dendrimer branch; in preferred embodiments, such conjugation is indirect (e.g., an intervening moiety exists between the dendrimer branch and the ligand) rather than direct (e.g., no intervening moiety exists between the dendrimer branch and the ligand).

Indirect attachment of a ligand to a dendrimer may exist where a scaffold compound intervenes. In preferred embodiments, ligands have functional utility for specific applications, e.g., for therapeutic, targeting, imaging, or drug delivery function(s). The terms "ligand", "conjugate", and "functional group" may be used interchangeably.

As used herein, the term "one-pot synthesis reaction" or equivalents thereof, e.g., "1- pot", "one pot", etc., refers to a chemical synthesis method in which all reactants are present in a single vessel. Reactants may be added simultaneously or sequentially, with no limitation as to the duration of time elapsing between introduction of sequentially added reactants.

As used herein, the term "amino alcohol" or "amino-alcohol" refers to any organic compound containing both an amino and an aliphatic hydroxy 1 functional group (e.g., which may be an aliphatic or branched aliphatic or alicyclic or hetero-alicyclic compound containing an amino group and one or more hydroxyl(s)). The generic structure of an amino alcohol may be expressed as NH2-R-(OH)m wherein m is an integer, and wherein R comprises at least two carbon molecules (e.g., at least 2 carbon molecules, 10 carbon molecules, 25 carbon molecules, 50 carbon molecules).

As used herein, the term "scaffold" refers to a compound to which other moieties are attached (e.g., conjugated). In some embodiments, a scaffold is conjugated to bioactive functional conjugates (e.g., a therapeutic agent, a targeting agent, a trigger agent, an imaging agent). In some embodiments, a scaffold is conjugated to a dendrimer (e.g., a PAMAM dendrimer). In some embodiments, conjugation of a scaffold to a dendrimer and/or a functional conjugate(s) is direct, while in other embodiments conjugation of a scaffold to a dendrimer and/or a functional conjugate(s) is indirect, e.g., an intervening linker is present between the scaffold compound and the dendrimer, and/or the scaffold and the functional conjugate(s).

As used herein, the term "Baker-Huang dendrimer" or "Baker-Huang PAMAM dendrimer" refers to a dendrimer comprised of branching units of structure:

Figure imgf000017_0001

wherein R comprises a carbon-containing functional group (e.g., CF3). In some

embodiments, the branching unit is activated to its HNS ester. In some embodiments, such activation is achieved using TSTU. In some embodiments, EDA is added. In some embodiments, the dendrimer is further treated to replace, e.g., CF3 functional groups with NH2 functional groups; for example, in some embodiments, a CF3 -containing version of the dendrimer is treated with K2CO3 to yield a dendrimer with terminal NH2 groups (for example, as shown in Scheme 2). In some embodiments, terminal groups of a Baker-Huang dendrimer are further derivatized and/or further conjugated with other moieties. For example, one or more functional ligands (e.g., for therapeutic, targeting, imaging, or drug delivery function(s)) may be conjugated to a Baker-Huang dendrimer, either via direct conjugation to terminal branches or indirectly (e.g., through linkers, through other functional groups (e.g., through an OH- functional group)). In some embodiments, the order of iterative repeats from core to surface is amide bonds first, followed by tertiary amines, with ethylene groups intervening between the amide bond and tertiary amines. In preferred embodiments, a Baker-Huang dendrimer is synthesized by convergent synthesis methods.

DETAILED DESCRIPTION OF THE INVENTION

Polyamidoamine (PAMAM) dendrimers have received much attention and shown promise for biomedical applications, especially as drug carriers (Hong et al. (2007)

Chemistry & Biol. 14: 107-115; Kukowska-Latallo et al. (2005) Cance Res. 65:5317-5324; each herein incorporated by reference in their entirety) due to their unique structure, physical and chemical properties, and biological behaviors. PAMAM dendrimers are highly branched and narrowly dispersed synthetic macromolecules with well-defined chemical structures. They can be easily modified and conjugated with multiple functionalities such as targeting molecules, imaging agents, and drugs. They are water soluble, biocompatible, and are rapidly cleared from the blood through the kidneys (e.g., for dendrimers with diameter less than 5 nm) (Peer et al. (2007) Nature Nanotechnol. 2:751-760; herein incorporated by reference in its entirety). However, challenges remain in synthesis of dendrimers and dendrimer-based pro-drugs. For example, classical synthesis methods of dendrimer platforms include many repetitive steps (e.g., PAMAM dendrimer of generation 1 requires four synthetic steps, and each subsequent generation thereafter requires an additional two synthetic steps), complicating synthesis methods. Additional challenges in PAMAM dendrimer synthesis include cross-linking of byproducts, batch-to-batch inconsistencies, and high cost, all of which limit practical application of dendrimers.

The present invention provides compositions and methods to overcome these problems without compromising beneficial features of PAMAM dendrimers. In some embodiments of the present invention, hydroxyl-terminated PAMAM dendrimers (referred to herein as HO-PAMAM) and methods of synthesis thereof are provided wherein each terminal branch of a dendrimer bears, e.g., multiple hydroxyl groups, or an oligo (ethylene glycol) group. In some embodiments, HO-PAMAM dendrimers are synthesized via reaction of ester- terminated PAMAM dendrimer of N and half generation (G.n.5, wherein N is an integer) and amino-alcohol through an amide bond (Figure 1). Alternatively, in some embodiments, the dendrimer is synthesized by reaction of amino-terminated PAMAM dendrimer of generation N (wherein N is an integer) and saccharide acid or saccharide lactone through an amide bond (Figure 3).

The hydroxyl terminated PAMAM dendrimers of the present invention provide significant improvements over existing technologies. For example, the presence of multiple hydroxyl groups or oligo(ethylene glycol) groups on the surface of the dendrimer

significantly improves water solubility of the composition. Additionally, conjugation yield is improved such that lower generation dendrimers may be used for making conjugates

(prodrugs) in comparison to classical synthesis methods, which typically utilize generation 5 PAMAM dendrimers. Lower-generation PAMAM dendrimers offer improved water solubility and smaller size than higher generation PAMAM dendrimers. Thus, some embodiments of the present invention provide simplified synthesis (e.g., requiring fewer steps) of HO-PAMAM dendrimers of lower generation and corresponding conjugates. For example, generation 3 (G.3) glucamine-terminated dendrimer bears 160 surface hydroxyl groups, exceeding the 128 amino groups present on the surface of generation 5 (G.5) PAMAM dendrimer. In some embodiments, methods of the present invention allow synthesis of G.3 glucamine-terminated dendrimer in only eight steps, while synthesis of G.5 PAMAM dendrimer requires 12 steps. Additionally, HO-PAMAM dendrimer does not require end-capping before or after conjugation, whereas the amino groups of classically- synthesized PAMAM dendrimer requires capping in order to reduce amino group-induced toxicity. Furthermore, in some embodiments, the reaction between amino-alcohol (e.g.

glucamine) and ester bond on the dendrimer and the reaction between PAMAM dendrimer and saccharide acid or saccharide lactone does not result in cross-linking reactions, which are problematic occurrences in classical synthesis schemes when an alkylene diamine such as ethylene diamine and 1 ,4-butylene diamine are used for making PAMAM dendrimer.

In certain embodiments, the present invention provides a composition comprising a dendrimer (e.g., a PAMAM dendrimer), wherein one or more terminal branches of the dendrimer comprises two or more OH groups. In some embodiments, the dendrimer has a generation between 0 and 3. In some embodiments, the one or more terminal branches of the dendrimer comprising two or more OH groups is represented by the following formula:

Figure imgf000020_0001

wherein X is a moiety such as an amino alcohol comprising two or more hydroxyl groups, a saccharide lactone comprising three or more hydroxyl groups, and a saccharide acid comprising three or more hydroxyl groups. In some embodiments, the amino alcohol is represented by the following formula:

Figure imgf000020_0002

wherein m is an integer between 2 and 6, wherein R is a chemical group comprising two or more carbon molecules (e.g., 2 carbon molecules, 5 carbon molecules, 10 carbon molecules, 15 carbon molecules, 25 carbon molecules, 30 carbon molecules, 50 carbon molecules). In some embodiments, the chemical group comprising two or more carbon molecules is an alkyl group comprising two or more carbon molecules. In some embodiments, the amino alcohol is a type such as glucamine or isomers thereof, 1 -amino- 1-deoxy-D-manitol or isomers thereof, glucosamine or isomers thereof, l-amino-2,5-anhydro-l-deoxy-D-mannitol or isomers thereof, tri(hydroxyl)aminomethane or isomers thereof, 3-amino-l,2-propanediol or isomers thereof, and amino-oligo(ethylene glycol). In some embodiments, the saccharide lactone comprising three or more hydroxyl groups and the saccharide acid comprising three or more hydroxyl groups is represented by the following formula:

Figure imgf000020_0003

wherein m is an integer equal to or higher than 3, and wherein R is a chemical group comprising two or more carbon molecules (e.g., 2 carbon molecules, 5 carbon molecules, 10 carbon molecules, 15 carbon molecules, 25 carbon molecules, 30 carbon molecules, 50 carbon molecules). In some embodiments, m is between 3 and 10. In some embodiments, the saccharide lactone is a type such as D-(+)-gluconic acid δ-lactone, D-gulonic acid γ- lactone, L-gulonic acid γ-lactone, D-(+)-glucuronic acid γ-lactone, α,β-glucooctanoic γ- lactone, and D-glucoheptono-l,4-lactone. In some embodiments, the saccharide acid is a type such as D-glucuronic acid, D-gluconic acid, and lactobionic acid. In some

embodiments, the dendrimer is conjugated with one or more functional ligands such as a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent. In some embodiments, conjugation of the one or more functional ligands with the dendrimer is represented by the following formula: X (Functional Ligand)y

, wherein y is at least 1.

In some embodiments, HO-PAMAM dendrimers may be further functionalized by conjugation with desired ligands (e.g., therapeutic agents, targeting agents, trigger agents, imaging agents). In some embodiments, HO-PAMAM dendrimer, folic acid (FA, or alternatively other targeting molecules), methotrexate (MTX, or alternatively other therapeutic compounds), and an appropriate imaging molecule may be easily attached to the dendrimer to form a targeted anticancer prodrug in a single step. In some embodiments, such one-step conjugation is facilitated by ester coupling agents (e.g., 2-chloro-l- methylpyridinium iodide and 4-(dimethylamino) pyridine) (see, e.g., U.S. Patent App. No. 61/226,993, herein incorporated by reference in its entirety).

Therefore, HO-PAMAMs, especially low generation (e.g., G.2, G.2.5, G.3) HO- PAMAM dendrimers have many benefits as drug carriers over traditional PAMAM

dendrimer platforms (e.g., amine-terminated G.5 PAMAM dendrimers). Such benefits include but are not limited to reduced synthetic steps and thereby cost of synthesis due to reliance on lower-generation dendrimer platforms; lower toxicity; no requirement for capping of terminal groups before or after conjugation; ability to conjugate functional ligands (e.g., therapeutic agents, targeting agents, imaging agents, trigger agents) to HO-PAMAM dendrimers using "one -pot" synthesis methods (see, e.g., U.S. Patent App. No. 61/226,993, herein incorporated by reference in its entirety); elimination of undesired cross-linked byproducts resulting in a narrower molecular weight distribution; higher water solubility; enhanced reproducibility of synthesis reactions; and improved scaling of synthesis reactions.

In some embodiments, amino-alcohols that find use in methods of the present invention include but are not limited to glucamine or its isomers; 1 -amino- 1-deoxy-D-manitol or its isomers; glucosamine or its isomer; l-amino-2,5-anhydro-l-deoxy-D-mannitol or its isomers; tri(hydroxyl)aminomethane or its isomers; 3-amino-l,2-propanediol or its isomers; and amino-oligo(ethylene glycol). In some embodiments, saccharide acids that find use in methods of the present invention include but are not limited to D-glucuronic acid; D-gluconic acid; and lactobionic acid. In some embodiments, saccharide lactones that find use in methods of the present invention include but are not limited to D-(+)-gluconic acid δ-lactone; D-gulonic acid γ-lactone; L-gulonic acid γ-lactone; D-(+)-glucuronic acid γ-lactone; α,β- glucooctanoic γ-lactone; and D-glucoheptono-l,4-lactone. In some embodiments, PAMAM dendrimers of low generation (e.g., G. l, G.2, G.3, G.0.5, G. l .5, and G.2.5) are used as starting materials to synthesize HO-PAMAM

dendrimers. In some embodiments, HO-PAMAM dendrimers are synthesized by reaction of an ester-terminated PAMAM dendrimer of N and half (G.N.5) generation with an amino- alcohol (e.g., as exemplified in Figure 1). In some embodiments, HO-PAMAM dendrimers are synthesized by reaction of an amino-terminated PAMAM denderimer of N generation with a saccharide acid or saccharide lactone (e.g., as exemplified in Figure 3).

In certain embodiments, the present invention provides a method of synthesizing a dendrimer (e.g., a PAMAM dendrimer) comprising at least one terminal branch comprising two or more hydroxyl groups, comprising: a) providing a dendrimer comprising at least one ester-terminated terminal branch; b) providing an amino alcohol comprising two or more hydroxyl groups; c) reacting the ester-terminated terminal branch of the dendrimer with the amino alcohol resulting in the formation of an amide bond, wherein the resulting dendrimer comprises at least one terminal branch comprising two or more hydroxyl groups. In certain embodiments, the present invention provides a method of synthesizing a dendrimer (e.g., a PAMAM dendrimer) comprising at least one terminal branch comprising two or more hydroxyl groups, comprising: a) providing an NH2-terminated dendrimer; b) providing a reactant such as a saccharide lactone and a saccharide acid; and c) reacting said NH2- terminated dendrimer with the saccharide lactone or the saccharide acid resulting in the formation of an amide bond.

Figure 2 shows an example of the synthesis of glucamine -terminated G.3 PAMAM dendrimer and conjugation of folic acid, methotrexate, and glucamine-terminated G.3 PAMAM dendrimer. Starting with methyl ester-terminated G.2.5 PAMAM dendrimer, the amino group of glucamine attacks the ester bond of the G.2.5 PAMAM denderimer to form the glucamine-terminated G.3 PAMAM dendrimer though an amide bond. In this reaction, no intra/inter-cross-linking occurs. Each terminal group of the new dendrimer contains 5 hydroxyl groups. Under the theoretical assumption that the reaction proceeds to completion, 160 hydroxyl groups occur on the surface of the dendrimer. Thereafter, FA and MTX can be easily attached to the hydroxyl through an ester bond by a "one-pot" reaction using coupling agents such as 2-chloro-l-methylpyridinium iodide and 4-(dimethylamino) pyridine (see, e.g., U.S. Patent App. No. 61/226,993, herein incorporated by reference in its entirety).

Figure 3 shows the synthesis of a HO-PAMAM dendrimer by reaction of amino- terminated PAMAM dendrimer with saccharide acid or saccharide lactone. The amino group of PAMAM dendrimer reacts with the carboxyl group of saccharide acid to form a new dendrimer, HO-PAMAM, through an amide bond using coupling agent such as DCC or EDC or another carbodiimide coupling agent. In an alternative embodiment, the amino group of PAMAM dendrimer directly attacks the lactone group of the saccharide lactone to generate a HO-PAMAM dendrimer through an amide bond. Neither of these embodiments produces undesirable cross-linking products. Functional ligands such as a therapeutic molecule (e.g., including but not limited to methotrexate (MTX), doxorubicin, SN-38 (see, e.g., U.S. Patent App. No. 61/221,596; herein incorporated by reference in its entirety) and other therapeutic molecules as described herein), a targeting molecule (e.g., including but not limited to folic acid (FA), RGD, Epidermal Growth Factor, an antibody, and other targeting molecules as described herein), and an imaging agent (e.g., including but not limited to fluoroscein isothiocyanate (FITC), a fluorescent tag, an MRI imaging agent, and other imaging molecules as described herein) are then attached to the HO-PAMAM dendrimer via an ester bond (as shown in Figures 4 and 5). The numbers of the ligands attached on the PAMAM dendrimer may be calculated using data generated by MALDI-TOF, GPC, and 1H NMR, as shown herein.

The present invention is not limited to the use of particular types and/or kinds of dendrimers. Indeed, dendrimeric polymers have been described extensively (See, e.g., Tomalia, Advanced Materials 6:529 (1994); Angew, Chem. Int. Ed. Engl, 29: 138 (1990); incorporated herein by reference in their entireties). Dendrimer polymers are synthesized as defined spherical structures typically ranging from 1 to 20 nanometers in diameter. Methods for manufacturing a G5 PAMAM dendrimer with a protected core are known (U.S. Patent App. No. 12/403,179; herein incorporated by reference in its entirety). In preferred embodiments, the protected core diamine is NH2-CH2-CH2-NHPG. Molecular weight and the number of terminal groups increase exponentially as a function of generation (the number of layers) of the polymer. In some embodiments of the present invention, half generation PAMAM dendrimers are used. For example, when an ethylenediamine (EDA) core is used for dendrimer synthesis, alkylation of this core through Michael addition results in a half- generation molecule with ester terminal groups; amidation of such ester groups with excess EDA results in creation of a full-generation, amine-terminated dendrimer (Majoros et al., Eds. (2008) Dendrimer-based Nanomedicine, Pan Stanford Publishing Pte. Ltd., Singapore, p. 42). Different types of dendrimers can be synthesized based on the core structure that initiates the polymerization process.

The dendrimer core structures dictate several characteristics of the molecule such as the overall shape, density and surface functionality (See, e.g., Tomalia et al., Chem. Int. Ed. Engl, 29:5305 (1990)). Spherical dendrimers can have ammonia as a trivalent initiator core or ethylenediamine (EDA) as a tetravalent initiator core. Recently described rod-shaped dendrimers (See, e.g., Yin et al, J. Am. Chem. Soc, 120:2678 (1998)) use polyethyleneimine linear cores of varying lengths; the longer the core, the longer the rod. Dendritic

macromolecules are available commercially in kilogram quantities and are produced under current good manufacturing processes (GMP) for biotechnology applications.

Dendrimers may be characterized by a number of techniques including, but not limited to, electrospray-ionization mass spectroscopy, 13C nuclear magnetic resonance spectroscopy, 1H nuclear magnetic resonance spectroscopy, size exclusion chromatography with multi-angle laser light scattering, ultraviolet spectrophotometry, capillary

electrophoresis and gel electrophoresis. These tests assure the uniformity of the polymer population and are important for monitoring quality control of dendrimer manufacture for GMP applications and in vivo usage.

Numerous U.S. Patents describe methods and compositions for producing dendrimers. Examples of some of these patents are given below in order to provide a description of some dendrimer compositions that may be useful in the present invention, however it should be understood that these are merely illustrative examples and numerous other similar dendrimer compositions could be used in the present invention.

U.S. Pat. No. 4,507,466, U.S. Pat. No. 4,558,120, U.S. Pat. No. 4,568,737, and U.S. Pat. No. 4,587,329 each describe methods of making dense star polymers with terminal densities greater than conventional star polymers. These polymers have greater/more uniform reactivity than conventional star polymers, i.e. 3rd generation dense star polymers. These patents further describe the nature of the amidoamine dendrimers and the 3- dimensional molecular diameter of the dendrimers.

U.S. Pat. No. 4,631,337 describes hydrolytically stable polymers. U.S. Pat. No.

4,694,064 describes rod-shaped dendrimers. U.S. Pat. No. 4,713,975 describes dense star polymers and their use to characterize surfaces of viruses, bacteria and proteins including enzymes. Bridged dense star polymers are described in U.S. Pat. No. 4,737,550. U.S. Pat. No. 4,857,599 and U.S. Pat. No. 4,871,779 describe dense star polymers on immobilized cores useful as ion-exchange resins, chelation resins and methods of making such polymers.

U.S. Pat. No. 5,338,532 is directed to starburst conjugates of dendrimer(s) in association with at least one unit of carried agricultural, pharmaceutical or other material. This patent describes the use of dendrimers to provide means of delivery of high

concentrations of carried materials per unit polymer, controlled delivery, targeted delivery and/or multiple species such as e.g., drugs antibiotics, general and specific toxins, metal ions, radionuclides, signal generators, antibodies, interleukins, hormones, interferons, viruses, viral fragments, pesticides, and antimicrobials.

U.S. Pat. No. 6,471,968 describes a dendrimer complex comprising covalently linked first and second dendrimers, with the first dendrimer comprising a first agent and the second dendrimer comprising a second agent, wherein the first dendrimer is different from the second dendrimer, and where the first agent is different than the second agent.

Other useful dendrimer type compositions are described in U.S. Pat. No. 5,387,617, U.S. Pat. No. 5,393,797, and U.S. Pat. No. 5,393,795 in which dense star polymers are modified by capping with a hydrophobic group capable of providing a hydrophobic outer shell. U.S. Pat. No. 5,527,524 discloses the use of amino terminated dendrimers in antibody conjugates.

PAMAM dendrimers are highly branched, narrowly dispersed synthetic

macromolecules with well-defined chemical structures. PAMAM dendrimers can be easily modified and conjugated with multiple functionalities such as targeting molecules, imaging agents, and drugs (Thomas et al. (2007) Poly(amidoamine) Dendrimer-based Multifunctional Nanoparticles, in Nanobiotechnology: Concepts, Methods and Perspectives, Merkin, Ed., Wiley- VCH; herein incorporated by reference in its entirety). They are water soluble, biocompatible, and cleared from the blood through the kidneys (Peer et al. (2007) Nat.

Nanotechnol. 2:751-760; herein incorporated by reference in its entirety) which eliminates the need for biodegradability. Because of these desirable properties, PAMAM dendrimers have been widely investigated for drug delivery (Esfand et al. (2001) Drug Discov. Today 6:427-436; Patri et al. (2002) Curr. Opin. Chem. Biol. 6:466-471; Kukowska-Latallo et al. (2005) Cancer Res. 65:5317-5324; Quintana et al. (2002) Pharmaceutical Res. 19: 1310-1316; Thomas et al. (2005) J. Med. Chem. 48:3729-3735; each herein incorporated by reference in its entirety), gene therapy (KukowskaLatallo et al. (1996) PNAS 93:4897-4902; Eichman et al. (2000) Pharm. Sci. Technolo. Today 3:232-245; Luo et al. (2002) Macromol. 35:3456- 3462; each herein incorporated by reference in its entirety), and imaging applications (Kobayashi et al. (2003) Bioconj. Chem. 14:388-394; herein incorporated by reference in its entirety).

The use of dendrimers as metal ion carriers is described in U.S. Pat. No. 5,560,929. U.S. Pat. No. 5,773,527 discloses non-crosslinked polybranched polymers having a comb- burst configuration and methods of making the same. U.S. Pat. No. 5,631,329 describes a process to produce polybranched polymer of high molecular weight by forming a first set of branched polymers protected from branching; grafting to a core; deprotecting first set branched polymer, then forming a second set of branched polymers protected from branching and grafting to the core having the first set of branched polymers, etc.

U.S. Pat. No. 5,902,863 describes dendrimer networks containing lipophilic organosilicone and hydrophilic polyanicloamine nanscopic domains. The networks are prepared from copolydendrimer precursors having PAMAM (hydrophilic) or

polyproyleneimine interiors and organosilicon outer layers. These dendrimers have a controllable size, shape and spatial distribution. They are hydrophobic dendrimers with an organosilicon outer layer that can be used for specialty membrane, protective coating, composites containing organic organometallic or inorganic additives, skin patch delivery, absorbants, chromatography personal care products and agricultural products.

U.S. Pat. No. 5,795,582 describes the use of dendrimers as adjuvants for influenza antigen. Use of the dendrimers produces antibody titer levels with reduced antigen dose. U.S. Pat. No. 5,898,005 and U.S. Pat. No. 5,861,319 describe specific immunobinding assays for determining concentration of an analyte. U.S. Pat. No. 5,661,025 provides details of a self- assembling polynucleotide delivery system comprising dendrimer polycation to aid in delivery of nucleotides to target site. This patent provides methods of introducing a polynucleotide into a eukaryotic cell in vitro comprising contacting the cell with a

composition comprising a polynucleotide and a dendrimer polyeation non-covalently coupled to the polynucleotide.

Dendrimer-antibody conjugates for use in in vitro diagnostic applications have previously been demonstrated (See, e.g., Singh et al, Clin. Chem., 40:1845 (1994)), for the production of dendrimer-chelant-antibody constructs, and for the development of boronated dendrimer-antibody conjugates (for neutron capture therapy); each of these latter compounds may be used as a cancer therapeutic (See, e.g., Wu et al, Bioorg. Med. Chem. Lett., 4:449

(1994); Wiener et al, Magn. Reson. Med. 31 : 1 (1994); Barth et al, Bioconjugate Chem. 5:58 (1994); and Barth et al).

Some of these conjugates have also been employed in the magnetic resonance imaging of tumors (See, e.g., Wu et al, (1994) and Wiener et al, (1994), supra). Results from this work have documented that, when administered in vivo, antibodies can direct dendrimer-associated therapeutic agents to antigen-bearing tumors. Dendrimers also have been shown to specifically enter cells and carry either chemotherapeutic agents or genetic therapeutics. In particular, studies show that cisplatin encapsulated in dendrimer polymers has increased efficacy and is less toxic than cisplatin delivered by other means (See, e.g., Duncan and Malik, Control Rel. Bioact. Mater. 23: 105 (1996)).

Dendrimers have also been conjugated to fluorochromes or molecular beacons and shown to enter cells. They can then be detected within the cell in a manner compatible with sensing apparatus for evaluation of physiologic changes within cells (See, e.g., Baker et al, Anal. Chem. 69:990 (1997)). Finally, dendrimers have been constructed as differentiated block copolymers where the outer portions of the molecule may be digested with either enzyme or light-induced catalysis (See, e.g., Urdea and Hom, Science 261 :534 (1993)). This allows the controlled degradation of the polymer to release therapeutics at the disease site and provides a mechanism for an external trigger to release the therapeutic agents.

In experiments conducted during the course of developing embodiments for the present invention, dendrimers containing functional components were developed having a therapeutic agent (e.g., a chemotherapeutic agent) conjugated with one or more functional groups (e.g., an imaging agent) and a targeting agent (e.g., folic acid)). The present invention is compatible with additional functional components (e.g., trigger agents (e.g., for release under hypoxic conditions)). In some embodiments, synthesis of the functionalized dendrimer is facilitated by use of PAMAM dendrimers bearing multiple hydroxyls (HO-PAMAMS) that may be further linked to functional ligands and in one-step (e.g., "one pot") synthesis reactions.

Accordingly, the present invention provides synthesis methods, compositions and applications for efficient, site-specific drug delivery using functionalized dendrimers comprising therapeutic agents conjugated with one or more functional groups (e.g., imaging agents, targeting agents, and trigger agents). In particular, the present invention relates to dendrimers comprising one or more functional groups conjugated with a therapeutic agent (e.g., a chemotherapeutic agent), methods of synthesizing the same, as well as systems and methods utilizing the therapeutic and diagnostic compositions (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutics, imaging, and/or targeting agents (e.g., in disease (e.g., cancer) diagnosis and/or therapy, etc.)). For example, in some embodiments, the novel therapeutic and diagnostic compositions comprise a therapeutic agent (e.g., a chemotherapeutic agent (e.g., methotrexate) conjugated with an imaging agent and a targeting agent (e.g., folic acid). As described in more detail below, examples of functional groups include, but are not limited to, targeting groups, trigger groups, and imaging groups.

The present invention is not limited to the use of particular therapeutic agents. In some embodiments, the therapeutic agents are effective in treating autoimmune disorders and/or inflammatory disorders (e.g., arthritis). Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide,

methotrexate, sulfasalazine, hydroxychloroquine), biologic agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-a inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloprotease inhibitors. In some embodiments, the therapeutic agents include, but are not limited to, infliximab, adalimumab, etanercept, parenteral gold or oral gold.

In some embodiments, the therapeutic agents are effective in treating cancer (see, e.g., U.S. Patent Nos. 6,471,968, 7,078,461, and U.S. Patent Application Serial Nos. 09/940,243, 10/431,682, 11/503,742, 11/661,465, 11/523,509, 12/403,179, 12/106,876, and 11/827,637; and U.S. Provisional Patent Application Serial Nos. 61/256,759, 61/140,840, 61/091,608, 61/097,780, 61/101,461, 61/237,172, 61/229,168, 61/221,596, and 61/251,244; each herein incorporated by reference in their entireties).

In some embodiments, the ligand (e.g., therapeutic agent) is conjugated to a trigger agent. The present invention is not limited to particular types or kinds of trigger agents.

In some embodiments, sustained release (e.g., slow release over a period of 24-48 hours) of the therapeutic agent is accomplished through conjugating the therapeutic agent (e.g., directly) (e.g., indirectly through one or more additional functional groups) to a trigger agent that slowly degrades in a biological system (e.g., amide linkage, ester linkage, ether linkage). In some embodiments, constitutively active release of the therapeutic agent is accomplished through conjugating the therapeutic agent to a trigger agent that renders the therapeutic agent constitutively active in a biological system (e.g., amide linkage, ether linkage).

In some embodiments, release of the therapeutic agent under specific conditions is accomplished through conjugating the therapeutic agent (e.g., directly) (e.g., indirectly through one or more additional functional groups) to a trigger agent that degrades under such specific conditions (e.g., through activation of a trigger molecule under specific conditions that leads to release of the therapeutic agent). For example, once a conjugate (e.g., a therapeutic agent conjugated with a trigger agent and a targeting agent) arrives at a target site in a subject (e.g., a tumor, or a site of inflammation), components in the target site (e.g., a tumor associated factor, or an inflammatory or pain associated factor) interact with the trigger agent thereby initiating cleavage of the therapeutic agent from the trigger agent. In some embodiments, the trigger agent is configured to degrade (e.g., release the therapeutic agent) upon exposure to a tumor-associated factor (e.g., hypoxia and pH, an enzyme (e.g., glucuronidase and/or plasmin), a cathepsin, a matrix metalloproteinase, a hormone receptor (e.g., integrin receptor, hyaluronic acid receptor, luteinizing hormone-releasing hormone receptor, etc.), cancer and/or tumor specific DNA sequence), an inflammatory associated factor (e.g., chemokine, cytokine, etc.) or other moiety.

In some embodiments, the present invention provides a therapeutic agent conjugated with a trigger agent that is sensitive to (e.g., is cleaved by) hypoxia (e.g., indolequinone). Hypoxia is a feature of several disease states, including cancer, inflammation and rheumatoid arthritis, as well as an indicator of respiratory depression (e.g., resulting from analgesic drugs).

Advances in the chemistry of bioreductive drug activation have led to the design of various hypoxia-selective drug delivery systems in which the pharmacophores of drugs are masked by reductively cleaved groups. In some embodiments, the trigger agent is utilizes a quinone, N-oxide and/or (hetero)aromatic nitro groups. For example, a quinone present in a conjugate is reduced to phenol under hypoxia conditions, with spontaneous formation of lactone that serves as a driving force for drug release. In some embodiments, a

heteroaromatic nitro compound present in a conjugate (e.g., a therapeutic agent conjugated (e.g., directly or indirectly) with a trigger agent) is reduced to either an amine or a

hydroxylamine, thereby triggering the spontaneous release of a therapeutic agent. In some embodiments, the trigger agent degrades upon detection of reduced p02 concentrations (e.g., through use of a redox linker).

The concept of pro-drug systems in which the pharmacophores of drugs are masked by reductively cleavable groups has been widely explored by many research groups and pharmaceutical companies (see, e.g., Beall, H.D., et al., Journal of Medicinal Chemistry, 1998. 41(24): p. 4755-4766; Ferrer, S., D.P. Naughton, and M.D. Threadgill, Tetrahedron, 2003. 59(19): p. 3445-3454; Naylor, M.A., et al, Journal of Medicinal Chemistry, 1997. 40(15): p. 2335-2346; Phillips, R.M., et al, Journal of Medicinal Chemistry, 1999. 42(20): p. 4071-4080; Zhang, Z., et al, Organic & Biomolecular Chemistry, 2005. 3(10): p. 1905-1910; each of which are herein incorporated by reference in their entireties). Several such hypoxia activated pro-drugs have been advanced to clinical investigations, and work in relevant oxygen concentrations to prevent cerebral damage. The present invention is not limited to particular hypoxia-activated trigger agents. In some embodiments, the hypoxia-activated trigger agents include, but are not limited to, indolequinones, nitroimidazoles, and

nitroheterocycles (see, e.g., Damen, E.W.P., et al, Bioorganic & Medicinal Chemistry, 2002. 10(1): p. 71-77; Hay, M.P., et al, Journal of Medicinal Chemistry, 2003. 46(25): p. 5533- 5545; Hay, M.P., et al., Journal of the Chemical Society-Perkin Transactions 1, 1999(19): p. 2759-2770; each herein incorporated by reference in their entireties).

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or associates with a tumor-associated enzyme. For example, in some embodiments, the trigger agent that is sensitive to (e.g., is cleaved by) and/or associates with a glucuronidase.

Glucuronic acid can be attached to several anticancer drugs via various linkers. These anticancer drugs include, but are not limited to, doxorubicin, paclitaxel, docetaxel, 5- fluorouracil, 9-aminocamtothecin, as well as other drugs under development. These prodrugs are generally stable at physiological pH and are significantly less toxic than the parent drugs.

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or associates with brain enzymes. For example, trigger agents such as indolequinone are reduced by brain enzymes such as, for example, diaphorase (DT-diaphorase) (see, e.g., Damen, E.W.P., et al., Bioorganic & Medicinal Chemistry, 2002. 10(1): p. 71-77; herein incorporated by reference in its entirety). For example, in such embodiments, the antagonist is only active when released during hypoxia to prevent respiratory failure.

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or associates with a protease. The present invention is not limited to any particular protease. In some embodiments, the protease is a cathepsin. In some embodiments, a trigger comprises a Lys-Phe-PABC moiety (e.g., that acts as a trigger). In some embodiments, a Lys-Phe-PABC moiety linked to doxorubicin, mitomycin C, and paclitaxel are utilized as a trigger- therapeutic conjugate in a dendrimer conjugate provided herein (e.g., that serve as substrates for lysosomal cathepsin B or other proteases expressed (e.g., overexpressed) in tumor cells. In some embodiments, utilization of a 1 ,6-elimination spacer/linker is utilized (e.g., to permit release of therapeutic drug post activation of trigger).

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or associates with plasmin. The serine protease plasmin is over expressed in many human tumor tissues. Tripeptide specifiers (e.g., including, but not limited to, Val-Leu-Lys) have been identified and linked to anticancer drugs through elimination or cyclization linkers.

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or associates with a matrix metalloprotease (MMP). In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or that associates with β-Lactamase (e.g., a β-Lactamase activated cephalosporin-based pro-drug).

In some embodiments, the trigger agent is sensitive to (e.g., is cleaved by) and/or activated by a receptor (e.g., expressed on a target cell (e.g., a tumor cell)).

In some embodiments, the trigger agent that is sensitive to (e.g., is cleaved by) and/or activated by a nucleic acid. Nucleic acid triggered catalytic drug release can be utilized in the design of chemotherapeutic agents. Thus, in some embodiments, disease specific nucleic acid sequence is utilized as a drug releasing enzyme-like catalyst (e.g., via complex formation with a complimentary catalyst-bearing nucleic acid and/or analog). In some embodiments, the release of a therapeutic agent is facilitated by the therapeutic component being attached to a labile protecting group, such as, for example, cisplatin or methotrexate being attached to a photolabile protecting group that becomes released by laser light directed at cells emitting a color of fluorescence (e.g., in addition to and/or in place of target activated activation of a trigger component of a dendrimer conjugate). In some embodiments, the therapeutic device also may have a component to monitor the response of the tumor to therapy. For example, where a therapeutic agent of the dendrimer induces apoptosis of a target cell (e.g., a cancer cell (e.g., a prostate cancer cell)), the caspase activity of the cells may be used to activate a green fluorescence. This allows apoptotic cells to turn orange, (combination of red and green) while residual cells remain red. Any normal cells that are induced to undergo apoptosis in collateral damage fluoresce green.

In some embodiments, the dendrimer and/or ligand (e.g., therapeutic agent) is conjugated (e.g., directly or indirectly) to a targeting agent. The present invention is not limited to any particular targeting agent. In some embodiments, targeting agents are conjugated to the therapeutic agents for delivery of the therapeutic agents to desired body regions (e.g., to the central nervous system (CNS); to a tumor). The targeting agents are not limited to targeting specific body regions.

In some embodiments, the targeting agent is a moiety that has affinity for a tumor associated factor. For example, a number of targeting agents are contemplated to be useful in the present invention including, but not limited to, RGD sequences, low-density lipoprotein sequences, a NAALADase inhibitor, epidermal growth factor, and other agents that bind with specificity to a target cell (e.g., a cancer cell)).

The present invention is not limited to cancer and/or tumor targeting agents. Indeed, multifunctional dendrimers can be targeted (e.g., via a linker conjugated to the dendrimer wherein the linker comprises a targeting agent) to a variety of target cells or tissues (e.g., to a biologically relevant environment) via conjugation to an appropriate targeting agent. For example, in some embodiments, the targeting agent is a moiety that has affinity for an inflammatory factor (e.g., a cytokine or a cytokine receptor moiety (e.g., TNF-a receptor)). In some embodiments, the targeting agent is a sugar, peptide, antibody or antibody fragment, hormone, hormone receptor, or the like.

In some embodiments of the present invention, the targeting agent includes but is not limited to an antibody, receptor ligand, hormone, vitamin, and antigen; however, the present invention is not limited by the nature of the targeting agent. In some embodiments, the antibody is specific for a disease-specific antigen. In some embodiments, the disease-specific antigen comprises a tumor-specific antigen. In some embodiments, the receptor ligand includes, but is not limited to, a ligand for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, glycoprotein, and VEGFR. In some embodiments, the receptor ligand is folic acid.

Antibodies can be generated to allow for the targeting of antigens or immunogens

(e.g., tumor, tissue or pathogen specific antigens) on various biological targets (e.g., pathogens, tumor cells, normal tissue). Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.

In some embodiments, the targeting agent is an antibody. In some embodiments, the antibodies recognize, for example, tumor-specific epitopes (e.g., TAG-72 (See, e.g., Kjeldsen et al, Cancer Res. 48:2214-2220 (1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and 5,512,443; each herein incorporated by reference in their entireties); human carcinoma antigen (See, e.g., U.S. Pat. Nos. 5,693,763; 5,545,530; and 5,808,005; each herein incorporated by reference in their entireties); TP1 and TP3 antigens from osteocarcinoma cells (See, e.g., U.S. Pat. No. 5,855,866; herein incorporated by reference in its entirety); Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells (See, e.g., U.S. Pat. No. 5,110,911; herein incorporated by reference in its entirety); "KC-4 antigen" from human prostrate adenocarcinoma (See, e.g., U.S. Pat. Nos. 4,708,930 and 4,743,543; each herein incorporated by reference in their entireties); a human colorectal cancer antigen (See, e.g., U.S. Pat. No. 4,921,789; herein incorporated by reference in its entirety); CA125 antigen from cystadenocarcinoma (See, e.g., U.S. Pat. No. 4,921,790; herein incorporated by reference in its entirety); DF3 antigen from human breast carcinoma (See, e.g., U.S. Pat. Nos. 4,963,484 and 5,053,489; each herein incorporated by reference in their entireties); a human breast tumor antigen (See, e.g., U.S. Pat. No. 4,939,240: herein incorporated by reference in its entirety); p97 antigen of human melanoma (See, e.g., U.S. Pat. No. 4,918,164: herein incorporated by reference in its entirety); carcinoma or orosomucoid-related antigen (CORA)( See, e.g., U.S. Pat. No.

4,914,021; herein incorporated by reference in its entirety); a human pulmonary carcinoma antigen that reacts with human squamous cell lung carcinoma but not with human small cell lung carcinoma (See, e.g., U.S. Pat. No. 4,892,935; herein incorporated by reference in its entirety); T and Tn haptens in glycoproteins of human breast carcinoma (See, e.g., Springer et al., Carbohydr. Res. 178:271-292 (1988); herein incorporated by reference in its entirety), MSA breast carcinoma glycoprotein termed (See, e.g., Tjandra et al, Br. J. Surg. 75:811-817 (1988); herein incorporated by reference in its entirety); MFGM breast carcinoma antigen (See, e.g., Ishida et al, Tumor Biol. 10: 12-22 (1989); herein incorporated by reference in its entirety); DU-PAN-2 pancreatic carcinoma antigen (See, e.g., Lan et al., Cancer Res. 45:305- 310 (1985); herein incorporated by reference in its entirety); CA125 ovarian carcinoma antigen (See, e.g., Hanisch et al, Carbohydr. Res. 178:29-47 (1988); herein incorporated by reference in its entirety); YH206 lung carcinoma antigen (See, e.g., Hinoda et al, (1988) Cancer J. 42:653-658 (1988); herein incorporated by reference in its entirety).

In some embodiments, the targeting agents target the central nervous system (CNS). In some embodiments, where the targeting agent is specific for the CNS, the targeting agent is transferrin (see, e.g., Daniels, T.R., et al, Clinical Immunology, 2006. 121(2): p. 159-176; Daniels, T.R., et al., Clinical Immunology, 2006. 121(2): p. 144-158; each herein

incorporated by reference in their entireties). Transferrin has been utilized as a targeting vector to transport, for example, drugs, liposomes and proteins across the blood-brain barrier (BBB) by receptor mediated transcytosis (see, e.g., Smith, M.W. and M. Gumbleton, Journal of Drug Targeting, 2006. 14(4): p. 191-214; herein incorporated by reference in its entirety). In some embodiments, the targeting agents target neurons within the central nervous system (CNS). In some embodiments, where the targeting agent is specific for neurons within the CNS, the targeting agent is a synthetic tetanus toxin fragment (e.g., a 12 amino acid peptide (Tet 1) (HLNILSTLWKYR)) (see, e.g., Liu, J.K., et al, Neurobiology of Disease, 2005. 19(3): p. 407-418; herein incorporated by reference in its entirety).

In some embodiments, the dendrimer and/or ligand (e.g., therapeutic agent) is conjugated (e.g., directly or indirectly) to an imaging agent. A multiplicity of imaging agents find use in the present invention. In some embodiments, a multifunctional dendrimer comprises at least one imaging agent that can be readily imaged. The present invention is not limited by the nature of the imaging component used. In some embodiments of the present invention, imaging modules comprise surface modifications of quantum dots (See e.g., Chan and Nie, Science 281 :2016 (1998)) such as zinc sulfide-capped cadmium selenide coupled to biomolecules (Sooklal, Adv. Mater., 10: 1083 (1998)).

In some embodiments, once a component(s) of a targeted multifunctional dendrimer has attached to (or been internalized into) a target cell (e.g., tumor cell and or inflammatory cell), one or more modules serves to image its location. In some embodiments, chelated paramagnetic ions, such as Gd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA), are conjugated to the multifunctional dendrimer. Other paramagnetic ions that may be useful in this context include, but are not limited to, gadolinium, manganese, copper, chromium, iron, cobalt, erbium, nickel, europium, technetium, indium, samarium, dysprosium, ruthenium, ytterbium, yttrium, and holmium ions and combinations thereof.

Dendrimeric gadolinium contrast agents have even been used to differentiate between benign and malignant breast tumors using dynamic MRI, based on how the vasculature for the latter type of tumor images more densely (Adam et al, Ivest. Rad. 31 :26 (1996)). Thus, MRI provides a particularly useful imaging system of the present invention.

Multifunctional dendrimers allow functional microscopic imaging of tumors and provide improved methods for imaging. The methods find use in vivo, in vitro, and ex vivo. For example, in one embodiment, dendrimer functional groups are designed to emit light or other detectable signals upon exposure to light. Although the labeled functional groups may be physically smaller than the optical resolution limit of the microscopy technique, they become self-luminous objects when excited and are readily observable and measurable using optical techniques. In some embodiments of the present invention, sensing fluorescent biosensors in a microscope involves the use of tunable excitation and emission filters and multiwavelength sources (See, e.g., Farkas et al, SPEI 2678:200 (1997); herein incorporated by reference in its entirety). In embodiments where the imaging agents are present in deeper tissue, longer wavelengths in the Near-infrared (NMR) are used (See e.g., Lester et al, Cell Mol. Biol. 44:29 (1998); herein incorporated by reference in its entirety). Biosensors that find use with the present invention include, but are not limited to, fluorescent dyes and molecular beacons.

In some embodiments of the present invention, in vivo imaging is accomplished using functional imaging techniques. Functional imaging is a complementary and potentially more powerful techniques as compared to static structural imaging. Functional imaging is best known for its application at the macroscopic scale, with examples including functional Magnetic Resonance Imaging (fMRI) and Positron Emission Tomography (PET). However, functional microscopic imaging may also be conducted and find use in in vivo and ex vivo analysis of living tissue. Functional microscopic imaging is an efficient combination of 3-D imaging, 3-D spatial multispectral volumetric assignment, and temporal sampling: in short a type of 3-D spectral microscopic movie loop. Interestingly, cells and tissues auto fluoresce. When excited by several wavelengths, providing much of the basic 3-D structure needed to characterize several cellular components (e.g., the nucleus) without specific labeling.

Oblique light illumination is also useful to collect structural information and is used routinely. As opposed to structural spectral microimaging, functional spectral microimaging may be used with biosensors, which act to localize physiologic signals within the cell or tissue. For example, in some embodiments, biosensor-comprising pro-drug complexes are used to image upregulated receptor families such as the folate or EGF classes. In such embodiments, functional biosensing therefore involves the detection of physiological abnormalities relevant to carcinogenesis or malignancy, even at early stages. A number of physiological conditions may be imaged using the compositions and methods of the present invention including, but not limited to, detection of nanoscopic biosensors for pH, oxygen concentration, Ca2+ concentration, and other physiologically relevant analytes.

In some embodiments, the present invention provides multifunctional dendrimers having a biological monitoring component. The biological monitoring or sensing component of a multifunctional dendrimer is one that can monitor the particular response in a target cell (e.g., tumor cell) induced by an agent (e.g., a therapeutic agent provided by a multifunctional dendrimer). While the present invention is not limited to any particular monitoring system, the invention is illustrated by methods and compositions for monitoring cancer treatments. In preferred embodiments of the present invention, the agent induces apoptosis in cells and monitoring involves the detection of apoptosis. In some embodiments, the monitoring component is an agent that fluoresces at a particular wavelength when apoptosis occurs. For example, in a preferred embodiment, caspase activity activates green fluorescence in the monitoring component. Apoptotic cancer cells, which have turned red as a result of being targeted by a particular signature with a red label, turn orange while residual cancer cells remain red. Normal cells induced to undergo apoptosis (e.g., through collateral damage), if present, will fluoresce green.

In these embodiments, fluorescent groups such as fluorescein are employed in the imaging agent. Fluorescein is easily attached to the dendrimer surface via the isothiocyanate derivatives, available from MOLECULAR PROBES, Inc. This allows the multifunctional dendrimer or components thereof to be imaged with the cells via confocal microscopy. Sensing of the effectiveness of the multifunctional dendrimer or components thereof is preferably achieved by using fluorogenic peptide enzyme substrates. For example, apoptosis caused by the therapeutic agent results in the production of the peptidase caspase-1 (ICE). CALBIOCHEM sells a number of peptide substrates for this enzyme that release a fluorescent moiety. A particularly useful peptide for use in the present invention is:

MCA-Tyr-Glu-Val-Asp-Gly-Trp-Lys-(DNP)-NH2 (SEQ ID NO: 1) where MCA is the (7- methoxycoumarin-4-yl)acetyl and DNP is the 2,4-dinitrophenyl group (See, e.g., Talanian et al., J. Biol. Chem., 272: 9677 (1997); herein incorporated by reference in its entirety). In this peptide, the MCA group has greatly attenuated fluorescence, due to fluorogenic resonance energy transfer (FRET) to the DNP group. When the enzyme cleaves the peptide between the aspartic acid and glycine residues, the MCA and DNP are separated, and the MCA group strongly fluoresces green (excitation maximum at 325 nm and emission maximum at 392 nm). In some embodiments, the lysine end of the peptide is linked to pro-drug complex, so that the MCA group is released into the cytosol when it is cleaved. The lysine end of the peptide is a useful synthetic handle for conjugation because, for example, it can react with the activated ester group of a bifunctional linker such as Mal-PEG-OSu. Thus the appearance of green fluorescence in the target cells produced using these methods provides a clear indication that apoptosis has begun (if the cell already has a red color from the presence of aggregated quantum dots, the cell turns orange from the combined colors).

Additional fluorescent dyes that find use with the present invention include, but are not limited to, acridine orange, reported as sensitive to DNA changes in apoptotic cells (see, e.g., Abrams et al., Development 117:29 (1993); herein incorporated by reference in its entirety) and czs-parinaric acid, sensitive to the lipid peroxidation that accompanies apoptosis (see, e.g., Hockenbery et al, Cell 75:241 (1993); herein incorporated by reference in its entirety). It should be noted that the peptide and the fluorescent dyes are merely exemplary. It is contemplated that any peptide that effectively acts as a substrate for a caspase produced as a result of apoptosis finds use with the present invention.

In some embodiments, conjugation between a dendrimer (e.g., terminal arm of a dendrimer) and a functional group (e.g., ligand) or between functional groups is

accomplished through use of a 1,3-dipolar cycloaddition reaction ("click chemistry"). 'Click chemistry' involves, for example, the coupling of two different moieties (e.g., a therapeutic agent and a functional group) (e.g., a first functional group and a second functional group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or equivalent thereof) on the surface of the first moeity and an azide moiety (e.g., present on a triazine composition of the present invention) (or equivalent thereof) (or any active end group such as, for example, a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.) on the second moiety. 'Click' chemistry is an attractive coupling method because, for example, it can be performed with a wide variety of solvent conditions including aqueous environments. For example, the stable triazole ring that results from coupling the alkyne with the azide is frequently achieved at quantitative yields and is considered to be biologically inert (see, e.g., Rostovtsev, V. V.; et al, Angewandte Chemie-International Edition 2002, 41, (14), 2596; Wu, P.; et al, Angewandte Chemie-International Edition 2004, 43, (30), 3928-3932; each herein incorporated by reference in their entireties).

In some embodiments, conjugation between a dendrimer (e.g., a terminal arm of a dendrimer) and a functional ligand is accomplished during a "one-pot" reaction. The term "one-pot synthesis reaction" or equivalents thereof, e.g., "1-pot", "one pot", etc., refers to a chemical synthesis method in which all reactants are present in a single vessel. Reactants may be added simultaneously or sequentially, with no limitation as to the duration of time elapsing between introduction of sequentially added reactants. In some embodiments, a one- pot reaction occurs wherein a hydroxyl-terminated dendrimer (e.g., HO-PAMAM dendrimer) is reacted with one or more functional ligands (e.g., a therapeutic agent, a pro-drug, a trigger agent, a targeting agent, an imaging agent) in one vessel, such conjugation being facilitated by ester coupling agents (e.g., 2-chloro-l-methylpyridinium iodide and 4-(dimethylamino) pyridine) (see, e.g., U.S. Patent App. No. 61/226,993, herein incorporated by reference in its entirety).

The present invention also includes methods involving co-administration of the multifunctional dendrimers and components thereof described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering multifunctional dendrimers of this invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In some embodiments, the multifunctional dendrimers described herein are administered prior to the other active agent(s). The agent or agents to be co-administered depends on the type of condition being treated. For example, when the condition being treated is cancer, the additional agent can be a chemotherapeutic agent or radiation. The additional agents to be co-administered, such as anticancer agents, can be any of the well-known agents in the art, including, but not limited to, those that are currently in clinical use. The determination of appropriate type and dosage of radiation treatment is also within the skill in the art or can be determined with relative ease. The present invention is not limited by the type of therapeutic agent delivered via multifunctional dendrimers of the present invention and/or co-administered with

multifuncaitional dendrimers of the present invention. For example, a therapeutic agent may be any agent selected from the group comprising, but not limited to, a pain relief agent, a pain relief agent antagonist, a chemotherapeutic agent, an anti-oncogenic agent, an anti-angiogenic agent, a tumor suppressor agent, an anti-microbial agent, or an expression construct comprising a nucleic acid encoding a therapeutic protein.

Indeed, in some embodiments of the present invention, methods and compositions are provided for the treatment of inflammatory diseases (e.g., dendrimers conjugated with therapeutic agents configured for treating inflammatory diseases). Inflammatory diseases include but are not limited to arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, degenerative arthritis, polymyalgia rheumatic, ankylosing spondylitis, reactive arthritis, gout, pseudogout, inflammatory joint disease, systemic lupus erythematosus, polymyositis, and fibromyalgia. Additional types of arthritis include achilles tendinitis, achondroplasia, acromegalic arthropathy, adhesive capsulitis, adult onset Still's disease, anserine bursitis, avascular necrosis, Behcet's syndrome, bicipital tendinitis, Blount's disease, brucellar spondylitis, bursitis, calcaneal bursitis, calcium pyrophosphate dihydrate deposition disease (CPPD), crystal deposition disease, Caplan's syndrome, carpal tunnel syndrome,

chondrocalcinosis, chondromalacia patellae, chronic synovitis, chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, Cogan's syndrome, corticosteroid-induced osteoporosis, costostemal syndrome, CREST syndrome, cryoglobulinemia, degenerative joint disease, dermatomyositis, diabetic finger sclerosis, diffuse idiopathic skeletal hyperostosis (DISH), discitis, discoid lupus erythematosus, drug-induced lupus, Duchenne's muscular dystrophy, Dupuytren's contracture, Ehlers-Danlos syndrome, enteropathic arthritis, epicondylitis, erosive inflammatory osteoarthritis, exercise-induced compartment syndrome, Fabry's disease, familial Mediterranean fever, Farber's lipogranulomatosis, Felty's syndrome, Fifth's disease, fiat feet, foreign body synovitis, Freiberg's disease, fungal arthritis, Gaucher's disease, giant cell arteritis, gonococcal arthritis, Goodpasture's syndrome, granulomatous arteritis, hemarthrosis, hemochromatosis, Henoch-Schonlein purpura, Hepatitis B surface antigen disease, hip dysplasia, Hurler syndrome, hypermobility syndrome, hypersensitivity vasculitis, hypertrophic osteoarthropathy, immune complex disease, impingement syndrome, Jaccoud's arthropathy, juvenile ankylosing spondylitis, juvenile dermatomyositis, juvenile rheumatoid arthritis, Kawasaki disease, Kienbock's disease, Legg-Calve-Perthes disease, Lesch-Nyhan syndrome, linear scleroderma, lipoid dermatoarthritis, Lofgren's syndrome, Lyme disease, malignant synovioma, Marfan' s syndrome, medial plica syndrome, metastatic carcinomatous arthritis, mixed connective tissue disease (MCTD), mixed cryoglobulinemia, mucopolysaccharidosis, multicentric reticulohistiocytosis, multiple epiphyseal dysplasia, mycoplasmal arthritis, myofascial pain syndrome, neonatal lupus, neuropathic arthropathy, nodular panniculitis, ochronosis, olecranon bursitis, Osgood-Schlatter's disease, osteoarthritis, osteochondromatosis, osteogenesis imperfecta, osteomalacia, osteomyelitis, osteonecrosis, osteoporosis, overlap syndrome, pachydermoperiostosis Paget's disease of bone, palindromic rheumatism, patellofemoral pain syndrome, Pellegrini-Stieda syndrome, pigmented villonodular synovitis, piriformis syndrome, plantar fasciitis, polyarteritis nodos, Polymyalgia rheumatic, polymyositis, popliteal cysts, posterior tibial tendinitis, Pott's disease, prepatellar bursitis, prosthetic joint infection, pseudoxanthoma elasticum, psoriatic arthritis, Raynaud's phenomenon, reactive arthritis/Reiter's syndrome, reflex sympathetic dystrophy syndrome, relapsing polychondritis, retrocalcaneal bursitis, rheumatic fever, rheumatoid vasculitis, rotator cuff tendinitis, sacroiliitis, salmonella osteomyelitis, sarcoidosis, saturnine gout,

Scheuermann's osteochondritis, scleroderma, septic arthritis, seronegative arthritis, shigella arthritis, shoulder-hand syndrome, sickle cell arthropathy, Sjogren's syndrome, slipped capital femoral epiphysis, spinal stenosis, spondylolysis, staphylococcus arthritis, Stickler syndrome, subacute cutaneous lupus, Sweet's syndrome, Sydenham's chorea, syphilitic arthritis, systemic lupus erythematosus (SLE), Takayasu's arteritis, tarsal tunnel syndrome, tennis elbow, Tietse's syndrome, transient osteoporosis, traumatic arthritis, trochanteric bursitis, tuberculosis arthritis, arthritis of Ulcerative colitis, undifferentiated connective tissue syndrome (UCTS), urticarial vasculitis, viral arthritis, Wegener's granulomatosis, Whipple's disease, Wilson's disease, and yersinial arthritis.

In some embodiments, the dendrimers of the present invention configured for treating autoimmune disorders and/or inflammatory disorders (e.g., rheumatoid arthritis) are conjugated with one or more therapeutic agents configured for treating autoimmune disorders and/or inflammatory disorders (e.g., rheumatoid arthritis). In some embodiments, the conjugated dendrimers of the present invention configured for treating autoimmune disorders and/or inflammatory disorders (e.g., rheumatoid arthritis) are co-administered to a subject

(e.g., a human suffering from an autoimmune disorder and/or an inflammatory disorder) with a therapeutic agent configured for treating autoimmune disorders and/or inflammatory disorders (e.g., rheumatoid arthritis). Examples of such agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologic agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol),

immunomodulators (e.g., anakinra, abatacept), and glucocorticoids (e.g., prednisone, methylprednisone), IL-1 inhibitors, and metalloprotease inhibitors..

In some embodiments, the disease is cancer. The present invention is not limited by the type of cancer treated using the compositions and methods of the present invention.

Indeed, a variety of cancer can be treated including, but not limited to, prostate cancer, colon cancer, breast cancer, lung cancer and epithelial cancer.

In some embodiments, the disease is a neoplastic disease, selected from, but not limited to, leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic, (granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,

lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, and neuroblastomaretinoblastoma. In some embodiments, the disease is an inflammatory disease selected from the group consisting of, but not limited to, eczema, inflammatory bowel disease, rheumatoid arthritis, asthma, psoriasis, ischemia/reperfusion injury, ulcerative colitis and acute respiratory distress syndrome. In some embodiments, the disease is a viral disease selected from the group consisting of, but not limited to, viral disease caused by hepatitis B, hepatitis C, rotavirus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV- II), human T-cell lymphotropic virus type I (HTLV-I), human T-cell lymphotropic virus type II (HTLV-II), AIDS, DNA viruses such as hepatitis type B and hepatitis type C virus;

parvoviruses, such as adeno-associated virus and cytomegalovirus; papovaviruses such as papilloma virus, polyoma viruses, and SV40; adenoviruses; herpes viruses such as herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), and Epstein-Barr virus; poxviruses, such as variola (smallpox) and vaccinia virus; and R A viruses, such as human

immunodeficiency virus type I (HIV -I), human immunodeficiency virus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I), human T-cell lymphotropic virus type II (HTLV-II), influenza virus, measles virus, rabies virus, Sendai virus, picornaviruses such as poliomyelitis virus, coxsackieviruses, rhinoviruses, reoviruses, togaviruses such as rubella virus (German measles) and Semliki forest virus, arboviruses, and hepatitis type A virus.

In some embodiments, the dendrimer is conjugated with one or more anti-cancer agents. In some embodiments, the dendrimer is co-administered with one or more anticancer agents. Examples of anti-cancer agents include, but are not limited to, Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin; Ametantrone Acetate;

Aminoglutethimide; Amsacrine; Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;

Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan;

Cabergoline; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefmgol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine;

Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; Geimcitabine Hydrochloride; Gemtuzumab

Ozogamicin; Gold Au 198; Goserelin Acetate; Guanacone; Hydroxyurea; Idarubicin

Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-lb; Iproplatin; Irinotecan

Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;

Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;

Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Methoxsalen; Metoprine;

Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin;

Mytomycin C; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid;

Nocodazole; Nogalamycin; Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate

Disodium; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer

Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin

Hydrochloride; Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin; Safmgol;

Safmgol Hydrochloride; Samarium/Lexidronam; Semustine; Simtrazene; Sparfosate Sodium;

Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Squamocin;

Squamotacin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin;

Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfm;

Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq;

Tiazofurin; Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene

Citrate; Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Valrubicin;

Vapreotide; Verteporfm; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate;

Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;

Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;

Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxyformycin; 9- aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2-chloro-2'-arabino- fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-

G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine);

cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N-nitrosourea

(MNU); N, N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N'-cyclohex- yl- N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N~ nitrosourea

(MeCCNU); N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nit- rosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C;

AZQ; adozelesin; Cisp latin; Carboplatin; Ormaplatin; Oxaliplatin; CI -973; DWA 2114R;

JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6- Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-amino camptothecin;

Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone;

losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); and 2- chlorodeoxyadenosine (2-Cda). Other anti-cancer agents include, but are not limited to, Antiproliferative agents (e.g., Piritrexim Isothionate), Antiprostatic hypertrophy agent (e.g., Sitogluside), Benign prostatic hyperplasia therapy agents (e.g., Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g., Pentomone), and Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F 18; Fluorodopa F 18; Insulin I 125; Insulin 1 131; Iobenguane I 123; lodipamide Sodium 1 131; Iodoantipyrine 1 131; lodocholesterol 1 131; lodohippurate Sodium I 123; lodohippurate Sodium I 125; lodohippurate Sodium 1 131; Iodopyracet I 125;

Iodopyracet 1 131; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin 1 131;

Iothalamate Sodium I 125; Iothalamate Sodium 1 131; Iotyrosine 1 131; Liothyronine I 125; Liothyronine 1 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg 203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99m Antimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc 99m Disofenin; Technetium Tc 99m Etidronate;

Technetium Tc 99m Exametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate; Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc 99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99m Mertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate; Technetium Tc 99m

Pentetate Calcium Trisodium; Technetium Tc 99m Sestamibi; Technetium Tc 99m

Siboroxime; Technetium Tc 99m Succimer; Technetium Tc 99m sulfur Colloid; Technetium Tc 99m Teboroxime; Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide;

Thyroxine I 125; Thyroxine 1 131; Tolpovidone 1 131; Triolein I 125; and Triolein 1 131.

Additional anti-cancer agents include, but are not limited to anti-cancer

Supplementary Potentiating Agents: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca++ antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);

antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as Cremaphor EL. Still other anticancer agents include, but are not limited to, annonaceous acetogenins; asimicin; rolliniastatin;

guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel; gemcitabine;

methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU; FUDR; FdUMP; Hydroxyurea; Docetaxel; discodermolide; epothilones; vincristine; vinblastine; vinorelbine; meta-pac;

irinotecan; SN-38; 10-OH campto; topotecan; etoposide; adriamycin; fiavopiridol; Cis-Pt; carbo-Pt; bleomycin; mitomycin C; mithramycin; capecitabine; cytarabine; 2-C1- 2'deoxyadenosine; Fludarabine-P04; mitoxantrone; mitozolomide; Pentostatin; and Tomudex. One particularly preferred class of anticancer agents are taxanes (e.g., paclitaxel and docetaxel). Another important category of anticancer agent is annonaceous acetogenin.

In some embodiments, the medical condition and/or disease is pain (e.g., chronic pain, mild pain, recurring pain, severe pain, etc.). In some embodiments, the conjugated

dendrimers of the present invention are configured to deliver pain relief agents to a subject (see, e.g., U.S. Patent Application No. 12/570,977; herein incorporated by reference in its entirety) (e.g., via conjugation with a pain relief agent and/or a pain relief agent antagonist. In some embodiments, the dendrimer conjugates are configured to deliver pain relief agents and pain relief agent antagonists to counter the side effects of pain relief agents (see, e.g., U.S. Patent Application No. 12/570,977; herein incorporated by reference in its entirety). The dendrimer conjugates are not limited to treating a particular type of pain and/or pain resulting from a disease (see, e.g., U.S. Patent Application No. 12/570,977; herein

incorporated by reference in its entirety). Examples include, but are not limited to, pain resulting from trauma (e.g., trauma experienced on a battlefield, trauma experienced in an accident (e.g., car accident)). In some embodiments, the dendrimer conjugates of the present invention are configured such that they are readily cleared from the subject (e.g., so that there is little to no detectable toxicity at efficacious doses) (see, e.g., U.S. Patent Application No. 12/570,977; herein incorporated by reference in its entirety).

In some embodiments, the dendrimer is conjugated with one or more pain relief agents. In some embodiments, the dendrimer is co-administered with one or more pain relief agents. In some embodiments, the pain relief agents include, but are not limited to, analgesic drugs, anxiolytic drugs, anesthetic drugs, antipsychotic drugs, hypnotic drugs, sedative drugs, and muscle relaxant drugs (see, e.g., U.S. Patent Application No. 12/570,977; herein incorporated by reference in its entirety).

In some embodiments, the analgesic drugs include, but are not limited to, nonsteroidal anti-inflammatory drugs, COX-2 inhibitors, and opiates. In some embodiments, the non-steroidal anti-inflammatory drugs are selected from the group consisting of Acetylsalicylic acid (Aspirin), Amoxiprin, Benorylate/Benorilate, Choline magnesium salicylate, Diflunisal, Ethenzamide, Faislamine, Methyl salicylate, Magnesium salicylate, Salicyl salicylate, Salicylamide, arylalkanoic acids, Diclofenac, Aceclofenac, Acemethacin, Alclofenac, Bromfenac, Etodolac, Indometacin, Nabumetone, Oxametacin, Proglumetacin, Sulindac, Tolmetin, 2-arylpropionic acids, Ibuprofen, Alminoprofen, Benoxaprofen,

Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen, Flunoxaprofen,

Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Suprofen, Tiaprofenic acid), N-arylanthranilic acids, Mefenamic acid, Flufenamic acid, Meclofenamic acid, Tolfenamic acid, pyrazolidine derivatives,

Phenylbutazone, Ampyrone, Azapropazone, Clofezone, Kebuzone, Metamizole,

Mofebutazone, Oxyphenbutazone, Phenazone, Sulfinpyrazone, oxicams, Piroxicam,

Droxicam, Lornoxicam, Meloxicam, Tenoxicam, sulphonanilides, nimesulide, licofelone, and omega-3 fatty acids. In some embodiments, the COX-2 inhibitors are selected from the group consisting of Celecoxib, Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib, and Valdecoxib. In some embodiments, the opiate drugs are selected from the group consisting of natural opiates, alkaloids, morphine, codeine, thebaine, semi-synthetic opiates, hydromorphone,

hydrocodone, oxycodone, oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine, dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine, Nalbuphine, Pentazocine, meperidine, diamorphine, ethylmorphine, fully synthetic opioids, fentanyl, pethidine, Oxycodone, Oxymorphone, methadone, tramadol, Butorphanol,

Levorphanol, propoxyphene, endogenous opioid peptides, endorphins, enkephalins, dynorphins, and endomorphins.

In some embodiments, the anxiolytic drugs include, but are not limited to,

benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium),

Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam

(Restoril, Normison, Planum, Tenox, and Temaze, Triazolam, serotonin 1 A agonists, Buspirone (BuSpar), barbituates , amobarbital (Amytal), pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental, Methylphenobarbital, Metharbital, Barbexaclone), hydroxyzine, cannabidiol, valerian, kava (Kava Kava), chamomile, Kratom, Blue Lotus extracts, Sceletium tortuosum (kanna) and bacopa monniera.

In some embodiments, the anesthetic drugs include, but are not limited to, local anesthetics, procaine, amethocaine, cocaine, lidocaine, prilocaine, bupivacaine,

levobupivacaine, ropivacaine, dibucaine, inhaled anesthetics, Desflurane, Enflurane, Halothane, Isoflurane, Nitrous oxide, Sevoflurane, Xenon, intravenous anesthetics,

Barbiturates, amobarbital (Amytal), pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental, Methylphenobarbital, Metharbital, Barbexaclone)), Benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium),

Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam

(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam, Etomidate, Ketamine, and Propofol.

In some embodiments, the antipsychotic drugs include, but are not limited to, butyrophenones, haloperidol, phenothiazines, Chlorpromazine (Thorazine), Fluphenazine (Prolixin), Perphenazine (Trilafon), Prochlorperazine (Compazine), Thioridazine (Mellaril), Trifluoperazine (Stelazine), Mesoridazine, Promazine, Triflupromazine (Vesprin),

Levomepromazine (Nozinan), Promethazine (Phenergan)), thioxanthenes, Chlorprothixene, Flupenthixol (Depixol and Fluanxol), Thiothixene (Navane), Zuclopenthixol (Clopixol & Acuphase)), clozapine, olanzapine, Risperidone (Risperdal), Quetiapine (Seroquel),

Ziprasidone (Geodon), Amisulpride (Solian), Paliperidone (Invega), dopamine, bifeprunox, norclozapine (ACP-104), Aripiprazole (Abilify), Tetrabenazine, and Cannabidiol.

In some embodiments, the hypnotic drugs include, but are not limited to, Barbiturates, Opioids, benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam

(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam, nonbenzodiazepines,

Zolpidem, Zaleplon, Zopiclone, Eszopiclone, antihistamines, Diphenhydramine, Doxylamine, Hydroxyzine, Promethazine, gamma-hydroxybutyric acid (Xyrem), Glutethimide, Chloral hydrate, Ethchlorvynol, Levomepromazine, Chlormethiazole, Melatonin, and Alcohol.

In some embodiments, the sedative drugs include, but are not limited to, barbituates, amobarbital (Amytal), pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental, Methylphenobarbital, Metharbital, Barbexaclone),

benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium),

Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam

(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam, herbal sedatives,

ashwagandha, catnip, kava (Piper methysticum), mandrake, marijuana, valerian, solvent sedatives, chloral hydrate (Noctec), diethyl ether (Ether), ethyl alcohol (alcoholic beverage), methyl trichloride (Chloroform), nonbenzodiazepine sedatives, eszopiclone (Lunesta), zaleplon (Sonata), Zolpidem (Ambien), zopiclone (Imovane, Zimovane)), clomethiazole (clomethiazole), gamma-hydroxybutyrate (GHB), Thalidomide, ethchlorvynol (Placidyl), glutethimide (Doriden), ketamine (Ketalar, Ketaset), methaqualone (Sopor, Quaalude), methyprylon (Noludar), and ramelteon (Rozerem).

In some embodiments, the muscle relaxant drugs include, but are not limited to, depolarizing muscle relaxants, Succinylcholine, short acting non-depolarizing muscle relaxants, Mivacurium, Rapacuronium, intermediate acting non-depolarizing muscle relaxants, Atracurium, Cisatracurium, Rocuronium, Vecuronium, long acting non- depolarizing muscle relaxants, Alcuronium, Doxacurium, Gallamine, Metocurine,

Pancuronium, Pipecuronium, and d-Tubocurarine.

In some embodiments, the dendrimer is conjugated with one or more pain relief agent antagonists. In some embodiments, the dendrimer is co-administered with one or more pain relief agent antagonists (see, e.g., U.S. Patent Application No. 12/570,977; herein

incorporated by reference in its entirety).

In some embodiments, the pain relief agent antagonists include drugs that counter the effect of a pain relief agent (e.g., an anesthetic antagonist, an analgesic antagonist, a mood stabilizer antagonist, a psycholeptic drug antagonist, a psychoanaleptic drug antagonist, a sedative drug antagonist, a muscle relaxant drug antagonist, and a hypnotic drug antagonist). In some embodiments, pain relief agent antagonists include, but are not limited to, a respiratory stimulant, Doxapram, BIMU-8, CX-546, an opiod receptor antagonist, Naloxone, naltrexone, nalorphine, levallorphan, cyprodime, naltrindole, norbinaltorphimine,

buprenorphine, a benzodiazepine antagonist, flumazenil, a non-depolarizing muscle relaxant antagonist, and neostigmine.

Where clinical applications are contemplated, in some embodiments of the present invention, the dendrimer conjugates are prepared as part of a pharmaceutical composition in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. However, in some embodiments of the present invention, a straight dendrimer formulation may be administered using one or more of the routes described herein.

In preferred embodiments, the dendrimer conjugates are used in conjunction with appropriate salts and buffers to render delivery of the compositions in a stable manner to allow for uptake by target cells. Buffers also are employed when the dendrimer conjugates are introduced into a patient. Aqueous compositions comprise an effective amount of the dendrimer conjugates to cells dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Except insofar as any conventional media or agent is incompatible with vectors, cells, or tissues, its use in therapeutic compositions is

contemplated. Supplementary active ingredients may also be incorporated into the compositions.

In some embodiments of the present invention, the active compositions include classic pharmaceutical preparations. Administration of these compositions according to the present invention is via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

The active dendrimer conjugates may also be administered parenterally or

intraperitoneally or intratumorally. Solutions of the active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

In some embodiments, a therapeutic agent is released from dendrimer conjugates within a target cell (e.g., within an endosome). This type of intracellular release (e.g., endosomal disruption of a linker-therapeutic conjugate) is contemplated to provide additional specificity for the compositions and methods of the present invention. The present invention provides dendrimers with multiple (e.g., 100-150) reactive sites for the conjugation of linkers and/or functional groups comprising, but not limited to, therapeutic agents, targeting agents, imaging agents and biological monitoring agents.

The compositions and methods of the present invention are contemplated to be equally effective whether or not the dendrimer conjugates of the present invention comprise a fluorescein (e.g. FITC) imaging agent. Thus, each functional group present in a dendrimer composition is able to work independently of the other functional groups. Thus, the present invention provides dendrimer conjugates that can comprise multiple combinations of targeting, therapeutic, imaging, and biological monitoring functional groups.

The present invention also provides a very effective and specific method of delivering molecules (e.g., therapeutic and imaging functional groups) to the interior of target cells (e.g., cancer cells). Thus, in some embodiments, the present invention provides methods of therapy that comprise or require delivery of molecules into a cell in order to function (e.g., delivery of genetic material such as siR As).

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, dendrimer conjugates are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570- 1580). In some embodiments of the present invention, the active particles or agents are formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so.

Multiple doses may be administered.

Additional formulations that are suitable for other modes of administration include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used.

Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%- 2%. Vaginal suppositories or pessaries are usually globular or oviform and weighing about 5 g each. Vaginal medications are available in a variety of physical forms, e.g., creams, gels or liquids, which depart from the classical concept of suppositories. In addition, suppositories may be used in connection with colon cancer. The dendrimer conjugates also may be formulated as inhalants for the treatment of lung cancer and such like.

In some embodiments, the present invention also provides kits comprising one or more of the reagents and tools necessary to generate a dendrimer conjugated with one or more triazine compositions (e.g., scaffolds) (e.g., triazine compositions capable of click chemistry for use in one-step synthesis of functionalized dendrimers) (e.g., triazine compositions having one or more functional groups), and methods of using such dendrimers.

Examples

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

Previous experiments involving dendrimer related technologies are located in U.S. Patent Nos. 6,471,968, 7,078,461; U.S. Patent Application Serial Nos. 09/940,243, 10/431,682, 11,503,742, 11,661,465, 11/523,509, 12/403,179, 12/106,876, 11/827,637, 10/039,393, 10/254,126, 09/867,924, 12/570,977, and 12/645,081; U.S. Provisional Patent Application Serial Nos.61/256,699, 61/226,993, 61/140,480, 61/091,608, 61/097,780, 61/101,461, 61/251,244, 60/604,321, 60/690,652, 60/707,991, 60/208,728, 60/718,448, 61/035,949, 60/830,237, and 60/925,181; and International Patent Application Nos.

PCT/US2010/051835, PCT/US2010/050893; PCT/US2010/042556, PCT/US2001/015204, PCT/US2005/030278, PCT/US2009/069257, PCT/US2009/036992, PCT/US2009/059071, PCT/US2007/015976, and PCT/US2008/061023, each herein incorporated by reference in their entireties.

Example 2

Synthesis of HO-PAMAM Dendrimers

Synthesis 1. Saccharide-terminated G.3 PAMAM dendrimer

G.3 PAMAM dendrimer (691 mg, 0.10 mmol) was dissolved in 10 mL of DMSO in a 25 mL flask. To the solution was added D-glucoheptono-l,4-lactone (999 mg, 4.80 mmol). The mixture was stirred at room temperature under nitrogen overnight. The mixture was diluted to 50 mL by adding 40 mL of deionized water, and then dialyzed against water (4 x 4 L) with cellulose dialysis membrane (MWCO = 3,500) for 48 hours. The product was dried by lyophilization for 3 days to yield 1300 mg (95.8%) of saccharide-terminated G.3 dendrimer.

Synthesis 2. Conjugation of methotrexate and saccharide-terminated G.3 PAMAM dendrimer

A solution of saccharide-terminated G.3 PAMAM dendrimer (100 mg, 7.37 x 10"3 mmol) and methotrexate (33.48 mg, 7.37 x 10~2 mmol) in dimethyl sulfoxide (15 mL) was stirred at room temperature under nitrogen. To the solution was added 2-chloro-l- methylpyridinium iodide (23.3 mg, 8.84 x 10~2 mmol) and 4-(dimethylamino)pyridine (21.6 mg, 1.77 x 10"1 mmol). The mixture was stirred overnight and then dissolved in deionized water (55 mL). The product was purified by dialysis against PBS buffer (3 x 4L) and then water (3 x 4L) over 48 hours. The final product was dried by lyophilization (3 days) to yield 110 mg (82.4%) of the conjugate. Synthesis 3. Conjugate of methotrexate, folic acid, and saccharide-terminated G.3

PAMAM dendrimer

A solution of saccharide-terminated G.3 PAMAM dendrimer (100 mg, 7.37 x 10"3 mmol), methotrexate (33.48 mg, 7.37 x 10~2 mmol), and folic acid (6.5 mg, 1.47 x 10~2 mmol) in dimethyl sulfoxide (15 mL) was stirred at room temperature under nitrogen. To the solution was added 2-chloro-l-methylpyridinium iodide (28.0 mg, 1.06 x 10"1 mmol) and 4- (dimethylamino)pyridine (26.0 mg, 2.12 x 10"1 mmol). The mixture was stirred overnight and then dissolved in deionized water (55 mL). The product was purified by dialysis against PBS buffer (3 x 4L) and then water (3 x 4L) over 48 hours. The final product was dried by lyophilization (3 days) to yield 80 mg (57.1%) of the conjugate.

Product analysis

Nuclear Magnetic Resonance Spectrometry H NMR)

1H NMR was used to confirm the surface modification of the PAMAM dendrimer with amino-alcohol or saccharide and the attachment of FA and MTX to the HO-PAMAM dendrimer (Figure 6). The 1H NMR spectrum of G.3 PAMAM dendrimer shows 6 broad peaks corresponding to the protons of the amide bond (CONH) at 7.91 ppm, amino groups (NH2), internal methylene groups (CH2) and those CH2 next to amino groups at 3.06, 2.65, 2.56, 2.42, and 2.19 ppm. The 1H NMR spectrum of saccharide-modified G.3 PAMAM dendrimer (G.3-(OH)6) clearly shows many new peaks at 8.09, 7.97, 7.82, 5.56, 4.86, 4.48, 4.43, 3.96 ppm, and multiple overlapped peaks between 3.86 to 3.30 ppm which attributed by saccharide moiety as compared with that of G.3-NH2. Compared with the spectrum of G.3- (OH)6, the spectra of G.3-MTX and G.3-FA-MTX show additional peaks. Some of the peaks overlap with the peaks of G.3-(OH)6. The H-7 of conjugated MTX on G.3-MTX appears at 8.57 ppm. The H-7 peaks of both conjugated FA and MTX are present at 8.63 ppm and 8.57 ppm, respectively. They are sharp and well separated single peaks. The integration ratio of these two peaks represents the molar ratio of MTX and FA on the conjugate of G.3-FA-MTX because both MTX and FA contain only one H-7. Therefore, the actual numbers of FA and MTX conjugated to the surface of the conjugate can be calculated using this integration ratio and molecular weight gain from G.3-(OH)6 to G.3-FA-MTX, as measured by MALDI-TOF or GPC. Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI- TOF)

The average molecular weights of dendrimers and conjugates were evaluated by MALDI-TOF mass spectrometry. Figure 7 shows the MALDI-TOF mass spectra of G.3- NH2, G.3-(OH)6, G.3-MTX5.2 and G.3-FAi.7-MTX4.3. The molecular weight increases for each species along the synthetic pathway was clearly seen, demonstrating that saccharide attachment and conjugation of MTX or FA and MTX to the G.3-(OH)6 occurred. The molecular weight gain of G.3-(OH)6 from G.3-NH2 is due to the attachment of the saccharide group. The average number of saccharide groups attached to G.3-(OH)6 was calculated by the difference of average molecular weight between G.3-(OH)6 and G.3-NH2 divided by the molecular weight of the saccharide group. The molecular weight increase from G.3-(OH)6 to G.3-MTX or G.3-FA-MTX was due to the addition of MTX or both FA and MTX. The number of MTXs attached to the conjugate (G.3-MTX) was calculated by the difference between conjugate and G.3-(OH)6 divided by the molecular mass of MTX minus 18

(MWMTX- 18). The number of FAs and MTXs attached to the conjugate (G.3-FA-MTX) was calculated using formulas (1) and (2), which are based on the molecular weight gain from

G.3-(OH)6 to G.3-FA-MTX, as measured by MALDI-TOF, and the integration ratio of H-7 of conjugated FA and MTX. Calculation results are listed in Table 1.

AMW FFA/(MTX + FA)

NFA = (1)

MWFA - 18

Figure imgf000053_0001

MWMTX -18

Note: NFA: molecules of folic acid attached to saccharide-modified PAMAM dendrimer molecule; NMTX: molecules of MTX attached to saccharide-modified PAMAM dendrimer molecule; AMW: molecular weight gain of conjugate from hydroxyl-terminated PAMAM dendrimer; FFA: 1H NMR integration fraction of H-7 (FA over FA and MTX); FMTX: 1H NMR integration fraction of H-7 (MTX over FA and MTX). MWFA: molecular weight of FA;

MWMTX: molecular weight of MTX. Table 1. Average molecular weight measured by MALDI-TOF and number of MTX and FA attached on conjugates.

Figure imgf000054_0001

High performance liquid chromatography (HPLC)

HPLC was used to evaluate the purity of dendrimers and conjugates. Small molecule impurities such as unreacted FA, MTX, coupling reagent, and by-products were very easily differentiated from the dendrimers and conjugates (G.3-NH2, G.3-(OH)6, G.3-MTX, G.3-FA- MTX) by HPLC due to the significant difference in retention time. Figure 8 shows two representative chromatograms of the conjugates (G.3-MTX5.2 and G.3-FA1.7-MTX4.3) under UV 280 nm which showed a single symmetric peak of the conjugate and no peaks of free FA, MTX and other impurities.

Example 3

Biological studies on the synthesized dendrimer conjugates In vitro binding studies assessed by flow cytometry

FA-receptor (FAR)-specificity for cellular binding of G.3 HO-PAMAM-FA was determined. To analyze this, a competition experiment was performed using G3-FA with a fluorescently labeled (FITC, FI) G.5-dendrimer containing FA as the targeting agent and MTX as the drug (a nanodevice that has previously been shown to bind and internalize into the FAR-expressing KB cells; Thomas et al. (2005) J. Med. Chem. 48:3729-3735; herein incorporated by reference in its entirety). As shown in Figure 9, the binding of the "G5-FI- FA-MTX" was competitively inhibited by the newly synthesized G3-FA, demonstrating FAR-specific binding. The FAR-specific binding of the G3-FA and G3-MTX was further confirmed by down-regulating the FAR using prolonged incubation with high concentrations of these ligands (Figure 10). Cytotoxicity studies by XTT assay

The cytotoxicity of several synthesized G3-MTX conjugates was determined by XTT assay as described previously (Thomas et al. (2005) J. Med. Chem. 48:3729-3735; herein incorporated by reference in its entirety). As shown in Figure 11, all the synthesized G3- MTX conjugates were cytotoxic for the KB cells, with potency equal to or greater than a batch of G5-FA-MTX (Cambrex 123-34) used as a gold-standard reference for monitoring in vitro and in vivo tumor cell cytotoxicity. These results also indicate that the multivalent binding and internalization of the G3-MTX conjugates through the FAR in the absence of the targeting agent FA is sufficient to induce cytotoxicity of FAR-expressing tumor cells

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, in vitro fertilization, development, or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:
1. A composition comprising a dendrimer, wherein one or more terminal branches of said dendrimer comprises two or more OH groups.
2. The composition of claim 1, wherein said dendrimer is a PAMAM dendrimer.
3. The composition of claim 2, wherein said PAMAM dendrimer has a generation between 0 and 3.
4. The composition of claim 1, wherein said one or more terminal branches of said dendrimer comprising two or more OH groups is represented by the following formula:
Figure imgf000056_0001
wherein X is selected from the group consisting of an amino alcohol comprising two or more hydroxyl groups, a saccharide lactone comprising three or more hydroxyl groups, and a saccharide acid comprising three or more hydroxyl groups.
5. The composition of claim 4, wherein said amino alcohol is represented by the following formula:
«V T0H ) m
wherein m is an integer between 2 and 6, wherein R is a chemical group comprising two or more carbon molecules.
6. The composition of claim 5, wherein said chemical group comprising two or more carbon molecules is an alkyl group comprising two or more carbon molecules.
7. The composition of claim 4, wherein said amino alcohol is selected from the group consisting of glucamine or isomers thereof, 1 -amino- 1-deoxy-D-manitol or isomers thereof, glucosamine or isomers thereof, l-amino-2,5-anhydro-l-deoxy-D-mannitol or isomers thereof, tri(hydroxyl)aminomethane or isomers thereof, 3-amino-l,2-propanediol or isomers thereof, and amino-oligo(ethylene glycol).
8. The composition of claim 4, wherein said saccharide lactone comprising three or more hydroxyl groups and said saccharide acid comprising three or more hydroxyl groups is represented by the following formula:
Figure imgf000057_0001
wherein m is an integer equal to or higher than 3, and wherein R is a chemical group comprising two or more carbon molecules.
9. The composition of claim 8, wherein m is between 3 and 10.
10. The composition of claim 8, wherein said saccharide lactone is selected from the group consisting of D-(+)-gluconic acid δ-lactone, D-gulonic acid γ-lactone, L-gulonic acid γ-lactone, D-(+)-glucuronic acid γ-lactone, α,β-glucooctanoic γ-lactone, and D-glucoheptono- 1,4-lactone.
11. The composition of claim 8, wherein said saccharide acid is selected from the group consisting of D-glucuronic acid, D-gluconic acid, and lactobionic acid.
12. The composition of claim 1, wherein said dendrimer is conjugated with one or more functional ligands selected from the group consisting of a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent.
13. The composition of claim 4, wherein said dendrimer is conjugated with one or more functional ligands selected from the group consisting of a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent.
14. The composition of claim 13, wherein said conjugation of said one or more functional ligand with said dendrimer is represented by the following formula: X (Functional Ligand)y
, wherein y is at least 1.
15. The composition of claim 14, wherein y is between 1 and 10.
16. The composition of claims 12 and 13, wherein said therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-oncogenic agents, anti-angiogenic agents, tumor suppressor agents, anti-microbial agents, expression constructs comprising a nucleic acid encoding a therapeutic protein, pain relief agents, pain relief agent antagonists, agents designed to treat arthritis, agents designed to treat inflammatory bowel disease, agents designed to treat an autoimmune disease, and agents designed to treat inflammatory pelvic disease.
17. The composition of claim 16, wherein said chemotherapeutic agent is methotrexate.
18. The composition of claims 12 and 13, wherein said targeting agent is selected from the group consisting of an agent binding a receptor selected from the group consisting of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR; an antibody that binds to a polypeptide selected from the group consisting of p53, Mucl, a mutated version of p53 that is present in breast cancer, HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, and MSA breast carcinoma glycoprotein; an antibody selected from the group consisting of human carcinoma antigen, TP1 and TP3 antigens from osteocarcinoma cells, Thomsen-Friedenreich (TF) antigen from
adenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigen from cystadenocarcinoma, DF3 antigen from human breast carcinoma, and p97 antigen of human melanoma, carcinoma or orosomucoid- related antigen; transferrin; and a synthetic tetanus toxin fragment.
19. The composition of claims 12 and 13, wherein said targeting agent is folic acid.
20. The composition of claims 12 and 13, wherein said trigger agent is configured for a function selected from the group consisting of permitting a delayed release of a functional group from the dendrimer, permitting a constitutive release of the therapeutic agent from the dendrimer, permitting a release of a functional group from the dendrimer under conditions of acidosis, permitting a release of a functional group from a dendrimer under conditions of hypoxia, and permitting a release of the therapeutic agent from a dendrimer in the presence of a brain enzyme.
21. The composition of claims 12 and 13, wherein said imaging agent is selected from the group consisting of fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and cis- parinaric acid.
22. The composition of claim 1, further comprising nanomaterials selected from the group consisting of gold nanoparticles, iron oxide nanoparticles, polymers, silica, albumin, quantum dots, and carbon nanotubes.
23. A method of synthesizing a dendrimer comprising at least one terminal branch comprising two or more hydroxyl groups, comprising:
a) providing a dendrimer comprising at least one ester-terminated terminal branch; b) providing an amino alcohol comprising two or more hydroxyl groups;
c) reacting said ester-terminated terminal branch of said dendrimer with said amino alcohol resulting in the formation of an amide bond, wherein said resulting dendrimer comprises at least one terminal branch comprising two or more hydroxyl groups.
24. The method of claim 23, wherein said dendrimer is a PAMAM dendrimer.
25. The method of claim 23, wherein said amino alcohol is selected from the group consisting of glucamine or isomers thereof, 1 -amino- 1-deoxy-D-manitol or isomers thereof, glucosamine or isomers thereof, l-amino-2,5-anhydro-l-deoxy-D-mannitol or isomers thereof, tri(hydroxyl)aminomethane or isomers thereof, 3-amino-l,2-propanediol or isomers thereof, and amino-oligo(ethylene glycol).
26. The method of claim 23, wherein said ester-terminated dendrimer is of generation N and one half, wherein N is an integer.
27. The method of claim 26, wherein the value of N does not exceed 5.
28. A method of synthesizing a dendrimer comprising at least one terminal branch comprising two or more hydroxyl groups, comprising:
a) providing an NH2-terminated dendrimer;
b) providing a reactant selected from the group consisting of a saccharide lactone and a saccharide acid;
c) reacting said NH2-terminated dendrimer with said saccharide lactone or said saccharide acid resulting in the formation of an amide bond.
29. The method of claim 28, wherein said dendrimer is a PAMAM dendrimer.
30. The method of claim 28, wherein said saccharide lactone is selected from the group consisting of D-(+)-gluconic acid δ-lactone, D-gulonic acid γ-lactone, L-gulonic acid γ- lactone, D-(+)-glucuronic acid γ-lactone, α,β-glucooctanoic γ-lactone, and D-glucoheptono- 1,4-lactone.
31. The method of claim 28, wherein said saccharide acid is selected from the group consisting of D-glucuronic acid, D-gluconic acid, and lactobionic acid.
32. The method of claims 25 or 28, further comprising conjugation with a functional ligand selected from the group consisting of a therapeutic agent, a pro-drug, a targeting agent, a trigger agent, and an imaging agent.
33. The method of claim 32, wherein said therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-oncogenic agents, anti-angiogenic agents, tumor suppressor agents, anti-microbial agents, expression constructs comprising a nucleic acid encoding a therapeutic protein, pain relief agents, pain relief agent antagonists, agents designed to treat arthritis, agents designed to treat inflammatory bowel disease, agents designed to treat an autoimmune disease, and agents designed to treat inflammatory pelvic disease.
34. The method of claim 33, wherein said chemotherapeutic agent is methotrexate.
35. The method of claim 32, wherein said targeting agent is selected from the group consisting of an agent binding a receptor selected from the group consisting of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR; an antibody that binds to a polypeptide selected from the group consisting of p53, Mucl, a mutated version of p53 that is present in breast cancer, HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, and MSA breast carcinoma glycoprotein; an antibody selected from the group consisting of human carcinoma antigen, TP1 and TP3 antigens from osteocarcinoma cells, Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigen from
cystadenocarcinoma, DF3 antigen from human breast carcinoma, and p97 antigen of human melanoma, carcinoma or orosomucoid-related antigen; transferrin; and a synthetic tetanus toxin fragment.
36. The method of claim 32, wherein said targeting agent is folic acid.
37. The method of claim 32, wherein said trigger agent is configured for a function selected from the group consisting of permitting a delayed release of a functional group from the dendrimer, permitting a constitutive release of the therapeutic agent from the dendrimer, permitting a release of a functional group from the dendrimer under conditions of acidosis, permitting a release of a functional group from a dendrimer under conditions of hypoxia, and permitting a release of the therapeutic agent from a dendrimer in the presence of a brain enzyme.
38. The method of claim 32, wherein said imaging agent is selected from the group consisting of fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and cis- parinaric acid.
PCT/US2010/054202 2009-10-30 2010-10-27 Hydroxyl-terminated dendrimers WO2011053618A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US25675909P true 2009-10-30 2009-10-30
US61/256,759 2009-10-30

Publications (2)

Publication Number Publication Date
WO2011053618A2 true WO2011053618A2 (en) 2011-05-05
WO2011053618A3 WO2011053618A3 (en) 2011-09-22

Family

ID=43922967

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/054202 WO2011053618A2 (en) 2009-10-30 2010-10-27 Hydroxyl-terminated dendrimers

Country Status (1)

Country Link
WO (1) WO2011053618A2 (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8252834B2 (en) 2008-03-12 2012-08-28 The Regents Of The University Of Michigan Dendrimer conjugates
US8889635B2 (en) 2008-09-30 2014-11-18 The Regents Of The University Of Michigan Dendrimer conjugates
CN104162175A (en) * 2014-06-18 2014-11-26 东华大学 Functionalized dendrimer-based SPECT-CT bimodal imaging contrast agent and preparation method thereof
US8912323B2 (en) 2009-10-30 2014-12-16 The Regents Of The University Of Michigan Multifunctional small molecules
US8945508B2 (en) 2009-10-13 2015-02-03 The Regents Of The University Of Michigan Dendrimer compositions and methods of synthesis
US9017644B2 (en) 2008-11-07 2015-04-28 The Regents Of The University Of Michigan Methods of treating autoimmune disorders and/or inflammatory disorders
US9402911B2 (en) 2011-12-08 2016-08-02 The Regents Of The University Of Michigan Multifunctional small molecules
CN106796244A (en) * 2014-03-28 2017-05-31 瑟乐斯佩克株式会社 Data acquisition method for determining likelihood that ovarian endometriotic cyst is cancerous, and diagnostic device for same
WO2017075171A3 (en) * 2015-10-27 2017-07-20 The Johns Hopkins University Pamam dendrimer based cest imaging agents and uses thereof
WO2017203437A1 (en) 2016-05-23 2017-11-30 Ineb - Instituto Nacional De Engenharia Biomédica Biodegradable dendritic structure, methods and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020123609A1 (en) * 2000-09-29 2002-09-05 The Regents Of The University Of California Dendrimeric support or carrier macromolecule
US20050008571A1 (en) * 1997-09-23 2005-01-13 George Newkome Detection and functionalization of dendrimers
US20050214247A1 (en) * 2002-04-19 2005-09-29 Sunil Shaunak Glycodendrimers having biological activity
US20080045689A1 (en) * 2004-08-11 2008-02-21 Basf Aktiengesellschaft Patents, Trademarks And Licenses Method for Producing Highly-Branched Polyester Amides
US20090012035A1 (en) * 2007-06-29 2009-01-08 Government Of The United States Of America, Represented By The Secretary, Depa Dendrimer conjugates of agonists and antagonists of the gpcr superfamily

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050008571A1 (en) * 1997-09-23 2005-01-13 George Newkome Detection and functionalization of dendrimers
US20020123609A1 (en) * 2000-09-29 2002-09-05 The Regents Of The University Of California Dendrimeric support or carrier macromolecule
US20050214247A1 (en) * 2002-04-19 2005-09-29 Sunil Shaunak Glycodendrimers having biological activity
US20080045689A1 (en) * 2004-08-11 2008-02-21 Basf Aktiengesellschaft Patents, Trademarks And Licenses Method for Producing Highly-Branched Polyester Amides
US20090012035A1 (en) * 2007-06-29 2009-01-08 Government Of The United States Of America, Represented By The Secretary, Depa Dendrimer conjugates of agonists and antagonists of the gpcr superfamily

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG ET AL.: 'Synthesis and Application of Carbohydrate-Containing Polymers' CHEM. MATER. vol. 14, 2002, pages 3232 - 3244 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8252834B2 (en) 2008-03-12 2012-08-28 The Regents Of The University Of Michigan Dendrimer conjugates
US8445528B2 (en) 2008-03-12 2013-05-21 The Regents Of The University Of Michigan Dendrimer conjugates
US8889635B2 (en) 2008-09-30 2014-11-18 The Regents Of The University Of Michigan Dendrimer conjugates
US8980907B2 (en) 2008-09-30 2015-03-17 The Regents Of The University Of Michigan Dendrimer conjugates
US9017644B2 (en) 2008-11-07 2015-04-28 The Regents Of The University Of Michigan Methods of treating autoimmune disorders and/or inflammatory disorders
US8945508B2 (en) 2009-10-13 2015-02-03 The Regents Of The University Of Michigan Dendrimer compositions and methods of synthesis
US8912323B2 (en) 2009-10-30 2014-12-16 The Regents Of The University Of Michigan Multifunctional small molecules
US9402911B2 (en) 2011-12-08 2016-08-02 The Regents Of The University Of Michigan Multifunctional small molecules
CN106796244A (en) * 2014-03-28 2017-05-31 瑟乐斯佩克株式会社 Data acquisition method for determining likelihood that ovarian endometriotic cyst is cancerous, and diagnostic device for same
CN104162175A (en) * 2014-06-18 2014-11-26 东华大学 Functionalized dendrimer-based SPECT-CT bimodal imaging contrast agent and preparation method thereof
CN104162175B (en) * 2014-06-18 2017-02-01 东华大学 Functionalized spect-ct bimodal imaging contrast agent and preparation method based dendrimers
WO2017075171A3 (en) * 2015-10-27 2017-07-20 The Johns Hopkins University Pamam dendrimer based cest imaging agents and uses thereof
WO2017203437A1 (en) 2016-05-23 2017-11-30 Ineb - Instituto Nacional De Engenharia Biomédica Biodegradable dendritic structure, methods and uses thereof

Also Published As

Publication number Publication date
WO2011053618A3 (en) 2011-09-22

Similar Documents

Publication Publication Date Title
Malkoch et al. Rapid and efficient synthesis of aliphatic ester dendrons and dendrimers
Lee et al. Designing dendrimers for biological applications
Singh et al. Folate and folate− PEG− PAMAM Dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice
Agrawal et al. Glycoconjugated peptide dendrimers-based nanoparticulate system for the delivery of chloroquine phosphate
Khandare et al. Dendrimer versus linear conjugate: influence of polymeric architecture on the delivery and anticancer effect of paclitaxel
Stiriba et al. Dendritic polymers in biomedical applications: from potential to clinical use in diagnostics and therapy
Tekade et al. Dendrimers in oncology: an expanding horizon
Wolinsky et al. Therapeutic and diagnostic applications of dendrimers for cancer treatment
Kurtoglu et al. Drug release characteristics of PAMAM dendrimer–drug conjugates with different linkers
Seow et al. Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles
Caminade et al. Dendrimers for drug delivery
Xiong et al. The therapeutic response to multifunctional polymeric nano-conjugates in the targeted cellular and subcellular delivery of doxorubicin
Ihre et al. Polyester dendritic systems for drug delivery applications: design, synthesis, and characterization
Yamamoto et al. Temperature-related change in the properties relevant to drug delivery of poly (ethylene glycol)–poly (d, l-lactide) block copolymer micelles in aqueous milieu
Hong et al. Interaction of poly (amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport
Crampton et al. Dendrimers as drug delivery vehicles: non‐covalent interactions of bioactive compounds with dendrimers
Kaminskas et al. Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated poly-l-lysine dendrimers
Zhou et al. Endosomal pH-activatable poly (ethylene oxide)-graft-doxorubicin prodrugs: synthesis, drug release, and biodistribution in tumor-bearing mice
Cheng et al. Pharmaceutical applications of dendrimers: promising nanocarriers for drug delivery
Kannan et al. Dynamics of cellular entry and drug delivery by dendritic polymers into human lung epithelial carcinoma cells
Tang et al. Block copolymer micelles with acid-labile ortho ester side-chains: synthesis, characterization, and enhanced drug delivery to human glioma cells
ES2365918T3 (en) Conjugates as polyacetal drug delivery system.
Röglin et al. A Synthetic “Tour de Force”: Well‐Defined Multivalent and Multimodal Dendritic Structures for Biomedical Applications
Botta et al. Relaxivity enhancement in macromolecular and nanosized GdIII‐based MRI contrast agents
Boyd et al. Cationic poly-L-lysine dendrimers: pharmacokinetics, biodistribution, and evidence for metabolism and bioresorption after intravenous administration to rats

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10827414

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10827414

Country of ref document: EP

Kind code of ref document: A2