# Patent

## Title
Multifunctional small molecules

## Abstract

The present invention relates to dendrimer synthesis. Specifically, the present invention relates to dendrimer compositions (e.g., scaffolds) capable of copper-free click conjugation for use in the synthesis of functionalized dendrimers, and methods of use of the same.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 U.S. National Phase Entry of pending International Patent Application No. PCT/US2012/066104, International Filing Date Nov. 20, 2012, which claims priority to U.S. Provisional Patent Application No. 61/568,521, filed Dec. 8, 2011, the contents of which are incorporated by reference in their entireties.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under W911NF-07-1-0437 awarded by the U. S. Army Research Office. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to dendrimer synthesis. Specifically, the present invention relates to dendrimer compositions (e.g., scaffolds) capable of copper-free click conjugation for use in the synthesis of functionalized dendrimers, and methods of use of the same.
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 on 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.
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) 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 with 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. 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 copper-free click conjugation for use in the synthesis of functionalized dendrimers are needed.
SUMMARY OF THE INVENTION
The present invention relates to dendrimer synthesis. Specifically, the present invention relates to dendrimer compositions (e.g., scaffolds) capable of copper-free click conjugation for use in the synthesis of functionalized dendrimers, and methods of use of the same.
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, PAMAM dendrimer, a Baker-Huang PAMAM dendrimer (see, e.g., U.S. Provisional Patent Application No. 61/251,244 and International Patent Application No. PCT/US2010/051835; each herein incorporated by reference in its entirety), a polypropylamine (POPAM) 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 some embodiments, the dendrimer is at least partially acetylated.
In certain preferred embodiments, so as to, for example, improve solubility (e.g., in water and/or methanol) (e.g., due to increased hydrogen bonding) a generation 5 or higher PAMAM dendrimer (e.g., amine-terminated or alkyne-terminated) is used.
The dendrimers used in the embodiments of the present invention 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 via copper-free click chemistry). 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 group. In some embodiments, the ligand is an azide ligand that includes an azido group. 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.
Functionalized dendrimers, such as PAMAM dendrimers conjugated with functional ligands (e.g., therapeutic agents, triggering agents, imaging agents, targeting agents) have many therapeutic and diagnostic applications. However, multi-step conjugation strategies used to attach different functional ligands to the surfaces of nanoparticles (e.g., dendrimers, PAMAM dendrimer branches) introduce higher polydispersity and require multiple processing steps, thereby complicating synthesis and negatively affecting properties such as therapeutic potency, pharmacokinetics, or effectiveness for multivalent targeting.
For example, multifunctional cancer therapeutics have been developed based on Generation 5 PAMAM dendrimers (G5) (see, e.g., Majoros, I. J.; et al., J Med Chem 2005, 48, 5892; Thomas, T. P.; et al., J Med Chem 2005, 48, 3729; Kukowska-Latallo, J. F.; et al., Cancer Res 2005, 65, 5317; Zhang, Y. H.; et al., Bioconjugate Chem 2010, 21, 489; each herein incorporated by reference in their entireties). The targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) were conjugated sequentially through amide- and ester-linkages, respectively (see, e.g., Majoros, I. J.; et al., J Med Chem 2005, 48, 5892; herein incorporated by reference in its entirety). Although this device was shown to bind and selectively kill KB tumor cells that over-express folate receptor (FR) in vitro and in vivo (see, e.g., Thomas, T. P.; et al., J Med Chem 2005, 48, 3729; Kukowska-Latallo, J. F.; et al., Cancer Res 2005, 65, 5317; each herein incorporated by reference in its entireties), the multi-step conjugation strategy that was employed resulted in a mixture of dendrimer populations having varying distributions of the targeting ligands (FA and MTX). The resulting polydispersity was more severe and problematic during large-scale synthesis of the conjugate, which hindered attempts to move into clinical trials. This was because, for example, the polydisperisty of the functionalized dendrimer-based therapeutics likely negatively affected properties such as therapeutic potency, pharmacokinetics, and effectiveness for multivalent targeting (see, e.g., Gillies, E. R.; et al., Drug Discov Today 2005, 10, 35; herein incorporated by reference its entirety). Moreover, multi-step conjugations make batch-to-batch reproducibility extremely challenging, as the characterization required after each step becomes progressively more difficult (see, e.g., Mullen, D. G.; et al., Bioconjugate Chem 2008, 19, 1748; Mullen, D. G.; Acs Nano 2010, 4, 657; each herein incorporated by reference in their entireties).
Triazine scaffolds (e.g., trivalent reagent 2,4,6-trichloro-1,3,5-triazines as a core scaffold) have been used to give access to multifunctional architectures, thereby enabling replacing several chemical entities with a single molecule via Cu(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition (CuAAC) (see, e.g., U.S. Patent Application Publication No. 2010/0158850; 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). The cytotoxicity of copper, however, is a drawback to such techniques as, for example, the copper is difficult to remove from the dendrimer following conjugation (see, e.g., van Dijk, M.; Bioconjug. Chem. 2009, 20, 2001-2016; herein incorporated by reference in its entirety). The present invention overcomes such issues and provides improved methods for the synthesis of well-defined multifunctional dendrimers. In particular, the present invention overcomes this problem through use of copper-free click conjugation.
“Copper-free 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 copper-free Huisgen 1,3-dipolar cycloaddition reaction between a cyclooctyne moeity (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 (see, e.g., Agard, ACS Chemical Biology, 1, 2006, 644-648; Baskin, PNAS 104, 2007, 16793-16797; Ornelas, JACS, 132, 2010, 3923-3931; each herein incorporated by reference in its entirety). ‘Copper-free click chemistry’ is an attractive coupling method because, for example, it can be performed with a wide variety of solvent conditions including aqueous environments. Moreover, copper-free click chemistry avoids copper related cytotoxicity issues found in Cu(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition.
Experiments conducted during the course of developing embodiments for the present invention demonstrated the synthesis of dendrimer compositions (e.g., scaffolds) capable of copper-free click conjugation for use in the synthesis of functionalized dendrimers, and methods of use of the same.
For example, generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer platforms having cyclooctyne ligands subsequently used for a copper-free Huisgen 1,3-dipolar cycloaddition (click reaction) with azido modified functional groups (e.g., methotrexate) was demonstrated. In such experiments, the G5 PAMAM dendrimer was first partially (70%) acetylated and then coupled with 20 cyclooctyne ligands through amide bonds. The remaining primary amine groups on the dendrimer surface were neutralized by acetylation. The platform was then ‘clicked’ with different numbers (5, 10, and 17) of γ-azido functionalized methotrexate. As described in Example 2, Scheme 3 displays a copper-free click reaction of the dendrimer platform with γ-azido-MTX. The copper-free click reactions were stoichiometric with excellent yields. Moreover, G5-MTXn, synthesized via copper-free click chemistry, was demonstrated as a folate receptor targeted drug-conjugate.




Accordingly, the present invention provides dendrimer structures wherein conjugation between a ligand and a functional group or between functional groups is accomplished through use of “copper-free click chemistry”.
In certain embodiments, the dendrimer platform is linked with a cyclooctyne ligand and the functional group (e.g., methotrexate) linked with an azide moiety. Indeed, solubility for functional groups within copper-free click conjugation is hindered through use of a function group/cyclooctyne construct (see, e.g., Ornelas, JACS, 132, 2010, 3923-3931; herein incorporated by reference in its entirety). In addition, in certain preferred embodiments, so as to, for example, improve solubility (e.g., in water and/or methanol) (e.g., due to increased hydrogen bonding) a generation 5 or higher PAMAM dendrimer (e.g., amine-terminated or alkyne-terminated) is used.
The present invention is not limited to particular azide moieties (or equivalent thereof). In some embodiments, the azide moiety comprises the formula N3−. For example, Example 2 and Scheme 2 describes the synthesis of an azide moiety (N3−) conjugated with a methotrexate compound.




The present invention is not limited to particular cyclooctyne moieties (or equivalents thereof). In some embodiments, the cyclooctyne moiety comprises the following formula:




The present invention is not limited to a particular manner of conjugating the azide moieties (or equivalents thereof) and/or the cyclooctyne moieties (or equivalents thereof) wither either a dendrimer structure and/or a functional group. In some embodiments, the azide moieties (or equivalents thereof) and/or the cyclooctyne moieties (or equivalents thereof) are conjugated with either a dendrimer and/or a functional group via a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.). However, as noted, in preferred embodiments, the dendrimer platform is linked with a cyclooctyne ligand and the functional group (e.g., methotrexate) linked with an azide moiety (e.g., as opposed to the dendrimer platform linked with the azide moiety and the functional group linked with the cyclooctyne ligand) (e.g., so as to increase solvent flexibility) (e.g., so as to reduce purification difficulty). As such, in preferred embodiments, the dendrimer platform is linked with the cyclooctyne ligand and the azide moiety linked with the functional group.
In experiments conducted during the development of embodiments for the present invention, cyclooctyne groups were conjugated with dendrimer structures via amide bonds. For example, as described in Example 2, the synthesis of the G5 PAMAM dendrimer with 20 cyclooctyne molecules via amide bonds on the surface is shown in Scheme 1:




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 alkyls.
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 anti-microbial 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 anti-inflammatory drug, an analgesic, an immunomodulator, a glucocorticoid, a TNF-α 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, finfliximab, 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-α 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 FA. 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, Muc1, 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 (SEQ ID NO: 2).
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 nitroheterocycle, 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. Examples of imaging agents include, but are not limited to, fluorescein isothiocyanate (FITC), 6-TAMARA, acridine orange, and cis-parinaric acid.
In certain embodiments, the present invention provides compositions comprising a generation 5 or higher PAMAM dendrimer conjugated with one or more cyclooctyne moieties. In some embodiments, the generation 5 or higher PAMAM dendrimer is conjugated with the one or more cyclooctyne moieties via amide bonds. In some embodiments, the cyclooctyne moieties are selected from the group consisting of




In some embodiments, the cyclooctyne moieties are conjugated with one or more ligands. In some embodiments, the one or more ligands are conjugated with azide moieties. In some embodiments, the cyclooctyne moieties are connected with the azide moeities via copper-free click chemistry. In some embodiments, the one or more ligands are independently selected from the group consisting of a therapeutic agent, a targeting agent, an imaging agent, and a trigger agent. In some embodiments, the one or more ligands are methotrexate and folic acid.

In certain embodiments, the present invention provides methods of synthesizing a functionalized dendrimer, the method comprising providing a generation 5 or higher PAMAM dendrimer having one or more cyclooctyne moieties and a one or more ligands having an azide moiety; and conjugating the generation 5 or higher PAMAM dendrimer with the ligands via copper-free click chemistry between the cyclooctyne moiety and the azide moiety. In some embodiments, the generation 5 or higher PAMAM dendrimer is conjugated with the one or more cyclooctyne moieties via amide bonds. In some embodiments, the one or more ligands are selected from the group consisting of a therapeutic agent, a targeting agent, an imaging agent, and a trigger agent. In some embodiments, the one or more ligands are methotrexate and folic 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows 1H NMR spectra of compound 2 (a), 4 (b), and 5 (c).
FIG. 2 shows 1H NMR of 9 (a), 10 (b), and 11 (c). Insets are the enlarged portion around pteridine ring proton peak at 8.61 ppm.
FIG. 3 shows 1H NMR and MS spectrum of compound 8.
FIG. 4 shows HPLC profile of Compound 8. HPLC analysis was carried out on a Waters Delta 600 HPLC system equipped with a Waters 2996 photodiode array detector, a Waters 717 Plus auto sampler, and a Waters Fraction collector III. The instrument was controlled by Empower 2 software. For analysis of the conjugates, a C5 silica-based RP-HPLC column (250×4.6 mm, 300 A°) connected to a C5 guard column (4×3 mm) was used. The mobile phase for elution of the conjugates was a linear gradient beginning with 100:0 (v/v) water/acetonitrile and ending with 20:80 (v/v) water/acetonitrile over 30 min at a flow rate of 1 mL/min. Trifluoroacetic acid (TFA) at 0.14 wt % concentration in water as well as in acetonitrile was used as a counterion to make the dendrimer surfaces hydrophobic.
FIG. 5 shows HPLC profiles of compounds 9, 10, and 11.
FIG. 6 shows a SPR sensorgram for G5, G5-MTX5 or G5-MTX10. SPR analysis was performed in a Biacore® X instrument (Pharmacia Biosensor AB, Uppsala, Sweden). Folate binding protein (FBP, bovine milk) was immobilized to a CM5 sensor chip where the carboxymethylated dextran-coated layer on the gold chip was conjugated with the protein following a standard amide coupling protocol (see, e.g., Hong, S.; et al., Chem. Biol. 2007, 14, 107-15; Plantinga, A.; et al., ACS Med. Chem. Lett. 2011, 2, 363-67; each herein incorporated by reference in their entireties). The conjugates were injected in HBS-EP buffer, at a flow rate of 20 μL/min (FA, MTX), or 30 μL/min (G5-MTX conjugates).
FIG. 7 shows rate constants and equilibrium dissociation constants (KO for binding of G5-MTXn (n=0, 5, 10) to the folate binding protein on the surface measured by SPR spectroscopy. The dissociation constant




  (
  
    
      K
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determined for each dendrimer conjugate refers to a mean value obtained from multiple independent measurements (n=8) per conjugate. a) An estimate based on the main dendrimer fraction for each conjugate that shows slower dissociation. b) Each dissociation constant





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      D
    
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represents a mean value calculated by averaging the kinetic data obtained from the analysis of eight individual sensorgrams acquired at four different concentrations (5.0 to 0.63 μM; each in duplicate);





  
    
       
      
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where KDmono=2.4×10−5 M (free MTX); d) valency (n)-corrected β value=(β÷n) where n is equal to 5 or 10.

FIG. 8 shows binding of G5-5T-MTX10 onto KB Cells. MTX and the fluorescent dye 5TAMRA (the 5T was conjugated on to the dendrimer using the click chemistry). A. Different concentrations of the conjugate was incubated with KB cells in FA-free medium for 4 h, and the mean FL1 fluorescence of 10,000 cells was determined in an Accuri flow cytometer. B. KB cells were pre-incubated with 15 μM free FA for 30 min, followed by incubation with 300 nM of the conjugate for 4 h in the presence of the FA.
FIG. 9 shows confocal microscopic images demonstrating the internalization of G5-5T-MTX10 in KB Cells. FR-positive KB and FR-negative B16-F10 cells were treated with 300 nM of the conjugate for 20 h and the fluorescence was measured in an Olympus confocal microscope. Some cells were pre-treated with 15 μM free FA prior to addition of the conjugate (upper right panel). The red and blue fluorescence indicates the conjugates and the nuclei stained with DAPI, respectively.
FIG. 10 shows inhibition of DHFR by G5-MTX10. The enzyme assay was performed using a DHFR enzyme assay kit (Sigma), using recombinant human DHFR, in the presence of the substrate DHF and the coenzyme NADPH, as per the vendor's protocol. The decrease in absorbance of NADPH was measured at 340 nm, and the slope of the time kinetics curve obtained was converted enzyme specific activity using 6.22 as the extinction coefficient of 1 mM NADPH solution.
FIG. 11 shows cytotoxicity of G5-MTX10 in FR-expressing KB (A) and RAW (B) cell lines, and in FR-negative B16-F10 cell line (C). The cells were incubated in the presence of different concentrations of the drugs in FA-free medium containing dialyzed serum for 3 days, and cell cytotoxicity was determined by XTT assay. The G5-FA3.3-MTX3.3 is used as a positive control, which was synthesized using a triazine linker with the FA conjugated through an amide bond and the MTX through an ester bond.



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, ayes, 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 hydrolyze 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.
A “hydrolytically 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 hydrolytically 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 “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 characterized. 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.
As used herein, the term “Baker-Huang dendrimer” or “Baker-Huang PAMAM dendrimer” refers to a dendrimer comprised of branching units of structure:




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.

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 “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, conjugation between a dendrimer (e.g., a terminal arm of a dendrimer) and a functional ligand is accomplished during a “one-pot” reaction. 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-1-methylpyridinium iodide and 4-(dimethylamino)pyridine) (see, e.g., International Patent Application No. PCT/US2010/042556, herein incorporated by reference in its entirety).
As used herein, the term “solvent” refers to a medium in which a reaction is conducted. Solvents may be liquid but are not limited to liquid form. Solvent categories include but are not limited to nonpolar, polar, protic, and aprotic.
As used herein, the term “dialysis” refers to a purification method in which the solution surrounding a substance is exchanged over time with another solution. Dialysis is generally performed in liquid phase by placing a sample in a chamber, tubing, or other device with a selectively permeable membrane. In some embodiments, the selectively permeable membrane is cellulose membrane. In some embodiments, dialysis is performed for the purpose of buffer exchange. In some embodiments, dialysis may achieve concentration of the original sample volume. In some embodiments, dialysis may achieve dilution of the original sample volume.
As used herein, the term “precipitation” refers to purification of a substance by causing it to take solid form, usually within a liquid context. Precipitation may then allow collection of the purified substance by physical handling, e.g. centrifugation or filtration.
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-1-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.
DETAILED DESCRIPTION OF THE INVENTION
The poly(amidoamine) (PAMAM) dendrimer has been an attractive drug delivery platform for several decades due to biocompatible, low polydispersity (see, e.g., Tomalia, D. A.; Int. Ed. 1990, 102, 119-157; herein incorporated by reference in its entirety), and the ability to make a wide variety of surface modifications, including direct conjugation to small molecules such as folic acid (see, e.g., Kukowska-Latallo, J. F.; Cancer Res. 2005, 65, 5317-5324; herein incorporated by reference in its entirety), antibodies (see, e.g., Patri, A. K.; Bioconjug. Chem. 2004, 15, 1174-1181; herein incorporated by reference in its entirety), and therapeutic drugs (see, e.g., Majoros, I. J.; Biomacromolecules 2006, 7, 572-579; herein incorporated by reference in its entirety). The synthesis of a functionalized PAMAM dendrimer is generally carried out using a sequential synthesis strategy, with purification and characterization steps required after the conjugation of each type of small molecule (see, e.g., Thomas, T. P.; Biomacromolecules 2008, 9, 603-609; Shukla, R.; Chem. Commun 2005, 5739-5741; each herein incorporated by reference in its entirety). While these processes yield multifunctional PAMAM dendrimer conjugates that are effective both in vitro (see, e.g., Thomas, T. P.; J. Med. Chem. 2005, 48, 3729-3735; herein incorporated by reference in its entirety) and in vivo (see, e.g., Myc, A.; Anticancer Drugs 2010, 21, 186-192; herein incorporated by reference in its entirety), it was determined that it was difficult to consistently replicate the number of small molecules conjugated to the surface of the dendrimer, both within and between batches.
Experiments conducted during the course of developing embodiments for the present invention developed a novel G5 PAMAM platform surface-modified with cyclooctyne groups and then further functionalized with small molecules through copper-free click chemistry. Cu(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition (CuAAC) has been widely used for bioconjugation due to several criteria including high yields, tolerance toward functional groups, and virtually no side-reactions (see, e.g., Meldal, M.; Chem. Rev. 2008, 108, 2952-3015; herein incorporated by reference in its entirety). The cytotoxicity of copper, however, is a potential drawback of the CuAAC reaction for the synthesis and/or functionalization of nanomaterials such as dendrimers for biomedical applications, and the copper is difficult to remove from the dendrimer after conjugation (see, e.g., van Dijk, M.; Bioconjug. Chem. 2009, 20, 2001-2016; herein incorporated by reference in its entirety). The present invention overcomes such issues and provides improved methods for the synthesis of well-defined multifunctional dendrimers. In particular, the present invention overcomes this problem through use of copper-free click conjugation (see, e.g., Sletten, E. M.; J. Am. Chem. Soc. 2010, 132, 11799-11805; herein incorporated by reference in its entirety). Indeed, the unusually high ring strain associated with cyclooctyne allows it to react selectively and rapidly with azide groups without byproducts and without the addition of a copper catalyst (see, e.g., Ornelas, C.; J. Am. Chem. Soc. 2010, 132, 3923-3931; herein incorporated by reference in its entirety).
Experiments conducted during the course of developing embodiments for the present invention demonstrated the synthesis of dendrimer compositions (e.g., scaffolds) capable of copper-free click conjugation for use in the synthesis of functionalized dendrimers, and methods of use of the same. In certain embodiments, the dendrimer platform is linked with a cyclooctyne ligand and the functional group (e.g., methotrexate) linked with an azide moiety (e.g., as opposed to the dendrimer platform linked with the azide moiety and the functional group linked with the cyclooctyne ligand) (e.g., so as to increase solvent flexibility) (e.g., so as to reduce purification difficulty). In certain preferred embodiments, so as to, for example, improve solubility (e.g., in water and/or methanol) (e.g., due to increased hydrogen bonding) a generation 5 or higher PAMAM dendrimer (e.g., amine-terminated or alkyne-terminated) is used. For example, generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer platforms having cyclooctyne ligands subsequently used for a copper-free Huisgen 1,3-dipolar cycloaddition (click reaction) with azido modified functional groups (e.g., methotrexate) was demonstrated. In such experiments, the G5 PAMAM dendrimer was first partially (70%) acetylated and then coupled with 20 cyclooctyne ligands through amide bonds. The remaining primary amine groups on the dendrimer surface were neutralized by acetylation. The platform was then ‘clicked’ with different numbers (5, 10, and 17) of γ-azido functionalized methotrexate. The copper-free click reactions were stoichiometric with excellent yields. Moreover, G5-MTXn, synthesized via copper-free click chemistry, was demonstrated as a folate receptor targeted drug-conjugate. Indeed, as described in Example 2, Scheme 3 displays a copper-free click reaction of the dendrimer platform with γ-azido-MTX.




In some embodiments, the present invention provides compositions facilitating one-step conjugation of functional groups to dendrimers (e.g., terminal arms of dendrimers) via copper-free click chemistry. In some embodiments, such compositions comprise multifunctional small molecule architectures (e.g., scaffolds) which permit conjugation to functional groups (e.g., therapeutic groups, imaging groups, targeting groups, pro-drugs complexes, trigger groups). In some embodiments, such functional group-conjugated compositions are used for one-step conjugation to dendrimers (e.g., to terminal branches of dendrimers or modified dendrimers). In some embodiments, compositions of the present invention comprise dendrimers conjugated with cyclooctyne moieties via amide bonds.
Accordingly, the present invention provides dendrimer structures wherein conjugation between a ligand and a functional group or between functional groups is accomplished through use of “copper-free click chemistry”. “Copper-free 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 copper-free Huisgen 1,3-dipolar cycloaddition reaction between a cyclooctyne moeity (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 (see, e.g., Agard, ACS Chemical Biology, 1, 2006, 644-648; Baskin, PNAS 104, 2007, 16793-16797; Ornelas, JACS, 132, 2010, 3923-3931; each herein incorporated by reference in its entirety). ‘Copper-free click chemistry’ is an attractive coupling method because, for example, it can be performed with a wide variety of solvent conditions including aqueous environments. Moreover, copper-free click chemistry avoids copper related cytotoxicity issues found in Cu(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition.
The present invention is not limited to particular azide moieties (or equivalent thereof). In some embodiments, the azide moiety comprises the formula N3−. For example, Example 2 and Scheme 2 describes the synthesis of an azide moiety (N3−) conjugated with a methotrexate compound.




The present invention is not limited to particular cyclooctyne moieties (or equivalents thereof). In some embodiments, the cyclooctyne moiety comprises the following formula:




The present invention is not limited to a particular manner of conjugating the azide moieties (or equivalents thereof) and/or the cyclooctyne moeities (or equivalents thereof) wither either a dendrimer structure and/or a functional group. In some embodiments, the azide moieties (or equivalents thereof) and/or the cyclooctyne moeities (or equivalents thereof) are conjugated with either a dendrimer and/or a functional group via a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.). However, as noted, in certain embodiments, the dendrimer platform is linked with a cyclooctyne ligand and the functional group (e.g., methotrexate) linked with an azide moiety (e.g., as opposed to the dendrimer platform linked with the azide moiety and the functional group linked with the cyclooctyne ligand) (e.g., so as to increase solvent flexibility) (e.g., so as to reduce purification difficulty). As such, in preferred embodiments, the dendrimer platform is linked with the cyclooctyne ligand and the azide moiety linked with the functional group.
In experiments conducted during the development of embodiments for the present invention, cyclooctyne groups were conjugated with dendrimer structures via amide bonds. For example, as described in Example 2, the synthesis of the G5 PAMAM dendrimer with 20 cyclooctyne molecules via amide bonds on the surface is shown in Scheme 1:




The present invention is not limited to the use of particular types and/or kinds of dendrimers (e.g., a dendrimer conjugated with at least one functional group). 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 application Ser. 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. In some embodiments, the PAMAM dendrimers are “Baker-Huang dendrimers” or “Baker-Huang PAMAM dendrimers” (see, e.g., U.S. Provisional Patent Application No. 61/251,244 and International Patent Application No. PCT/US2010/051835; each herein incorporated by reference in its entirety).
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 describes 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 polycation 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. Left., 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 certain preferred embodiments, so as to, for example, improve solubility (e.g., in water and/or methanol) (e.g., due to increased hydrogen bonding) a generation 5 or higher PAMAM dendrimer (e.g., amine-terminated or alkyne-terminated) is used.
Accordingly, in some embodiments, the present invention provides synthesis methods, compositions and applications for efficient, site-specific delivery using functionalized dendrimers with one or more functional groups (e.g., therapeutic agents, imaging agents, targeting agents, and trigger agents). In particular, in some embodiments, the present invention relates to dendrimers conjugated with a therapeutic agent (e.g., a chemotherapeutic agent) (e.g., via copper-free click chemistry), 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 PAMAM dendrimers conjugated with a therapeutic agent (e.g., a chemotherapeutic agent (e.g., MTX), an imaging agent, and a targeting agent (e.g., FA). 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), and glucocorticoids (e.g., prednisone, methylprednisone), TNF-α 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. Pat. Nos. 6,471,968, 7,078,461, and U.S. patent application Ser. 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 Ser. 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 with the dendrimer via 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 ligand (e.g., 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 a 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 a 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 pO2 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 pro-drugs 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 13-Lactamase (e.g., a 13-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 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, a dendrimer is conjugated (e.g., directly or indirectly (e.g., via copper-free click chemistry)) with a targeting agent. The present invention is not limited to any particular targeting agent. In some embodiments, targeting agents are conjugated to a dendrimer (e.g., directly or indirectly) for delivery 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-α 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) (SEQ ID NO: 2)) (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, a dendrimer is conjugated (e.g., directly or indirectly (e.g., via copper-free click chemistry)) 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 on 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 autofluoresce 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).
In some 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 cis-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 or between functional groups is accomplished through use of copper-free click chemistry. “Copper-free 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 copper-free Huisgen 1,3-dipolar cycloaddition reaction between a cyclooctyne moeity (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. ‘Copper-free click chemistry’ is an attractive coupling method because, for example, it can be performed with a wide variety of solvent conditions including aqueous environments. Moreover, copper-free click chemistry avoids copper related cytotoxicity issues found in Cu(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition.
The present invention is not limited to particular azide moieties (or equivalent thereof). In some embodiments, the azide moiety comprises the formula N3−. For example, Example 2 and Scheme 2 describes the synthesis of an azide moiety (N3−) conjugated with a methotrexate compound.




The present invention is not limited to particular cyclooctyne moieties (or equivalents thereof). In some embodiments, the cyclooctyne moiety comprises the following formula:




The present invention is not limited to a particular manner of conjugating the azide moieties (or equivalents thereof) and/or the cyclooctyne moeities (or equivalents thereof) wither either a dendrimer structure and/or a functional group. In some embodiments, the azide moieties (or equivalents thereof) and/or the cyclooctyne moeities (or equivalents thereof) are conjugated with either a dendrimer and/or a functional group via a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.). In certain embodiments, the dendrimer platform is linked with a cyclooctyne ligand and the functional group (e.g., methotrexate) linked with an azide moiety (e.g., as opposed to the dendrimer platform linked with the azide moiety and the functional group linked with the cyclooctyne ligand) (e.g., so as to increase solvent flexibility) (e.g., so as to reduce purification difficulty). As such, in preferred embodiments, the dendrimer platform is linked with the cyclooctyne ligand and the azide moiety linked with the functional group.
In experiments conducted during the development of embodiments for the present invention, cyclooctyne groups were conjugated with dendrimer structures via amide bonds. For example, as described in Example 2, the synthesis of the G5 PAMAM dendrimer with 20 cyclooctyne molecules via amide bonds on the surface is shown in Scheme 1:




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-1-methylpyridinium iodide and 4-(dimethylamino)pyridine) (see, e.g., International Patent Application No. PCT/US2010/042556, herein incorporated by reference in its entirety).
Functionalized nanoparticles (e.g., dendrimers) often contain moieties (including but not limited to ligands, functional ligands, conjugates, therapeutic agents, targeting agents, imaging agents, fluorophores) that are conjugated to the periphery. Such moieties may for example be conjugated to one or more dendrimer branch termini. Classical multi-step conjugation strategies used during the synthesis of functionalized dendrimers generate a stochastic distribution of products with differing numbers of ligands attached per dendrimer molecule, thereby creating a population of dendrimers with a wide distribution in the numbers of ligands attached. The low structural uniformity of such dendrimer populations negatively affects properties such as therapeutic potency, pharmacokinetics, or effectiveness for multivalent targeting. Difficulties in quantifying and resolving such populations to yield samples with sufficient structural uniformity can pose challenges. However, in some embodiments, use of separation methods (e.g., reverse phase chromatography) customized for optimal separation of dendrimer populations in conjunction with peak fitting analysis methods allows isolation and identification of subpopulations of functionalized dendrimers with high structural uniformity (see, e.g., U.S. Provisional Pat. App. No. 61/237,172; herein incorporated by reference in its entirety). In certain embodiments, such methods and systems provide a dendrimer product made by the process comprising: a) conjugation of at least one ligand type to a dendrimer to yield a population of ligand-conjugated dendrimers; b) separation of the population of ligand-conjugated dendrimers with reverse phase HPLC to result in subpopulations of ligand-conjugated dendrimers indicated by a chromatographic trace; and c) application of peak fitting analysis to the chromatographic trace to identify subpopulations of ligand-conjugated dendrimers wherein the structural uniformity of ligand conjugates per molecule of dendrimer within said subpopulation is, e.g., approximately 80% or more.
The present invention is not limited by the type of therapeutic agent delivered via multifunctional 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, costosternal 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, flat 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 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) 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).
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. 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. The dendrimer conjugates are not limited to treating a particular type of pain and/or pain resulting from a disease. 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).
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 RNA 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 composition is co-administered with an anti-cancer agent (e.g., 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; Cedefingol; 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-n1; Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; 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; Safingol; Safingol 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; Temoporfin; 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; Verteporfin; 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; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; C1-973; 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 I 131; Iobenguane I 123; Iodipamide Sodium I 131; Iodoantipyrine I 131; Iodocholesterol I 131; Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; Iodohippurate Sodium I 131; Iodopyracet I 125; Iodopyracet I 131; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate Sodium I 125; Iothalamate Sodium I 131; Iotyrosine I 131; Liothyronine I 125; Liothyronine I 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 I 131; Tolpovidone I 131; Triolein I 125; and Triolein I 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; flavopiridol; Cis-Pt; carbo-Pt; bleomycin; mitomycin C; mithramycin; capecitabine; cytarabine; 2-C1-2′deoxyadenosine; Fludarabine-PO4; 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 composition is co-administered with a pain relief agent. 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.
In some embodiments, the analgesic drugs include, but are not limited to, non-steroidal 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 lA 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 composition is co-administered with a pain relief agent antagonist. 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” refers 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 siRNAs).
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 functional groups (e.g., via copper-free click chemistry), 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. Pat. Nos. 6,471,968, 7,078,461; U.S. patent application Ser. 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 Ser. 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
The synthesis of the G5 PAMAM dendrimer with 20 cyclooctyne molecules on the surface is shown in Scheme 1.




Briefly, G5 PAMAM dendrimer (112 primary amine groups on surface based on potentiometric titration results) (see, e.g., Mullen, D. G.; ACS Nano 2010, 4, 657-670; herein incorporated by reference in its entirety) was first partially acetylated with acetic anhydride (75 equiv) in methanol in the presence of triethyl amine (TEA). This step enhances the solubility of dendrimer in organic solvents. After purification using a 10 KMW cut-off ultrafiltration membrane, compound 2 yielded a white solid after lyophilization. The cyclooctyne ligand 3 was synthesized by a method from recent reports (see, e.g., Ornelas, C.; J. Am. Chem. Soc. 2010, 132, 3923-3931; Bernardin, A.; Bioconjug. Chem. 2010, 21, 583-588; each herein incorporated by reference in its entirety). Partially acetylated dendrimer 2 and compound 3 (20 equiv) were then dissolved in anhydrous dimethyl sulfoxide (DMSO) with diisopropyl ethylamine (DIPEA). Py-Bop15 reagent in DMSO was added and the reaction mixture was allowed to stir overnight at room temperature. The remaining primary amine groups were neutralized by acetylation to minimize nonspecific binding. Compound 5 was purified via membrane filtration as described above. The overall recovery yield of 5 is 81% (2-5, two steps).
The average number of conjugated molecules per dendrimer was determined by 1H NMR (FIG. 1). Based on a literature method, the number of acetamide groups for compound 2 was calculated to be 72.13 The integration of methyl groups (NHCOCH3) (1.84 ppm) was set to 216 and this peak was used as an internal reference to determine the number of ligands (FIG. 1a). Based on the NMR integration values, 40 primary amine groups remained on the dendrimer surface that could be used to conjugate the cyclooctyne ligand. After the conjugation, the methyne proton signal (4.42 ppm) in the eight-member-ring of compound 3 was well separated from the dendrimer proton peaks in NMR spectrum that allowed integration of this peak and determination of the total cyclooctyne numbers (FIG. 1b). Based on the 1H NMR, a total of 20 cyclooctyne molecules were attached per dendrimer molecule. The remaining amino groups were then completely acetylated to give compound 5. In this case, if the integration of the methyne proton (4.42 ppm) is set to 20, the integration of the acetamide is 270 (FIG. 1 c), which is close to the theoretic number 276 (92 acetamide groups).
MTX is a well-known and extensively characterized antineoplastic agent that is active when conjugated to a dendrimer (see, e.g., Kamal, M. A.; Mol. Cell. Biochem. 1996, 159, 47-53; herein incorporated by reference in its entirety). Using a modified literature procedure, 17 c-azido MTX was synthesized (Scheme 2).




Briefly, 4-amino-4-deoxy-N10-methylpteroic acid (APA) was first reacted with Glu-a-OtBu using BOP15 reagent to give a-OtBu-MTX (see, e.g., Shukla, R.; Chem. Commun. 2005, 5739-5741; herein incorporated by reference in its entirety). Then the c-carboxylic acid was coupled with 3-azido-propyl amine in the presence of HATU (PyBop, bromotripyrrolidinophosphonium hexafluorophosphate; BOP reagent, benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexafluorophosphate). After exposure of a-carboxylic acid functional group with trifluoroacetic acid (TFA), compound 8 was obtained in excellent yield. All compounds were characterized with 1H NMR and electrospray mass spectra. Compound 8 was further analyzed with HPLC.
Using a copper-free click conjugations protocol, compound 8 was then clicked to the newly synthesized cyclooctyne dendrimer platform. (Scheme 3) Because MTX has a much lower affinity toward the alpha folate receptor than folic acid (see, e.g., Genestier, L.; Immunopharmacology 2000, 47, 247-257; herein incorporated by reference in its entirety) different stoichiometries of MTX were conjugated to the platform in order to eventually use this material to test therapeutic effects. This was based on the hypothesis that more MTX molecules on dendrimer surface will enhance the internalization of the nanoparticle because of multivalent binding, thus increases cytotoxicity. Initially, the dendrimer platform 5 was dissolved in methanol (2.0 mM); variable equivalents of c-azido MTX 8 (5, 10, and 18 equiv) in DMSO (40 mM) were added. The reactions were stirred for 24 h at room temperature shielded from light. After the removal of organic solvent, the residues were redissolved in phosphate buffer saline (1_PBS buffer, pH 7.4) and purified by 10 K centrifugal filters (PBS—2, DI water_6) and then lyophilized to give compounds 9, 10, and 11. The recovery yields of 9 and 10 were excellent (>85%). Compound 11 has a relatively poor solubility in water and PBS buffer, which resulted in a low recovery yield (30%).




The average numbers of MTX molecules in each conjugates were determined by 1H NMR (FIG. 2). The aromatic proton in the pteridine ring has a chemical shift at 8.61 ppm and it is well separated from other proton peaks. Comparison of the integration of this peak to the internal acetamide proton integration will determine the numbers of MTX attached. In this case, the integrations of the internal reference peaks were set to 276 which represent 92 acetamide groups. The numbers of MTX are 4.78, 9.88, and 16.87 for compounds 9, 10, and 11, respectively. These results indicate that the copper-free click reactions are highly efficient with >90% of c-azido MTX conjugated. HPLC profiles of these three compounds have more than 98% purity.
The results of this study also point to a number of advantages that the approach of having the cylcooctyne ligand attached to the dendrimer and the azide moiety coupled to methotrexate as compared to the opposite approach (azide moiety on dendrimer and cyclooctyne coupled to methotrexate). For example, some advantages of this approach (e.g., having the cylcooctyne ligand attached to the dendrimer and the azide moiety coupled to methotrexate) include, but are not limited to, improved solubility of reactants, greater flexibility in solvent systems, and reduced material heterogeneity. Poor solubility of the reactants in a dendrimer reaction would also have implications on the quality of mass transport and, in a situation where there is an excess of conjugation sites on the dendrimer relative to the amount of functional group being attached, have a negative effect on the material uniformity. Sub-optimal mass transport has been shown to be dramatically increase material heterogeneity of a dendrimer—functional group system for soluble functional groups that reacted under poor mixing conditions (see, e.g., Mullen D. et al. Macromolecules, 2010, 43, 6577-6587; herein incorporated by reference in its entirety).
Maintaining aqueous solubility of the cyclooctyne on the dendrimer is made possible through the use of a high generation (generation 5 in this example) dendrimer. This is because, for example, the dendrimer backbone has sufficient hydrogen bonding sites at generations 5 and higher to solubilize the cyclooctyne ligand in water or methanol.
Example 3
All 1H NMR spectra were measured on 400 or 500 MHz Varian vnmrs NMR system equipped with a multinuclear 5-mm probe. 1H chemical shifts are reported in parts per million from TMS. Mass spectra were performed on a Waters 1525 mass spectrometer. A thermometer was used without further calibration. Anhydrous solvents were obtained from Acros Organics or Sigma-Aldrich and used as received. Chemicals were purchased from Sigma-Aldrich. G5 PAMAM dendrimer was purchased from Dendritech, Inc. Midland, Mich. APA was purchased from Wako Chemicals, Inc. Richmond, Va. Silica gel 60, particle size 0.040-0.063 mm, 230-400 mesh ASTM was obtained from EM Sciences. Thin layer chromatography (TLC) was performed using Whatman Adsorption plates, 60 Å silica gel, 250 μm layer thickness. Deionized water (18.2MΩ) was made using a Nanopure Infinity DI water system.
G5-(Ac)72 (Compound 2).
G5 PAMAM dendrimer (208 mg, 7.61 μmol) was dissolved in anhydrous MeOH (30 mL). TEA (96.9 μL, 91 equ.) was added. A solution of acetic anhydride (52.6 μL, 75 equ.) in MeOH (8 mL) was added to the above mixture dropwise. After the addition, the reaction was stirred at room temperature over night. Solvent was removed using rotary evaporation. 1×PBS buffer (2.5 mL, pH=7.4) was added to the residue, and the solution was transferred to a 4 mL Amicon centrifugal filter S2 (10K cut-off). After centrifugation at 4,200 rpm for 25 minutes at 13° C., 1×PBS buffer (2.5 mL) was added to re-dissolve the material. This process was repeated two more times with PBS buffer and 6 times of DI water. The product was finally transferred to a small glass vial (5 mL) with DI water (2.0 mL) and lyophilized to give compound 2 as a fluffy white solid (202.3 mg, 87.5%). MALDI-Tof: MW.=33,233.9.
Compound 3 was synthesized according to Bernardin, A., et al., Bioconjug. Chem. 2010, 21, 583-588 (herein incorporated by reference in its entirety). Detected spectra have good agreements with published data.
G5-(Ac)72-(Cyclooctyne)20 (compound 4) Compound 2 (104.8 mg, 3.45 μmol) was dissolved in anhydrous DMSO (3.0 mL). DIPEA (130 μL) was added followed by the addition of compound 3 (12.6 mg, 69.04 μmol, 20 eqi. per dendrimer). PyBop reagent (36.0 mg, 69.04 μmol) in DMSO (2.5 mL) was then added dropwise. The reaction was stirred under argon overnight at room temperature, then diluted with 1×PBS buffer (80 mL) and concentrated with six, 4 mL Amicon centrifugal filters (10K cutoff).
The material was purified using the same method described for compound 2. After lyophilization, compound 4 was obtained as a white fluffy solid (101.4 mg, 89.8%). MALDI-Tof: MW.=34,233.0.
G5-(Ac)92-(Cyclooctyne)20 (compound 5) Compound 4 was (94.6 mg, 2.81 μmol) was dissolved in anhydrous MeOH (12 mL). TEA (30 μL) was added. A solution of acetic anhydride (16 μL, 60 equ.) in MeOH (2.0 mL) was added to the above mixture dropwise. After the addition, the reaction was stirred at room temperature over night. Solvent was removed using rotary evaporation. Compound 5 was purified with method described above as a white fluffy solid (85.8 mg, 90.2%). MALDI-Tof: MW.=34,448.4.
Compound 6 was synthesized according to Nagy, A., et al., Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6373-6376 (herein incorporated by reference in its entirety). Detected spectra were in agreement with published spectra.
Compound 7 Compound 6 (43.8 mg, 85.78 μmol) was dissolved in anhydrous DMF (0.5 mL). 3-azidopropyl amine (17.2 mg, 171.6 μmol) and DIPEA (45 μL) was then added. The mixture was cooled with an ice-water bath. HATU (39.1 mg, 102.9 μmol) was added as a solid. The reaction was stirred at 0° C. for an hour, then was allowed to warm to room temperature and stirred overnight. After solvent removal, the residue was purified on silica, eluting with 1%-10% MeOH/DCM (Rf=0.35) to give 36.3 mg (71.0%) of 7 as a pale white solid. 1H NMR (DMSO-d6, 500 MHz) δ 1.22 (m, 2H), 1.59 (s, 9H), 1.60 (m, 2H), 1.82-2.07 (m, 2H), 2.17 (m, 2H), 3.07 (m, 2H), 4.21 (m, 2H), 3.21 (s, 3H), 4.78 (s, 2H), 6.61 (s, br, 2H), 6.81 (d, 2H, J=9 Hz), 7.71 (d, 2H, J=9 Hz), 7.88 (m, 1H), 8.56 (s, 1H); 13C NMR (DMSO d6, 125 MHz) δ 26.46, 27.70, 28.39, 31.82, 35.84, 48.38, 52.01, 52.90, 54.87, 80.32, 111.07, 120.56, 121.16, 121.58, 128.71, 128.92, 146.99, 149.09, 150.88, 161.60, 162.72, 166.30, 171.57 ppm. MS(EI+) found (M+) 615.2 (M+Na), calc (C27H36N12O4 m/z) 615.3 (M+Na).
Compound 8 Compound 7 (30 mg, 50.6 μmol) was suspended in DCM (1.5 mL) then TFA (1.7 mL) was added. The clear solution was stirred at room temperature for 1.5 hours, after which the color of the solution turned to light pink. The mixture was transferred to two glass centrifugal tubes (20 mL). Diethyl ether (10 mL) was added to each tube slowly. A yellow solid precipitated out. After three times of DCM wash through centrifugation compound 8 was collected as a yellow solid. (25.4 mg, 93.7%) 1H NMR (CD3OD, 500 MHz) δ 1.36 (m, 2H), 1.70 (m, 2H), 2.11-2.28 (m, 2H), 2.36 (m, 2H), 3.20 (m, 2H), 3.29 (s, 3H), 3.42 (m, 2H), 4.26 (m, 1H), 4.93 (s, 2H), 6.86 (d, 2H, J=9 Hz), 7.77 (d, 2H, J=9 Hz), 8.61 (s, 1H); 13C NMR (DMSO-d6, 125 MHz) δ 26.94, 28.83, 32.43, 36.28, 48.82, 52.65, 55.27, 111.58, 121.85, 122.66, 129.39, 146.09, 149.20, 151.10, 151.66, 156.27, 163.17, 166.57, 172.12, 174.25 ppm. MS(EI+) found (M+) 537.2 (M+H), calc (C23H28N1204 m/z) 537.2 (M+H). Analytical HPLC retention time is 19.5 min. with >96% purity.
Example 4
This example describes the general procedure for copper-free click conjugation of dendrimer platform (compound 5) with γ-azido-MTX (compound 8). Dendrimer platform (compound 5) was dissolved in MeOH to make a 2.0 mM solution. γ-azido-MTX (compound 8) in DMSO (40 mM) was added. The copper-free click reaction was stirred at room temperature for 24 hours. Solvent was removed and the residue was re-suspended in 1×PBS buffer (pH=7.4). The solution was transferred to 4 mL Amicon centrifugal filters (10K cut-off). After centrifugation at 4,200 rpm for 25 minutes at 13° C., 1×PBS buffer was added to re-dissolve the material. This process was repeated two more times with PBS buffer and 6 times of DI water. Products were finally transferred to small glass vials (5 mL) with DI water (2.0 mL) and lyophilized to give compound 9, 10, and 11 as pale yellow solids.
Compound 9. Starting from 7.0 mg compound 5, 7.5 mg 9 (95% recovery yield) was obtained. 1HNMR shows there are 4.87 MTX molecules conjugated. MALDI-Tof: MW.=35,625.6. Analytical HPLC retention time is 24.0 min.
Compound 10. Starting from 7.0 mg compound 5, 6.1 mg 10 (85% recovery yield) was obtained. 1HNMR shows there are 9.88 MTX molecules conjugated. MALDI-Tof: MW.=38,807.7.
Analytical HPLC retention time is 23.0 min.
Compound 11. Starting from 6.8 mg compound 5, 2.6 mg 11 (30% recovery yield) was obtained. 1HNMR shows there are 16.87 MTX molecules conjugated. Analytical HPLC retention time is 22.8 min.
Example 5
This example describes in vitro studies for G5-MTXn synthesized via copper-free click chemistry.
G5-MTXn as a Folate Receptor Targeted Drug-Conjugate—In Vitro Studies
Surface Plasmon Resonance (SPR-Based Binding Studies):
The SPR spectroscopy has been previously applied for multivalent ligand-receptor interactions including FA-conjugated G5 PAMAM dendrimers (see, e.g., Hong, S.; et al., Chem. Biol. 2007, 14, 107-15; Arranz-Plaza, E.; et al., J. Am. Chem. Soc. 2002, 124, 13035-46; Adler, P.; et al., J. Biol. Chem. 1995, 270, 5164-71; Gestwicki, J. E.; et al., Anal. Biochem. 2002, 305, 149-55; Rao, J.; et al., J. Am. Chem. Soc. 1999, 121, 2629-30; Tassa, C.; et al., Bioconj. Chem. 2010, 21, 14-19; each herein incorporated by reference in their entireties). Folate binding protein (FBP)-presenting chip was prepared at the surface protein density of 11 ng/mm2 (≈2-3 FBP per 10 nm2) following an EDC-based amide coupling method as described previously (see, e.g., Hong, S.; et al., Chem. Biol. 2007, 14, 107-15; Plantinga, A.; et al., ACS Med. Chem. Lett. 2011, 2, 363-67; each herein incorporated by reference in their entireties). Binding studies of small molecule ligands (FA, MTX), and dendrimer-MTX conjugates (G5-MTXn; n=0, 5, 10) were performed using the FBP-presenting chip.
As shown in FIG. 1, the G5-control dendrimer (G5-MTX0) did not show any significant binding to the FBP (FIG. 1c). In contrast, the G5-MTX5 and G5-MTX10 bound in a concentration-dependent fashion (FIG. 1a, b). These results are indicative of specific adsorption to FBP by the MTX ligand attached to the dendrimer. The G5-MTX conjugates showed markedly slow dissociation, a hall mark for multivalent tight binding as consistently observed in numerous other multivalent systems (see, e.g., Hong, S.; et al., Chem. Biol. 2007, 14, 107-15; Arranz-Plaza, E.; et al., J. Am. Chem. Soc. 2002, 124, 13035-46; Adler, P.; et al., J. Biol. Chem. 1995, 270, 5164-71; each herein incorporated by reference in their entireties). Such a dissociation feature is also in agreement with G5-FA8.2, a multivalent dendrimer comparator presenting the FA ligand (see, e.g., Hong, S.; et al., Chem. Biol. 2007, 14, 107-15; herein incorporated by reference in its entirety).
In an approach to determine the equilibrium dissociation constants (KD) for G5-MTXn on a quantitative basis, the SPR data was analyzed by using the non-linear regression method as established for SPR analysis (see, e.g., Ober, R. J.; et al., Anal. Biochem. 2003, 312, 57-65; herein incorporated by reference in its entirety). This analytical method enabled the SPR data to be fit according to a linear combination of exponential functions, and to extract the estimates for their kinetic rate constants (kon, koff) and KD (FIG. 7). The binding avidity of G5-MTX5 (KD=28 nM) and G5-MTX10 (KD=5.5 nM) is 857 and 4360 times higher than free MTX molecule (KD=24 μM). This observation is fully consistent with the hypothesis that complete dissociation by a multivalent ligand occurs very slowly because all of the ligands tethered to a single multivalent particle have to dissociate simultaneously from multiple receptor sites (see, e.g., Arranz-Plaza, E.; et al., J. Am. Chem. Soc. 2002, 124, 13035-46; Adler, P.; et al., J. Biol. Chem. 1995, 270, 5164-71; Mammen, M.; et al., Angew. Chem., Int. Ed. 1998, 37, 2755; each herein incorporated by reference in its entirety). While not limited to a particular mechanism of understanding, these results indicate that the G5-MTXn conjugates bind much more tightly to the multivalent FR on the surface than a free MTX molecule. It further indicates that it is promising to target FAR-overexpressing cells using an MTX-based dual-acting (targeting agent and drug) multivalent platform.
In Vitro Cell Binding Studies:
In order to test the binding of G5-MTXn on to folate receptor (FR)-expressing cells, a fluorescently-tagged ‘G5-5T-MTX10’ conjugate was synthesized using 5TAMRA (5T) as the fluorescent dye. As shown in FIG. 8, the G5-5T-MTX10 bound to the FR-expressing KB cells in a dose-dependent fashion (A), and the binding was completely blocked in the presence of excess free FA, indicating receptor-mediated binding (B). Flow cytometric analysis also showed that the G5-5T-MTX10 conjugate failed to bind to the FR-negative B16-F10 cells up to 300 nM even after a 20 h incubation period.
The internalization of the conjugate was tested by confocal microscopy (FIG. 9). The G5-5T-MTX internalized into the FR-expressing KB cells, but not into the FR-negative B16 cells. A z-series analysis in KB cells confirmed the cytosolic presence of the conjugate in KB cells.
Dihydrofolate Reductase (DHFR) Enzyme Inhibition Studies:
The efficacy of the G5-MTX10 to inhibit the enzyme DHFR was examined in an in vitro enzymatic assay system. As shown in FIG. 10, the G5-MTX10 significantly inhibited the DHFR at 300 and 1000 nM concentrations.
Cytotoxicity Studies:
The cytotoxicity of the G5-MTX10 was tested in the FR-expressing KB and RAW264.7 cell lines, and in the FR-negative B16-F10 cell line. As given in FIG. 11 (A and B), in the cervical carcinoma KB and the macrophage cell line RAW, the conjugate induced a dose-dependent cytotoxicity with maximal inhibition between 30 and 100 nM, whereas the conjugate was non-cytotoxic in the FR-negative B16-F10 cell line up to 1000 nM (FIG. 11C). Another batch of a synthetic conjugate with 6 MTX (‘G5-MTX6’) also showed cytotoxicity in KB cells with about 15-20% lower potential vs. the G5-MTX10.
These studies show that multivalent G5-MTXn serves as a simple, but potent nanotherapuetic for the treatment of cancer as well as rheumatoid arthritis.
INCORPORATION BY REFERENCE
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

## Claims

1. A composition comprising a generation 5 or higher PAMAM dendrimer conjugated with one or more cyclooctyne moieties.

2. The composition of claim 1, wherein said generation 5 or higher PAMAM dendrimer is conjugated with said one or more cyclooctyne moieties via amide bonds.

3. The composition of claim 1, wherein said cyclooctyne moieties are selected from the group consisting of

4. The composition of claim 1, wherein said cyclooctyne moieties are conjugated with one or more ligands.

5. The composition of claim 4, wherein said one or more ligands are conjugated with azide moieties.

6. The composition of claim 5, wherein said cyclooctyne moieties are connected with said azide moeities via copper-free click chemistry.

7. The composition of claim 4, wherein said one or more ligands are independently selected from the group consisting of a therapeutic agent, a targeting agent, an imaging agent, and a trigger agent.

8. The composition of claim 4, wherein said one or more ligands are methotrexate and folic acid.

9. A method of synthesizing a functionalized dendrimer, said method comprising:
a) providing a generation 5 or higher PAMAM dendrimer having one or more cyclooctyne moieties and a one or more ligands having an azide moiety; and
b) conjugating said generation 5 or higher PAMAM dendrimer with said ligands via copper-free click chemistry between said cyclooctyne moiety and said azide moiety.

10. The method of claim 9, wherein said generation 5 or higher PAMAM dendrimer is conjugated with said one or more cyclooctyne moieties via amide bonds.

11. The method of claim 9, wherein said one or more ligands are selected from the group consisting of a therapeutic agent, a targeting agent, an imaging agent, and a trigger agent.

12. The method of claim 9, wherein said one or more ligands are methotrexate and folic acid.