WO2012058552A1 - Iron stabilized polymer micelles for drug delivery applications - Google Patents

Abstract

The present invention provides polymer micelles for drug delivery applications.

Description

IRON STABILIZED POLYMER MICELLES FOR DRUG DELIVERY APPLICATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional application serial number 61/408,043, filed October 29, 2010, the entirety of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of polymer chemistry and more particularly polymer micelle based drug delivery.

BACKGROUND OF THE INVENTION

[0003] The development of new therapeutic agents has dramatically improved the quality of life and survival rate of patients suffering from a variety of disorders. However, drug delivery innovations are needed to improve the success rate of these treatments. Specifically, delivery systems are still needed which effectively minimize premature excretion and/or metabolism of therapeutic agents and deliver these agents specifically to diseased cells thereby reducing their toxicity to healthy cells.

[0004] Rationally-designed, nanoscopic drug carriers, or "nanovectors," offer a promising approach to achieving these goals due to their inherent ability to overcome many biological barriers. Moreover, their multi-functionality permits the incorporation of cell-targeting groups, diagnostic agents, and a multitude of drugs in a single delivery system. Polymer micelles, formed by the molecular assembly of functional, amphiphilic block copolymers, represent one notable type of multifunctional nanovector.

[0005] Polymer micelles are particularly attractive due to their ability to deliver large payloads of a variety of drugs (e.g. small molecule, proteins, and DNA/RNA therapeutics), their improved in vivo stability as compared to other colloidal carriers (e.g. liposomes), and their nanoscopic size which allows for passive accumulation in diseased tissues, such as solid tumors, by the enhanced permeation and retention (EPR) effect. Using appropriate surface functionality, polymer micelles are further decorated with cell-targeting groups and permeation enhancers that can actively target diseased cells and aid in cellular entry, resulting in improved cell-specific delivery.

[0006] While self assembly represents a convenient method for the bottom-up design of nanovectors, the forces that drive and sustain the assembly of polymer micelles are concentration dependent and inherently reversible. In clinical applications, where polymer micelles are rapidly diluted following administration, this reversibility, along with high concentrations of micelle- destabilizing blood components (e.g. proteins, lipids, and phospholipids), often leads to premature dissociation of the drug-loaded micelle before active or passive targeting is effectively achieved. For polymer micelles to fully reach their cell-targeting potential and exploit their envisioned multi-functionality, in vivo circulation time must be improved. Drug delivery vehicles are needed, which are infinitely stable to post-administration dilution, can avoid biological barriers (e.g. reticuloendothelial system (RES) uptake), and deliver drugs in response to the physiological environment encountered in diseased tissues, such as solid tumors.

BRIEF DESCRIPTON OF THE DRAWINGS

[0007] Figure 1. Stability of Iron (III) Crosslinked Dox micelles.

[0008] Figure 2. Crosslinking optimization with iron (III) chloride.

[0009] Figure 3. Crosslinking kinetics with iron (III) chloride

[0010] Figure 4. pH-Dependent release of iron (III) crosslinked micelles vs. uncrosslinked micelles.

[0011] Figure 5. Comparison of crosslinking using iron (II) vs. iron (III)

[0012] Figure 6. pH Dependent Release of iron (II) vs. iron (III) crosslinked micelles.

[0013] Figure 7. Rat PK of Free DOX compared to uncrosslinked and iron (III) crosslinked micelles.

[0014] Figure 8. pH Dependent release of crosslinked Dox micelles.

[0015] Figure 9. Pulse-treatment cytotoxicity assay for free drug and targeted and untargeted crosslinked Dox micelles in A549 cells.

[0016] Figure 10. Pulse-treatment cytotoxicity assay for free drug and targeted and untargeted crosslinked Dox micelles in 8505C cells.

[0017] Figure 11. 72-hour cytotoxicity assay for free doxorubicin and crosslinked Dox micelles in Caki-1 and MG-63 cells. [0018] Figure 12. Iron (Il)-mediated crosslinking of daunorubucin in water or 20mM Tris buffer.

[0019] Figure 13. pH-dependent release of 2mM or 5mM iron (II) crosslinked and

uncrosslinked daunorubicin micelles.

[0020] Figure 14. Rat pharmacokinetics of free daunorubicin compared to uncrosslinked and iron (II) crosslinked daunorubicin micelles.

[0021] Figure 15. Repeat experiment of Rat pharmacokinetics of free daunorubicin compared to uncrosslinked and iron (II) crosslinked daunorubicin micelles.

[0022] Figure 16. ELISA for rat IgM from Example 55.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

1. General Description:

[0023] Polymer micelles are one type of nanovector formed by the aqueous assembly of block copolymers that are polymer chains containing both hydrophilic and hydrophobic portions. These structures often exist as spherical particles with a core-shell morphology and sub-micron diameter. Their size and structural uniformity impart a striking resemblance to virus particles, which are Nature's version of the perfect delivery system and are capable of highly efficient delivery to cells and tissue. It is believed that the nanoscopic size of viruses (approximately 20 to 400 nanometers in diameter) contributes to their ability to elude the body's natural defense mechanisms while proteins on the virus surface enable highly selective targeting and infection of specific cells. The design of nanovectors, such as block copolymer micelles, that effectively mimic the selectivity and evasiveness of viral particles remains a major goal of drug delivery research. Polymer micelles present a viable alternative due to the inherent modularity of block copolymers, which offer considerable tuning of the micelle size and surface functionality.

[0024] One advantage of the polymer micelle modularity is the ability to tune the core and shell components. This is particularly useful for drug delivery because the core of the assembly can serve as a reservoir for a variety of therapeutic agents while the hydrophilic shell imparts solubility and stability to the aqueous assemblies. From a pharmacokinetic viewpoint, the distribution of drug-loaded micelles is largely determined by the size and surface chemistry of the micelle and not by the drug itself. Thus, polymer micelles possessing a hydrophobic core are utilized for the encapsulation of potent, small molecule drugs that were previously shelved due to poor aqueous solubility. The isolation of hydrophobic chemotherapeutics in the micelle core has also provided new strategies to overcome multi-drug resistance (MDR) mechanisms in cancer cells. Polymer micelles with cationically charged, core-forming blocks are used to encapsulate biomolecules such as plasmid DNA and siRNA. Therapeutics of this type are normally susceptible to rapid in vivo degradation, and their encapsulation in polymer micelles improves their biodistribution profiles thus leading to future clinical successes.

[0025] According to one embodiment, the present invention provides a micelle comprising a multiblock copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly(amino acid block), and a hydrophobic D,L-mixed poly(amino acid) block, characterized in that said micelle has an inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic shell. It will be appreciated that the polymeric hydrophilic block corresponds to the hydrophilic shell, the optionally crosslinkable or crosslinked poly(amino acid block) corresponds to the optionally crosslinked outer core, and the hydrophobic D,L-mixed poly(amino acid) block corresponds to the inner core.

[0026] The "hydrophobic D,L-mixed poly(amino acid)" block, as described herein, consists of a mixture of D and L enantiomers to facilitate the encapsulation of hydrophobic moieties. It is well established that homopolymers and copolymers of amino acids, consisting of a single stereoisomer, may exhibit secondary structures such as the a-helix or β-sheet. See a-Aminoacid- N-Caroboxy-Anhydrides and Related Heterocycles, H.R. Kricheldorf, Springer- Verlag, 1987. For example, poly(L-benzyl glutatmate) typically exhibits an a-helical conformation; however this secondary structure can be disrupted by a change of solvent or temperature (see Advances in Protein Chemistry XVI, P. Urnes and P. Doty, Academic Press, New York 1961). The secondary structure can also be disrupted by the incorporation of structurally dissimilar amino acids such as β-sheet forming amino acids (e.g. proline) or through the incorporation of amino acids with dissimilar stereochemistry (e.g. mixture of D and L stereoisomers), which results in poly(amino acids) with a random coil conformation. See Sakai, R.; Ikeda; S.; Isemura, T. Bull Chem. Soc. Japan 1969, 42, 1332-1336, Paolillo, L.; Temussi, P.A.; Bradbury, E.M.; Crane-Robinson, C. Biopolymers 1972, 11, 2043-2052, and Cho, I.; Kim, J.B.; Jung, H.J. Polymer 2003, 44, 5497- 5500.

[0027] While the methods to influence secondary structure of poly(amino acids) have been known for some time, it has been suprisingly discovered that block copolymers possessing a random coil conformation are particularly useful for the encapsulation of hydrophobic molecules and nanoparticles when compared to similar block copolymers possessing a helical segment. Without wishing to be bound to any particular theory, it is believed that provided block copolymers having a coil-coil conformation allow for efficient packing and loading of hydrophobic moieties within the micelle core, while the steric demands of a rod-coil conformation for a helix-containing block copolymer results in less effective encapsulation.

[0028] One biological barrier to any drug delivery system and another issue which cell- responsive nanovectors addresess is the non-specific uptake by the reticuloendothelial system. The RES consists of a host of cells which are designed to remove cellular debris and foreign particles from the bloodstream. Like viruses, synthetic nanovectors are more apt at escaping RES detection by the nature of their size. In addition, the covalent attachment of poly(ethylene glycol) is a commonly used method to reduce opsonization and non-specific RES uptake of small molecule, protein, and nanoparticulate drug carriers. See Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokin. 2001, 40, 539-551; Bhadra, D.; Bhadra, S.; Jain, P.; Jain, N. K. Pharmazie

2002, 57, 5-29; Shenoy, D. B.; Amiji, M. A. Int. J. Pharm. 2005, 293, 261-270; and Torchilin, V. Adv. Drug. Del. Rev. 2002, 54, 235-252.

[0029] PEG has become a standard choice for the hydrophilic, corona-forming segment of block copolymer micelles, and numerous studies have confirmed its ability to reduce RES uptake of micellar delivery systems. See Kwon, G.; Suwa, S.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Cont. Rel. 1994, 29, 17-23; Caliceti, P.; Veronese, F. M. Adv. Drug Del. Rev.

2003, 55, 1261-1277; Ichikawa, K.; Hikita, T.; Maeda, N.; Takeuchi, Y.; Namba, Y.; Oku, N. Bio. Pharm. Bull. 2004, 27, and 443-444. The ability to tailor PEG chain lengths offers numerous advantages in drug carrier design since studies have shown that circulation times and RES uptake are influenced by the length of the PEG block. In general, longer PEG chains lead to longer circulation times and enhanced stealth properties. In a systematic study of PEG-b- poly(lactic-co-glycolic acid) (PLGA) micelles with PEG molecular weights ranging from 5,000 - 20,000 Da, Langer and coworkers found that micelles coated with 20,000 Da PEG chains were the least susceptible to liver uptake. After 5 hours of circulation, less than 30% of the micelles had accumulated in the liver. See Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603.

[0030] While PEGylation of nanovectors is an effective method to reduce RES uptake and extend in vivo circulation lifetime, other challenges exist which limit the ultimate effectiveness of colloidal drug carriers. One such barrier relates to their self assembly and subsequent in vivo stability. Self assembly represents a convenient, bottom-up approach to nanovector design. The hydrophobic forces that drive the aqueous assembly of colloidal drug carriers, such as polymer micelles and liposomes, are relatively weak, and these assembled structures dissociate below a finite concentration known as the critical micelle concentration ("CMC"). The CMC value of these systems is of great importance in clinical applications since drug-loaded colloidal carriers are diluted in the bloodstream following administration and rapidly reach concentrations below the CMC (μΜ or less). This often leads to premature drug release outside the targeted area, rendering the drug carrier and cell-targeting strategies ineffective.

[0031] In addition to advances in polymer micelle technology, significant efforts have been made in the development of stimuli-responsive polymeric materials that can respond to environmental pH changes. See Chatterjee, J.; Haik, Y.; Chen, C. J. J. App. Polym. Sci. 2004, 91, 3337-3341; Du, J. Z.; Amies, S. P. J. Am. Chem. Soc. 2005, 127, 12800 - 12801; and Twaites, B. R.; de las Heras Alarcon, C; Cunliffe, D.; Lavigne, M.; Pennadam, S.; Smith, J. R.; Gorecki, D. C; Alexander, C. J. Control. Release 2004, 97, 551-566. This is of importance for sensitive protein and nucleic acid-based drugs where escape from acidic intracellular compartments (i.e. endosome and lysosome) and cytoplasmic release are required to achieve therapeutic value. See Murthy, N.; Campbell, J.; Fausto, N.; Hoffman, A. S.; Stayton, P. S. J. Control. Release 2003, 89, 365-374; El-Sayed, M. E. H.; Hoffman, A. S.; Stayton, P. S. J. Control. Release 2005, 104, 417-427; and Liu, Y.; Wenning, L.; Lynch, M.; Reineke, T. J. Am. Chem. Soc. 2004, 126, 7422-7423. Acid-sensitive delivery systems that can successfully escape the endosome and transport small-molecule chemotherapeutic drugs into the cytoplasm are also of interest since these carriers can bypass many of the cellular mechanisms responsible for multidrug resistance. In some of these cases, the polymers are designed to respond to the significant pH gradient between the blood (pH 7.4) and the late-early endosome (pH ~ 5.0 - 6.0).

[0032] In contrast to shell-crosslinked micelles, the crosslinking of multiblock copolymer micelles in accordance with the present invention is accomplished without large dilution volumes because micelle-micelle coupling does not occur. Such crosslinking will enhance post- administration circulation time leading to more efficient passive drug targeting by the EPR effect and improved active targeting using cancer-specific targeting groups. In addition, stimuli- responsive crosslinking may offer another targeting mechanism to isolate the release of the chemotherapy drug exclusively within the tumor tissue and cancer cell cytoplasm.

[0033] To address these pressing issues and develop improved disease-fighting systems, the present application describes drug loaded polymer micelles that possess a crosslinked, or stabilized, outer core. This stabilization technology allows for improved circulation lifetimes in vivo. While we have reported that metal ions can be utilized to stabilize polymer micelles containing a carboxylic acid containing outer core, it was surprisingly found that iron ions perform exceptionally well at performing iron-acetate bonds and stabilizing the micelle to dilution in complex media.

[0034] In certain embodiments, the present invention provides iron-crosslinked micelles that effectively encapsulate therapeutic agents at pH 7.4 (blood) but dissociate and release the drug at acidic pH values ranging from 5.0 (endosomal pH) to 6.8 (extracellular tumor pH).

2. Definitions:

[0035] Compounds of this invention include those described generally above, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5^th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0036] As used herein, the term "multiblock copolymer" refers to a polymer comprising one synthetic polymer portion and two or more poly(amino acid) portions. Such multi-block copolymers include those having the format W-X'-X", wherein W is a synthetic polymer portion and X and X' are poly(amino acid) chains or "amino acid blocks". In certain embodiments, the multiblock copolymers of the present invention are triblock copolymers. As described herein, one or more of the amino acid blocks may be "mixed blocks", meaning that these blocks can contain a mixture of amino acid monomers thereby creating multiblock copolymers of the present invention. In some embodiments, the multiblock copolymers of the present invention comprise a mixed amino acid block and are tetrablock copolymers.

[0037] As used herein, the term "portion" or "block" refers to a repeating polymeric sequence of defined composition. A portion or a block may consist of a single monomer or may be comprise of on or more monomers, resulting in a "mixed block".

[0038] One skilled in the art will recognize that a monomer repeat unit is defined by parentheses around the repeating monomer unit. The number (or letter representing a numerical range) on the lower right of the parentheses represents the number of monomer units that are present in the polymer chain. In the case where only one monomer represents the block (e.g. a homopolymer), the block will be denoted solely by the parentheses. In the case of a mixed block, multiple monomers comprise a single, continuous block. It will be understood that brackets will define a portion or block. For example, one block may consist of four individual monomers, each defined by their own individual set of parentheses and number of repeat units present. All four sets of parentheses will be enclosed by a set of brackets, denoting that all four of these monomers combine in random, or near random, order to comprise the mixed block. For clarity, the randomly mixed block of [BCADDCBADABCDABC] would be represented in shorthand by

[0039] As used herein, the term "triblock copolymer" refers to a polymer comprising one synthetic polymer portion and two poly(amino acid) portions.

[0040] As used herein, the term "tetrablock copolymer" refers to a polymer comprising one synthetic polymer portion and either two poly(amino acid) portions, wherein 1 poly(amino acid) portion is a mixed block or a polymer comprising one synthetic polymer portion and three poly(amino acid) portions.

[0041] As used herein, the term "inner core" as it applies to a micelle of the present invention refers to the center of the micelle formed by the hydrophobic D,L-mixed poly(amino acid) block.. In accordance with the present invention, the inner core is not crosslinked. By way of illustration, in a triblock polymer of the format W-X'-X", as described above, the inner core corresponds to the X" block.

[0042] As used herein, the term "outer core" as it applies to a micelle of the present invention refers to the layer formed by the first poly(amino acid) block. The outer core lies between the inner core and the hydrophilic shell. In accordance with the present invention, the outer core is either crosslinkable or is cross-linked. By way of illustration, in a triblock polymer of the format W-X'-X", as described above, the outer core corresponds to the X' block. It is contemplated that the X' block can be a mixed block.

[0043] As used herein, the terms "drug-loaded" and "encapsulated", and derivatives thereof, are used interchangeably. In accordance with the present invention, a "drug-loaded" micelle refers to a micelle having a drug, or therapeutic agent, situated within the core of the micelle. This is also refered to as a drug, or therapeutic agent, being "encapsulated" within the micelle.

[0044] As used herein, the term "polymeric hydrophilic block" refers to a polymer that is not a poly(amino acid) and is hydrophilic in nature. Such hydrophilic polymers are well known in the art and include polyethyleneoxide (also referred to as polyethylene glycol or PEG), and derivatives thereof, poly(N-vinyl-2-pyrolidone), and derivatives therof, poly(N- isopropylacrylamide), and derivatives thereof, poly(hydroxyethyl acrylate), and derivatives thereof, poly(hydroxylethyl methacrylate), and derivatives thereof, and polymers of N-(2- hydroxypropoyl)methacrylamide (HMPA) and derivatives thereof.

[0045] As used herein, the term "poly(amino acid)" or "amino acid block" refers to a covalently linked amino acid chain wherein each monomer is an amino acid unit. Such amino acid units include natural and unnatural amino acids. In certain embodiments, each amino acid unit of the optionally a crosslinkable or crosslinked poly(amino acid block)is in the L- configuration. Such poly(amino acids) include those having suitably protected functional groups. For example, amino acid monomers may have hydroxyl or amino moieties which are optionally protected by a suitable hydroxyl protecting group or a suitable amine protecting group, as appropriate. Such suitable hydroxyl protecting groups and suitable amine protecting groups are described in more detail herein, infra. As used herein, an amino acid block comprises one or more monomers or a set of two or more monomers. In certain embodiments, an amino acid block comprises one or more monomers such that the overall block is hydrophilic. In still other embodiments, amino acid blocks of the present invention include random amino acid blocks, ie blocks comprising a mixture of amino acid residues.

[0046] As used herein, the term "D,L-mixed poly(amino acid) block" refers to a poly(amino acid) block wherein the poly(amino acid) consists of a mixture of amino acids in both the D- and L-configurations. In certain embodiments, the D,L-mixed poly(amino acid) block is hydrophobic. In other embodiments, the D,L-mixed poly(amino acid) block consists of a mixture of D-configured hydrophobic amino acids and L-configured hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising is hydrophobic.

[0047] Exemplary poly(amino acids) include poly(benzyl glutamate), poly(benzyl aspartate), poly(L-leucine-co-tyrosine), poly(D-leucine-co-tyrosine), poly(L-phenylalanine-co-tyrosine), poly(D-phenylalanine-co-tyrosine), poly(L-leucine-coaspartic acid), poly(D-leucine-co-aspartic acid), poly(L-phenylalanine-co-aspartic acid), poly(D-phenylalanine-co-aspartic acid).

[0048] As used herein, the phrase "natural amino acid side-chain group" refers to the side- chain group of any of the 20 amino acids naturally occuring in proteins. Such natural amino acids include the nonpolar, or hydrophobic amino acids, glycine, alanine, valine, leucine isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine is sometimes classified as nonpolar or hydrophobic and other times as polar. Natural amino acids also include polar, or hydrophilic amino acids, such as tyrosine, serine, threonine, aspartic acid (also known as aspartate, when charged), glutamic acid (also known as glutamate, when charged), asparagine, and glutamine. Certain polar, or hydrophilic, amino acids have charged side-chains. Such charged amino acids include lysine, arginine, and histidine. One of ordinary skill in the art would recognize that protection of a polar or hydrophilic amino acid side-chain can render that amino acid nonpolar. For example, a suitably protected tyrosine hydroxyl group can render that tyroine nonpolar and hydrophobic by virtue of protecting the hydroxyl group.

[0049] As used herein, the phrase "unnatural amino acid side-chain group" refers to amino acids not included in the list of 20 amino acids naturally occuring in proteins, as described above. Such amino acids include the D-isomer of any of the 20 naturally occuring amino acids. Unnatural amino acids also include homoserine, ornithine, and thyroxine. Other unnatural amino acids side-chains are well know to one of ordinary skill in the art and include unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including those that are N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, labelled, and the like.

[0050] As used herein, the term "tacticity" refers to the stereochemistry of the poly(amino acid) hydrophobic block. A poly(amino acid) block consisting of a single stereoisomer (e.g. all L isomer) is referred to as "isotactic". A poly(amino acid) consisting of a random incorporation of D and L amino acid monomers is referred to as an "atactic" polymer. A poly(amino acid) with alternating stereochemistry (e.g. ...DLDLDL...) is referred to as a "syndiotactic" polymer. Polymer tacticity is described in more detail in "Principles of Polymerization", 3rd Ed., G. Odian, John Wiley & Sons, New York: 1991, the entire contents of which are hereby incorporated by reference.

[0051] The term "aliphatic" or "aliphatic group", as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spiro-fused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbon atoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms. In still other embodiments, aliphatic groups contain 1-6 carbon atoms, and in yet other embodiments aliphatic groups contain 1-4 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0052] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as in neutron scattering experiments, as analytical tools or probes in biological assays.

[0053] As used herein, the term "detectable moiety" is used interchangeably with the term "label" and relates to any moiety capable of being detected (e.g., primary labels and secondary labels). A "detectable moiety" or "label" is the radical of a detectable compound.

[0054] "Primary" labels include radioisotope-containing moieties (e.g., moieties that contain

32 33 35 14

P, P, S, or C), mass-tags, and fluorescent labels, and are signal-generating reporter groups which can be detected without further modifications. [0055] Other primary labels include those useful for positron emission tomography including molecules containing radioisotopes (e.g. ¹⁸F) or ligands with bound radioactive metals (e.g. ⁶²Cu). In other embodiments, primary labels are contrast agents for magnetic resonance imaging such as gadolinium, gadolinium chelates, or iron oxide (e.g Fe₃0₄ and Fe₂0₃) particles. Similarly, semiconducting nanoparticles (e.g. cadmium selenide, cadmium sulfide, cadmium telluride) are useful as fluorescent labels. Other metal nanoparticles (e.g colloidal gold) also serve as primary labels.

[0056] Unless otherwise indicated, radioisotope-containing moieties are optionally substituted hydrocarbon groups that contain at least one radioisotope. Unless otherwise indicated, radioisotope-containing moieties contain from 1-40 carbon atoms and one radioisotope. In certain embodiments, radioisotope-containing moieties contain from 1-20 carbon atoms and one radioisotope.

[0057] The terms "fluorescent label", "fluorescent group", "fluorescent compound", "fluorescent dye", and "fluorophore", as used herein, refer to compounds or moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength. Examples of fluorescent compounds include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-Tetra- bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X. 3. Description of Exemplary Embodiments:

A . Drug Loaded Polymer Micelles

[0058] As described generally above, one embodiment of the present invention provides a drug loaded micelle comprised of a multiblock copolymer, consisting of a polymeric hydrophilic block, a crosslinkable poly(amino acid block) , and a hydrophobic D,L-mixed poly(amino acid) block, and a hydrophobic therapeutic agent; characterized in that said micelle has an inner core, a crosslinkable outer core, and a hydrophilic shell wherein the therapeutic agent resides in the hydrophobic inner core.

[0059] In certain embodiments, the present invention provides a stabilized, therapeutic agent loaded micelle comprised of a multiblock copolymer, consisting of a polymeric hydrophilic block, a crosslinkable poly(amino acid block) , and a hydrophobic D,L-mixed poly (amino acid) block, and a hydrophobic therapeutic agent wherein the polymer micelle is represented by Formula I:

wherein:

each n is independently 45-450;

each s is independently 1 or 2

each w is independently 0-30;

each x is independently 1-30; each y is independently 5-80;

each R_y is independently a mixture of 1 or more moieties that represent a natural or unnatural amino acid such that the overall mixture is hydrophobic; and

each R¹ is independently -N₃ -OCH₃ or wherein T is a targeting group moiety.

[0060] As defined generally above, each n group of formula I is independently 110-450. In certain embodiments, the present invention provides compounds of formula I, as described above, wherein n is about 225. In other embodiments, n is about 270. In other embodiments, n is about 350. In other embodiments, n is about 110. In other embodiments, n is about 450. In other embodiments, n is selected from 110 ± 10, 180 ± 10, 225 ± 10, 275 ± 10, 315 ± 10, or 450 ± 10.

[0061] In certain embodiments, each w group of formula I is independently about 0 to about 30. In certain embodiments, the w group of formula I is about 10. In other embodiments, w is about 20. In some embodiments, w is 0. In yet other embodiments, w is a range from 0 to 10. According to yet another embodiment, w is about 15. In other embodiments, w is about 5. In other embodiments, w is selected from 5 ± 3, 10 ± 3, 10 ± 5, 15 ± 5, or 20 ± 5.

[0062] In certain embodiments, each x group of formula I is independently about 1 to about 30. In certain embodiments, the x group of formula I is about 10. In other embodiments, x is about 20. According to yet another embodiment, x is about 15. In other embodiments, x is about 5. In other embodiments, x is selected from 3 ± 2, 5 ± 3, 10 ± 3, 10 ± 5, 15 ± 5, or 20 ± 5.

[0063] In certain embodiments, each y group of formula I is independently about 5 to about 80. In certain embodiments, the y group of formula I is about 20. In other embodiments, y is about 30. According to yet another embodiment, y is about 40. In other embodiments, y is about 60. In other embodiments, y is selected from 20 ± 5, 30 ± 5, 40 ± 5, 50 ± 5, or 60 ± 5.

[0064] As defined generally above, each R_y group of formula I is independently a mixture of 1 or more moieties that represent a natural or unnatural amino acid such that the overall mixture is hydrophobic. Suitable moieties include, but are not limited to those in Table 1 below. Table 1.

a b c d e

[0065] In some embodiments, each T targeting group moiety of formula I is independently a moiety selected from folate, a Her-2 binding peptide, a urokinase-type plasminogen activator receptor (uPAR) antagonist, a CXCR4 chemokine receptor antagonist, a GRP78 peptide antagonist, an RGD peptide, an RGD cyclic peptide, a luteinizing hormone -releasing hormone (LHRH) antagonist peptide, an aminopeptidase targeting peptide, a brain homing peptide, a kidney homing peptide, a heart homing peptide, a gut homing peptide, an integrin homing peptide, an angiogencid tumor endothelium homing peptide, an ovary homing peptide, a uterus homing peptide, a sperm homing peptide, a microglia homing peptide, a synovium homing peptide, a urothelium homing peptide, a prostate homing peptide, a lung homing peptide, a skin homing peptide, a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphona homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide. Such targeting groups are well knon in the art and are described in detail in WO 2008/134731. [0066] In some embodiments, the T targeting group is a moiety selected from a tumor homing group, a prostate specific membrane antigen homing peptide, an aminopeptidate N homing peptide, a Her-2 homing peptide, a breast cancer homing peptide, a VEGFRl homing peptide, or a CXCR4 homing peptide.

[0067] In certain embodiments, the present invention provides a stabilized, therapeutic agent loaded micelle comprised of a multiblock copolymer, consisting of a polymeric hydrophilic block, a crosslinkable poly(amino acid block) , and a hydrophobic D,L-mixed poly (amino acid) block, and a hydrophobic therapeutic agent wherein the polymer micelle is represented by Formula II:

each n is independently 45-450;

each w is independently 0-30;

each x is independently 1-30;

each y is independently 5-40;

each z is independently 5-40; and each R l is independently -N₃ -OCH₃ or ^{1 N}—^-N wherein T is a targeting group moiety. [0068] As defined generally above, each n group of formula II is independently 110-450. In certain embodiments, the present invention provides compounds of formula II, as described above, wherein n is about 225. In other embodiments, n is about 270. In other embodiments, n is about 350. In other embodiments, n is about 110. In other embodiments, n is about 450. In other embodiments, n is selected from 110 ± 10, 180 ± 10, 225 ± 10, 275 ± 10, 315 ± 10, or 450 ± 10.

[0069] In certain embodiments, each w group of formula II is independently about 0 to about 30. In certain embodiments, the w group of formula II is about 10. In other embodiments, w is about 20. In some embodiments, w is 0. In yet other embodiments, w is a range from 0 to 10. According to yet another embodiment, w is about 15. In other embodiments, w is about 5. In other embodiments, w is selected from 5 ± 3, 10 ± 3, 10 ± 5, 15 ± 5, or 20 ± 5.

[0070] In certain embodiments, each x group of formula II is independently about 1 to about 30. In certain embodiments, the x group of formula II is about 10. In other embodiments, x is about 20. According to yet another embodiment, x is about 15. In other embodiments, x is about 5. In other embodiments, x is selected from 3 ± 2, 5 ± 3, 10 ± 3, 10 ± 5, 15 ± 5, or 20 ± 5.

[0071] In certain embodiments, the y group of formula II is about 5 to about 40. In certain embodiments, the y group of formula II is about 10. In other embodiments, y is about 20. According to yet another embodiment, y is about 15. In other embodiments, y is about 30. In other embodiments, y is selected from 10 ± 3, 15 ± 3, 17 ± 3, 20 ± 5, or 30 ± 5.

[0072] In certain embodiments, the z group of formula II is about 5 to about 40. In certain embodiments, the z group of formula II is about 10. In other embodiments, z is about 20. According to yet another embodiment, z is about 15. In other embodiments, z is about 30. In other embodiments, z is selected from 10 ± 3, 15 ± 3, 17 ± 3, 20 ± 5, or 30 ± 5.

[0073] In some embodiments, each T targeting group moiety of formula I is independently a moiety selected from folate, a Her-2 binding peptide, a urokinase-type plasminogen activator receptor (uPAR) antagonist, a CXCR4 chemokine receptor antagonist, a GRP78 peptide antagonist, an RGD peptide, an RGD cyclic peptide, a luteinizing hormone -releasing hormone (LHRH) antagonist peptide, an aminopeptidase targeting peptide, a brain homing peptide, a kidney homing peptide, a heart homing peptide, a gut homing peptide, an integrin homing peptide, an angiogencid tumor endothelium homing peptide, an ovary homing peptide, a uterus homing peptide, a sperm homing peptide, a microglia homing peptide, a synovium homing peptide, a urothelium homing peptide, a prostate homing peptide, a lung homing peptide, a skin homing peptide, a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphona homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide. Such targeting groups are well knon in the art and are described in detail in WO 2008/134731.

[0074] In some embodiments, the T targeting group is a moiety selected from a tumor homing group, a prostate specific membrane antigen homing peptide, an aminopeptidate N homing peptide, a Her-2 homing peptide, a breast cancer homing peptide, a VEGFR1 homing peptide, or a CXCR4 homing peptide.

[0075] In certain embodiments, the hydrophobic therapeutic agent is paclitaxel. In other embodiments, the hydrophobic therapeutic agent is docetaxel. In certain embodiments, the hydrophobic therapeutic agent is vinorelbine. In certain embodiments, the hydrophobic therapeutic agent is letrozole. In other embodiments, the hydrophobic therapeutic agent is etoposide. In yet another embodiment, the hydrophobic therapeutic agent is cabazitaxel. In another embodiement, the hydrophobic agent is an indenoisoquinoline. In another embodiement, the hydrophobic agent is vitamin E succinate. In another embodiement, the hydrophobic agent is a taxane. In another embodiement, the therapeutic agent is an anthracycline.

4. General Methods for Providing Compounds of the Present Invention

[0076] Multiblock copolymers of the present invention are prepared by methods known to one of ordinary skill in the art and those described in detail in United States patent application serial number 11/325,020 filed January 4, 2006 and published as US 20060172914 on August 3, 2006, the entirety of which is hereby incorporated herein by reference. Generally, such multiblock copolymers are prepared by sequentially polymerizing one or more cyclic amino acid monomers onto a hydrophilic polymer having a terminal amine salt wherein said polymerization is initiated by said amine salt. In certain embodiments, said polymerization occurs by ring- opening polymerization of the cyclic amino acid monomers. In other embodiments, the cyclic amino acid monomer is an amino acid NCA, lactam, or imide. Details of preparing exemplary multiblock copolymers of the present invention are set forth in the Examplification, infra. [0077] Methods of preparing micelles are known to one of ordinary skill in the art. Micelles can be prepared by a number of different dissolution methods. In the direct dissolution method, the block copolymer is added directly to an aqueous medium with or without heating and micelles are spontaneously formed up dissolution. The dialysis method is often used when micelles are formed from poorly aqueous soluble copolymers. The copolymer is dissolved in a water miscible organic solvent such as N-methyl pyrollidinone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dimethylacetamide, and this solution is then dialyzed against water or another aqueous medium. During dialysis, micelle formation is induced and the organic solvent is removed. Alternatively, the block copolymer can be dissolved in in a water miscible organic solvent such as N-methyl pyrollidinone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dimethylacetamide and added dropwise to water or another aqueous medium. The micelles can then be isolated by filtration or lyophilization.

[0078] Emulsification methods can also be employed for micelle formation. For example, the block copolymer is dissolved in a water-immiscible, volatile solvent (e.g. dichloromethane) and added to water with vigorous agitation. As the solvent is removed by evaporation, micelles spontaneously form. Prepared micelles can then be filtered and isolated by lyophilization.

[0079] Micelles can be prepared by a number of different dissolution methods. In the direct dissolution method, the block copolymer is added directly to an aqueous medium, with or without heating, and micelles are spontaneously formed up dissolution. The dialysis method is often used when micelles are formed from poorly aqueous soluble copolymers. The copolymer is dissolved in a water miscible organic solvent such as N-methyl pyrollidinone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dimethylacetamide, and this solution is then dialyzed against water or another aqueous medium. During dialysis, micelle formation is induced and the organic solvent is removed. Alternatively, the block copolymer can be dissolved in in a water miscible organic solvent such as N-methyl pyrollidinone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dimethylacetamide and added dropwise to water or another aqueous medium. The micelles can then be isolated by filtration or lyophilization.

[0080] Crosslinking reactions designed for drug delivery preferably meet a certain set of requirements to be deemed safe and useful for in vivo applications. For example, in other embodiments, the crosslinking reaction would utilize non-cytotoxic reagents, would be insensitive to water, would not alter the drug to be delivered, and in the case of cancer therapy, would be reversible at pH levels commonly encountered in tumor tissue (pH ~ 6.8) or acidic organelles in cancer cells (pH - 5.0 - 6.0).

[0081] One embodiment of the present invention utilizes the iron-mediated coupling of carboxylic acids to crosslink the micelle together. The interaction between iron and carboxylic acids in biological systems is well known in the art. See Silver, "Chemistry of Iron" 1993. Without wishing to be bound to any particular theory, it is believed that the carboxylic acid will function as a ligand in the carboxylate form (i.e. high pH) but will readily disassociate when the proton ion concentration is sufficiently high (i.e. low pH). (Scheme 3) In some embodiments, the iron moiety is Fe²⁺. In some embodiments, the iron moiety is Fe³⁺.

Scheme 1

[0082] In one embodiment, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by addition of iron (II) chloride to the micelle solution. In another embodiment, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by addition of iron (III) chloride to the micelle. In certain embodiments, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by dissolving the micelles in TRIS buffer solution containing iron (II) chloride. In yet other embodiments, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by dissolving the micelles in TRIS buffer solution containing iron (III) chloride.

[0083] In certain embodiments, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by addition of iron (II) chloride to the micelle solution, followed by adjustment of the pH to 7-8. In other embodiments, drug-loaded micelles possessing carboxylic acid functionality in the outer core are crosslinked by addition of iron (III) chloride to the micelle solution, followed by adjustment of the pH to 7-8. 5. Uses, Methods, and Compositions

[0084] As described herein, micelles of the present invention having a drug encapsulated therein are useful for treating cancer. According to one embodiment, the present invention relates to the treatment of colorectal cancer. In another embodiement, the present invention relates to the treatment of pancreatic cancer. According to another embodiment, the present invention relates to a method of treating breast cancer. In another embodiement, the present invention relates to the treatment of prostate cancer. According to another embodiment, the present invention relates to a method of treating a cancer selected from ovary, cervix, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidney carcinoma, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, large intestine, rectum, brain and central nervous system, and leukemia, comprising administering a micelle in accordance with the present invention having an drug encapsulated therein.

[0085] P-glycoprotein (Pgp, also called multidrug resistance protein) is found in the plasma membrane of higher eukaryotes where it is responsible for ATP hydrolysis-driven export of hydrophobic molecules. In animals, Pgp plays an important role in excretion of and protection from environmental toxins; when expressed in the plasma membrane of cancer cells, it can lead to failure of chemotherapy by preventing the hydrophobic chemotherapeutic drugs from reaching their targets inside cells. Indeed, Pgp is known to transport hydrophobic chemotherapeutic drugs out of tumor cells. According to one aspect, the present invention provides a method for delivering a an drug to a cancer cell while preventing, or lessening, Pgp excretion of that chemotherapeutic drug, comprising administering a drug-loaded micelle comprising a multiblock polymer of the present invention loaded with an drug.

Compositions

[0086] According to another embodiment, the invention provides a composition comprising a micelle of this invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In certain embodiments, the composition of this invention is formulated for administration to a patient in need of such composition. In other embodiments, the composition of this invention is formulated for oral administration to a patient.

[0087] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0088] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0089] Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

[0090] Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and N⁺(Ci_₄ alkyl)₄ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0091] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0092] For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0093] The pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. In certain embodiments, pharmaceutically acceptable compositions of the present invention are enterically coated.

[0094] Alternatively, the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0095] The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0096] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

[0097] For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0098] For ophthalmic use, the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum. [0099] The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

[00100] In certain embodiments, the pharmaceutically acceptable compositions of this invention are formulated for oral administration.

[00101] The amount of the compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, the compositions should be formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of the drug can be administered to a patient receiving these compositions.

[00102] It will be appreciated that dosages typically employed for the encapsulated drug are contemplated by the present invention. In certain embodiments, a patient is administered a drug- loaded micelle of the present invention wherein the dosage of the drug is equivalent to what is typically administered for that drug. In other embodiments, a patient is administered a drug- loaded micelle of the present invention wherein the dosage of the drug is lower than is typically administered for that drug.

[00103] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition.

[00104] In order that the invention described herein may be more fully understood, the following examples are set forth. It will be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. EXEMPLIFICATION

Preparation of multiblock copolymers and drug loaded micelles of the Present Invention

[00105] As described generally above, multiblock copolymers of the present invention are prepared using the heterobifunctional PEGs described herein and in United States patent application serial number 11/256,735, filed October 24, 2005, published as WO2006/047419 on May 4, 2006 and published as US 20060142506 on June 29, 2006, the entirety of which is hereby incorporated herein by reference. The preparation of multiblock polymers in accordance with the present invention is accomplished by methods known in the art, including those described in detail in United States patent application serial number 11/325,020, filed January 4, 2006, published as WO2006/74202 on July 13, 2006 and published as US 20060172914 on August 3, 2006, the entirety of which is hereby incorporated herein by reference.

[00106] In each of the Examples below, where an amino acid, or corresponding NCA, is designated "D", then that amino acid, or corresponding NCA, is of the D-configuration. Where no such designation is recited, then that amino acid, or corresponding NCA, is of the L- configuration.

[00107] Particle size distribution was determined by dynamic light scattering. Lyopholyzed polymers were dissolved at 5 mg/mL in phosphate buffered saline at pH 7.4 and equilibrated overnight. Each sample was analyzed in a PSS NICOMP 380 with a 690 nm laser at a 90 degree angle or in a Wyatt Dynapro with a 658 nm laser. DLS sizing data was recorded from the volume weighted Gaussian distribution (Nicomp) or Regularization fit (DynaPro).

Example 1

[00108] Dibenzylamino Ethanol Benzyl chloride (278.5g, 2.2 mol), ethanol amine (60 mL, 1 mol), potassium carbonate (283. lg, 2.05mol) and ethanol (2 L) were mixed together in a 3L 3- neck flask, fitted with an overhead stirrer, a condenser and a glass plug. The apparatus was heated up to reflux for 36 hr, after which the insoluble solid was filtered through a medium frit. The filtrate was recovered and ethanol was removed by rotoary evaporation. The viscous liquid was redissolved in ether, the solid suspension removed by filtration and extracted twice against water. The ether solution was kept and the aqueous layer was extracted twice with dichloromethane (2 x 400 mL). The fraction were recombined, dried over MgSC^, stirred over carbon black for 15 min and filtered through a celite pad. Dichloromethane was removed and the solid was redissolved into a minimal amount of ether (combined volume of 300 mL with the first ether fraction, 300 mL). Hexanes ( 1700 mL) was added and the solution was heated up gently till complete dissolution of the product. The solution was then cooled down gently, placed in the fridge (+ 4°C) overnight and white crystals were obtained. The recrystallization was done a second time. 166.63g, 69% yield. 1H NMR (d₆-DMSO) δ 7.39-7.24 (10H), 4.42 (1H), 3.60 (4H), 3.52 (2H), 2.52 (2H).

Example 2

^{3) Μθ0Η} (Dibenzyl)N-PEG12k-OH

[00109] (Dibenzyl)-N-Poly(ethylene oxide)2₇o-OH Potassium is freshly cut under dry hexanes to remove all oxide. Potassium (3.13 g, 80 mmol) is weighed in a tared vial containing dry hexanes, then transferred with tweezers to a Schlenk flask with an Argon purge. The flask is then evacuated and any residual hexanes is allowed to evaporate, then the flask backfilled with Argon. Separately, recrystallized, sublimed naphthalene (12.30 g, 100 mmol) is added to a 250 mL round bottom flask. The flask and its contents are dried under vacuum for 15 minutes, then backfilled with Argon. Dry THF (200 mL) is then added to the Schlenk flask containing the potassium, and dry THF (200 mL) is added to the flask containing the naphthalene. Once the naphthalene is completely dissolved in the THF, the entire solution is transferred to the Schlenk flask. A green color begins to appear within 1 minute of the naphthalene solution addition. The solution is stirred overnight to allow for complete reaction, yielding -400 mL of a 0.2 M potassium naphtalenide solution. The solution is used within 48 hours of preparation. Any unused solution is quenched by the addition of isopropyl alcohol. [00110] The glassware was assembled while still warm. Vacuum was then applied to the assembly and the ethylene oxide line to about 10 mTorr. The setup was backfilled with argon. 2- Dibenzylamino ethanol from Example 1 (3.741 g, 40.4 mmol) was introduced via the sidearm of the jacketed flask under argon overpressure. Two vacuum/argon backfill cycles were applied to the whole setup. THF line was connected to the 14/20 side-arm and vacuum was applied to the whole setup. At this stage, the addition funnel was closed and left under vacuum. THF (4 L) was introduced via the side-arm in the round bottom flask under an argon overpressure. An aliquot of the THF added to the reaction vessel was collected and analyzed by Karl-Fisher colorometric titration to ensure water content of the THF is less than 6 ppm. Next, 2-dibenzylamino ethanol was converted to potassium 2-dibenzylamino ethoxide via addition of potassium naphthalenide (200 mL). Ethylene oxide (500 ml, 10.44 mol) was condensed under vacuum at - 30°C into the jacketed addition funnel, while the alkoxide solution was cooled to 10 °C. Once the appropriate amount of ethylene oxide was condensed, the flow of ethylene oxide was stopped, and the liquid ethylene oxide added directly to the cooled alkoxide solution. After complete ethylene oxide addition, the addition funnel was closed and the reaction flask backfilled with argon. While stirring, the following temperature ramp was applied to the reaction: 12 hrs at 20 °C, 1 hr from 20 °C to 40 °C and 3 days at 40 °C. The reaction went from a light green tint to a golden yellow color. Upon termination with an excess methanol, the solution color changed to light green. The solution was precipitated into ether and isolated by filtration. 459 g, 99 % yield was recovered after drying in a vacuum oven overnight. 1H NMR (d6-DMSO) δ 7.4-7.2 (10H), 4.55 (1H), 3.83-

3.21 (910 H) ppm.

Example 3

[00111] NH₂-Poly(ethylene oxide)₂₇₀-OH (Dibenzyl)-N-poly(ethylene oxide)₂₇₀-OH from Example 2 (455g, 39.56mmol) was split into two equal amounts and was introduced into two 2L flasks. A separate batch of (dibenzyl)-N-poly(ethylene oxide)₂₇o-OH (273g, 23.74mmol) was added to a third 2L flask. The following steps were repeated for each flask. The following steps were repeated for each flask. (Dibenzyl)-N-poly(ethylene oxide)₂₇o-OH (~225g), Pd(OH)₂/C (32 g, 45.6 mmol), ammonium formate (80 g, 1.27 mol) and ethanol (1.2 L) were mixed together in a 2L flask. The reaction was heated to 80 °C while stirring for 24 hrs. The reaction was cooled to room temperature and filtered through a triple layer Celite/MgSCVCelite pad. The MgSC^ powder is fine enough that very little Pd(OH)₂/C permeates through the pad. Celite helps prevent the MgS04 layer from cracking. At this stage, the three filtrates were combined, precipitated into ~ 30L of ether and filtered through a medium glass frit. The wet polymer was then dissolved into 4 L of water, 1 L of brine and 400mL of saturated K₂CO₃ solution. The pH was checked to be -11 by pH paper. The aqueous solution was introduced into a 12L extraction funnel, rinsed once with 4 L of ether and extracted 4 times with dichloromethane (6 L, 6 L, 6 L, 2 L). Dichloromethane fractions were recombined, dried over MgSC^ (3 kg) , filtered, concentrated to ~ 3 L by rotary evaporation and precipitated into diethyl ether (30 L). 555g, 75% yield of the title compound was recovered after filtration and evaporation to dryness in a vacuum oven. 1H

NMR (d6-DMSO) 4.55 (1H), 3.83-3.21 (910 H), 2.96 (2H) ppm.

Example 4

[00112] Boc-NH-Poly(ethylene oxide)₂₇₀-OH NH₂-Poly(ethylene oxide)₂₇₀-OH (555g, 48.26 mmol) from Example 3 was dissolved into 4L of DI water. A saturated solution of K₂C0₃ (120 mL) was added, to keep the pH basic (pH - 11 with pH paper). Di-tert-butyl dicarbonate (105g, 0.48mol) was added to the aqueous solution of NH₂-poly(ethylene oxide)₂₇o-OH and allowed to stir at room temperature overnight. At this stage, a 5 mL aliquot of the reaction was extracted with 10 mL of dichloromethane and the dichloromethane extract precipitated into ether. A 1H NMR was run to ensure completion of the reaction. Thereafter, the aqueous solution was placed into a 12L extraction funnel, was rinced once with ether (4L) and extracted three times with dichloromethane (6L, 6L and 6L). The organic fractions were recombined, dried over MgSC^ (3kg), filtered, concentrated to - 4L and precipitated into 30 L of ether. The white powder was filtered and dried overnight in a vacuum oven, giving 539g of the title compound in 97% yield.

1H NMR (d₆-DMSO) δ 6.75 (1H), 4.55 (1H), 3.83-3.21 (910 H), 3.06 (2H), 1.37 (9H) ppm. Example 5

[00113] Boc-NH-Poly(ethylene oxide)₂₇₀-N₃ Boc-NH-Poly(ethylene oxide)₂₇₀-OH (539g, 49.9 mmol) from Example 4 was placed into a 6 L jacketed flask and dried by azeotropic distillation from toluene (3L). It was then dissolved into 3L of dry dichloromethane under inert atmosphere. The solution was cooled to 0 °C, methanesulfonyl chloride (10.9 mL, 140.8 mmol) was added followed by triethylamine (13.1 mL, 94 mmol). The reaction was allowed to warm to room temperature and proceeded overnight under inert atmosphere. The solution was evaporated to dryness by rotary evaporation and used as-is for the next step.

[00114] NaN₃ (30.5g, 470 mmol) and 3 L of ethanol were added to the flask containing the polymer. The solution was heated to 80 °C and allowed to react overnight. It was then evaporated to dryness by rotary evaporation (bath temperature of 55 °C) and dissolved in 2 L of dichloromethane. The latter solution was the filtered through a Buchner funnel fitted with a Whatman paper #1 to remove most of the salts. The solution was concentrated down to ~ 1 L by rotary evaporation. The product was purified by silica gel flash column chromatography using a 8 in. diameter column with a coarse frit. About 7 L of dry silica gel were used. The column was packed with 1 :99 MeOH/CH₂Cl₂ and the product was loaded and eluted onto the column by pulling vacuum from the bottom of the column. The elution profile was the following: 1 :99 MeOH/CH₂Cl₂ for 1 column volume (CV), 3:97 MeOH/CH₂Cl₂ for 2 CV and 10:90 MeOH/CH₂Cl₂ for 6 CV. The different polymer-containing fractions were recombined (~ 40L of dichloromethane), concentrated by rotary evaporation and precipitated into a 10-fold excess of diethyl ether. The title compound was recovered by filtration as a white powder and dried overnight in vacuo, giving 446.4g, 82% yield. 1H NMR (d₆-DMSO) δ 6.75 (1H), 3.83-3.21 (910 H), 3.06 (2H), 1.37 (9H) ppm. M_n (MALDI-TOF) = 11,554 g/mol. PDI (DMF GPC) = 1.04 Example 6

N3-EO270-NH-BOC N₃-E027Q-NH₂/DFA

[00115] DFA^{+ "}NH₃-Poly(ethylene oxide)₂₇₀-N₃ Boc-NH-Poly(ethylene oxide)₂7o-N₃ (313g, 27.2 mmol) from Example 5 was weighed into a 2 L beaker, 600 mL of DFA, 600 mL of dichloromethane were added. The solution was stirred at room temperature for 32 hr and the polymer was recovered by two consecutive precipitation in ether ( 2 x 30 L). The white powder was dried overnight in a vacuum oven to afford the title compound. (306 g, 98% yield). 1H NMR (de-DMSO) δ 7.67 (3H), 6.13 (1H), 3.82 - 3.00 (1060H), 2.99 (2H).

Example 7

[00116] D-Leucine NCA H-D-Leu-OH (lOOg, 0.76 mol) was suspended in 1 L of anhydrous THF and heated to 50 °C while stirring heavily. Phosgene (20% in toluene) (500 mL, 1 mol) was added the amino acid suspension. After lh20 min, the amino acid dissolved, forming a clear solution. The solution was concentrated on the rotovap, transferred to a beaker, and hexane was added to precipitate the product. The white solid was isolated by filtration and dissolved in toluene (~ 700 mL) with a small amount of THF (~ 60 mL). The solution was filtered over a bed of Celite to remove any insoluble material. An excess of hexane (~ 4 L) was added to the filtrate to precipitate the product. The NCA was isolated by filtration and dried in vacuo. (91g, 79% yield) D-Leu NCA was isolated as a white, crystalline solid. 1H NMR (d₆-DMSO) δ 9.13 (1H), 4.44 (1H), 1.74 (1H), 1.55 (2H), 0.90 (6H) ppm. Example 8

[00117] tert- utyl Aspartate NCA H-Asp(OBu)-OH (120g, 0.63mol) was suspended in 1.2 L of anhydrous THF and heated to 50 °C while stirring heavily. Phosgene (20% in toluene) (500 mL, 1 mol) was added the amino acid suspension. After lh30 min, the amino acid dissolved, forming a clear solution. The solution was concentrated on the rotovap, transferred to a beaker, and hexane was added to precipitate the product. The white solid was isolated by filtration and dissolved in anhydrous THF. The solution was filtered over a bed of Celite to remove any insoluble material. An excess of hexane was added to precipitate the product. The NCA was isolated by filtration and dried in vacuo. 93g (68%) of Asp(OBu) NCA was isolated as a white, crystalline solid. 1H NMR (d₆-DMSO) δ 8.99 (1H), 4.61 (1H), 2.93 (1H), 2.69 (1H), 1.38 (9H) ppm.

Example 9

[00118] Benzyl Tyrosine NCA H-Tyr(OBzl)-OH (140g, 0.52 mol) was suspended in 1.5 L of anhydrous THF and heated to 50 °C while stirring heavily. Phosgene (20% in toluene) (500 mL, 1 mol) was added the amino acid suspension via cannulation. The amino acid dissolved over the course of approx. Ih30, forming a pale yellow solution. The solution was first filtered through a Buchner fitted with a Whatman paper #1 to remove any particles still in suspension. Then, the solution was concentrated by rotary evaporation, transferred to a beaker, and hexane was added to precipitate the product. The off-white solid was isolated by filtration and dissolved in anhydrous THF (~ 600 mL). The solution was filtered over a bed of Celite to remove any insoluble material. An excess of hexane (~ 6 L) was added to the filtrate to precipitate the product. The NCA was isolated by filtration and dried in vacuo. 114.05g, 74.3% of Tyr(OBzl) NCA was isolated as a off-white powder. 1H NMR (d₆-DMSO) δ 9.07 (IH), 7.49-7.29 (5H), 7.12-7.07 (2H), 6.98-6.94 (2H), 5.06 (2H), 4.74 (IH), 3.05-2.88 (2H) ppm.

Example 10

n ~ 270

N3"E0270~i>P{Asp{OBu)₁o"&-P((Tyr(OBz)₂₀-co-(D-Leu)_g3)~Ac [00119] N₃-Poly(ethylene oxide)₂₇o-6-Poly(Asp(OBu)io)-6-Poly(dLeu2o-co-Tyr(OBzl)₂o)- Ac

[00120] Step A: DFA^{" +}NH₃-Poly(ethylene oxide)₂₇₀-N₃ (294g, 25.6 mmol) from Example 6 was weighed into an oven-dried, 6L jacketed round-bottom flask, dissolved in toluene (2 L), and dried by azeotropic distillation. After distillation, the polymer was left under vacuum overnight before adding the NCA. Asp(OBu) NCA (55 g, 256 mmol) from Example 8 was added to the flask, the flask was evacuated under reduced pressure, and subsequently backfilled with nitrogen gas. Dry N-methylpyrrolidone (NMP) (1.8L) was introduced by cannula and the solution was heated to 60 °C. The reaction mixture was allowed to stir for 48 hours at 60 °C under nitrogen gas.

[00121] Step B: D-Leu NCA (82g, 0.522 mol) (Example 7) and Tyr (OBzl) NCA (155g, 0.522 mol) (Example 9) were dissolved under nitrogen gas into 360 ml of NMP into an oven- dried, round bottom flask and the mixture was subsequently cannulated to the polymerization reaction via a syringe. The solution was allowed to stir at 60°C for another three days and 12hrs at which point the reaction was complete (by HPLC). The solution was cooled to room temperature and 25 mL were precipitated into 1L of ether.

[00122] Step C: Diisopropylethylamine (DIPEA) (50 mL), dimethylaminopyridine (DMAP) (5g), and acetic anhydride (50 mL) were added to the rest of the solution. Stirring was continued overnight at room temperature. The polymer was precipitated into diethyl ether (50 L) and isolated by filtration. The title product was isolated by filtration and dried in vacuo to give the block copolymer as an off-white powder (426g, Yield = 73%). 1H NMR (d₆-DMSO) δ 8.43-7.62 (50H), 7.35 (100H), 7.1 (40H), 6.82 (40H), 4.96 (40H), 4.63-3.99 (50H), 3.74-3.2 (1500H), 3.06- 2.6 (60H), 1.36 (90H), 1.27-0.47 (180).

Example 11

[00123] N₃-Poly(ethylene oxide)₂₇o-6-Poly(Aspio)-6-Poly(dLeu₂o-c0-Tyr_2O)-Ac N₃-

Poly(ethylene oxide)₂₇o-¾-Poly(Asp(OBu)io)-¾-Poly(dLeu₂o-co-Tyr(OBzl)₂o)-Ac (420g, 20.5 mmol) from Example 10 was dissolved into 3 L of a solution of pentamethyl benzene (PMB, 0.5M) in trifluoroacetic acid (TFA). The reaction was allowed to stir for five hours at room temperature. The solution was precipitated into diethyl ether (50 L) and the solid was recovered by filtration through a 2L medium frit. The polymer was redissolved into 4L of dichloromethane and precipitated into diethyl ether (~50 L). The polymer was redissolved one more time into a 50:50 dichloromethane:isopropanol mixture and diethyl ether was poured on the top of the solution (~ 50L). The title compound was obtained as an off-white polymer after drying the product overnight in vacuo (309.3g, 83% yield). 1H NMR (d₆-DMSO) δ 12.2 (2H), 9.1 (13H), 8.51-7.71 (49H), 6.96 (29H), 6.59 (26H), 4.69-3.96 (59H), 3.81-3.25 (1040H), 3.06-2.65 (45H), 1.0-0.43 (139). ¹³C NMR (d₆-DMSO) δ 171.9, 171, 170.5, 170.3, 155.9, 130.6, 129.6, 127.9 115.3, 114.3, 70.7, 69.8, 54.5, 51.5, 50, 49.8, 49.4, 36.9, 36, 24.3, 23.3, 22.3, 21.2. IR (ATR) 3290, 2882, 1733, 1658, 1342, 1102, 962 cm^"1. M_n (MALDI-TOF) = 17,300 g/mol. PDI (DMF GPC) = 1.1

Example 12

Click of HER 2 - Alkyne onto N₃-E0270-¾-Poly(Asp(OBu)io)-¾-Poly(dLeu2o-co-Tyr(OBzl)₂o)-

Ac

HER 2 - Alkyne

. CuS0₄. 5H₂0

. (BimC4A)₃

. Na Ascorbate

. DMSO: H20 1 :1

. 50 °C, 2 days

. 25 mg palymer/mL of solution

[00124] N₃-E0270-¾-Poly(Asp(OBu)io)-¾-Poly(dLeu₂o-co-Tyr(OBzl)₂o)-Ac (299.3 mg, 16 μιηοΐ), HER 2 - Alkyne (26.2 mg, 32.9 μιηοΐ), sodium ascorbate (79.8 mg, 0.402 mmol), (BimC4A)3 (23.13 mg, 32.6 μπωΐ), CuS04 . 5H₂0 (3.94 mg, 15.8 μmol), DMSO (6 mL) and water (6 mL) were added into a 20 mL vial, capped and stirred for 48 hr at 50°C. The light brown solution was dialyzed (3500 MWCO bag) 3 times against DI water with EDTA (15g/L) and 2 times against DI water. The solution was freeze-dried and an off-white powder was obtained. (300 mg , 96 % yield). 1H NMR (D₂0) δ 7.28-6.49, 4.66-4.40, 3.97-3.46, 2.77-2.52 ppm. Example 13

N3-EG270-b-Poly(Asp₁₀^-Poly{d-LeU2o -Tyr2e)-Ac

UPAR - Alkyne

. CuS0₄. 5H₂0

. (BimC4A)₃

. Na Ascorbats

. D SO: H20 1 :1

. 50 °C, 2 days

. 25 mg polymer/mL of solution

[00125] UPAR-Poly(ethylene oxide)₂₇o-6-Poly(Aspio)-6-Poly(dLeu₂o-co-Tyr₂o)-Ac N₃-

Poly(ethylene oxide)₂₇o-£-Poly(Aspio)-£-Poly(dLeu₂o-co-Tyr₂₀)-Ac (306.2 mg, 16.4 μmol) from Example 11, alkynyl-UPAR (25.0 mg, 21.1 μιηοΐ), sodium ascorbate (86.9 mg, 0.44 mmol), (BimC4A)3 (23.4 mg, 33.1 μπωΐ), CuS0₄ . 5H₂0 (5.44 mg, 21.8 μιηοΐ), DMSO (6 mL) and water (6 mL) were added into a 20 mL vial, capped and stirred for 48 hr at 50°C. The light brown solution was dialyzed (3500 MWCO bag) 3 times against deionized water with EDTA (15g/L) and 2 times against deionized water. The solution was freeze-dried and the title compound was obtained as an off-white powder. (272.2 mg , 85 % yield). 1H NMR (D₂0) δ 8.16 (IH), 7.84 (IH), 7.44-6.72 (4H), 4.60-4.26 (18H), 4.06-3.41 (1040H), 2.99 (6H), 2.73 (IH), 2.58-1.69 (34H). Example 14

GRP 78^■ Alkyne

. CuS0₄. 5H-0

. (BimC4A)₃

. Ha Ascorbate

. DMSO: H20 1 :1

. 50 °C, 2 days

. 25 mg polymer/mL of solution

[00126] GRP78-Poly(ethylene oxide)₂₇o-6-Poly(Aspio)-6-Poly(dLeu₂o-co-Tyr₂o)-Ac N₃-

Poly(ethylene oxide)₂₇o-£-Poly(Aspio)-£-Poly(dLeu₂o-co-Tyr₂₀)-Ac (296.6 mg, 15.9 μιηοΐ) from Example 11, alkynyl-GRP 78 (32.5 mg, 20.7 μιηοΐ), sodium ascorbate (80.55 mg, 0.41 mmol), (BimC4A)3 (24.8 mg, 35 μιηοΐ), CuS0₄ . 5H₂0 (5.30 mg, 21.2 μιηοΐ), DMSO (6 mL) and water (6 mL) were added into a 20 mL vial, capped and stirred for 48 hr at 50°C. The light brown solution was dialyzed (3500 MWCO bag) 3 times against DI water with EDTA (15g/L) and 2 times against DI water. The solution was freeze-dried and an off-white powder was obtained. (244.3 mg , 92 % yield). 1H NMR (D₂0) δ 8.16 (1H), 7.44-6.72 (8H), 4.35 (3H), 4.06-3.41 (1040H), 2.97-2.62 (12H). Example 15

EGFR - Alkyne

L of solution

[00127] EGFR-Poly(ethylene oxide)₂₇o-6-Poly(Aspio)-6-Poly(dLeu₂o-co-Tyr₂o)-Ac N₃-

Poly(ethylene oxide)₂₇o-¾-Poly(Aspio)-£-Poly(dLeu₂o-co-Tyr₂₀)-Ac (877 mg, 46 μιηοΐ) from Example 11, alkynyl-EGFR (50 mg, 56.3 μιηοΐ), sodium ascorbate (232 mg, 1.17 mmol), (BimC4A)3 (66 mg, 94 μmol), CuS0₄ . 5H₂0 (12 mg, 47 μιηοΐ), DMSO (17 mL) and water (17 mL) were added into a 50 mL round bottom flask, capped and stirred for 48 hr at 50°C. The light brown solution was dialyzed (3500 MWCO bag) 3 times against DI water with EDTA (15g/L) and 2 times against DI water. The solution was freeze-dried and an off-white powder was obtained. (512 mg , 56 % yield). 1H NMR (D₂0) δ 8.16, 7.09, 6.82, 4.35, 4.06-3.41 (1040H), 3.27-2.73 (14H). Example 16

CMC Determination

[00128] The CMC of micelles prepared from block copolymers, as described above, were determined using the method described by Eisnberg. (Astafieva, I.; Zhong, X.F.; Eisenberg, A. "Critical Micellization Phenomena in Block Copolymer Polyelectrolyte Solutions" Macromolecules 1993, 26, 7339-7352.) To perform these experiments, a constant concentration of pyrene (5 x 10^~7 M) was equilibrated with varying concentrations of block copolymer (ca. 2 x 10² - 1 x 10^~4 mg/mL) in phosphate buffered saline at room temperature for 16 hours. Excitation spectra (recorded on a Perkin Elmer LS-55 spectrophotometer with excitation between 328 and 342 nm, emission at 390 nm, 2.5 nm slit width, 15 nm/min scan speed) were recorded for each polymer concentration and the fluorescence intensities recorded at 333 and 338 nm. Eisenberg has shown that the vibrational fine structure of pyrene is highly sensitive to the polarity of its environment. Specifically, the (0,0) excitation band of pyrene will shift from 333 nm in an aqueous environment to 338.5 nm in a hydrophobic environment. The ratio of peak intensities (I338 I333) reveals the hydrophobicity of the environment surrounding the pyrene. Values of ~ 2.0 correspond to a hydrophobic environment such as polystyrene or poly(benzyl glutamate), whereas values of ~ 0.35 correspond to an aqueous environment. Plotting this ratio vs. log of the block copolymer concentration allows for the graphical interpretation of the CMC value. A more quantitative number can be obtained by fitting a logarithmic (y=a ln(x) + b) regression to the data points between the two plateaus (at ~ 2 and -0.35). The CMC can be found by setting y=0.35 and solving for x (concentration in mg/mL).

Example 17

Preparation of DOX loaded micelles

[00129] N₃-E0270-¾-Poly(Aspio)-¾-Poly(dLeu₂o-co-Tyr₂o)-Ac (524 mg) (From Example 11) and water (300 mL) was added to a 1 L beaker and stirred until a homogeneous solution was present. Doxorubicin hydrochloride (62 mg) was suspended in triethylamine (60 uL) and dichloromethane (10 mL). The resulting doxorubicin suspension was added dropwise to the rapidly stirring aqueous solution. The resulting solution was covered with foil and allowed to stir for an additional eight hours. Over this period of time, a color change from purple to red was noted. The solution was filtered through a 0.22 μηι filter and then lyophilized to give 577 mg (93 % yield) as a red powder.

Example 18

Iron (III) Crosslinking of DOX loaded micelles

[00130] Dox loaded micelles (100 mg) (prepared as in Example 17) was dissolved in 20 mM Tris HC1 buffer supplemented with 5 mM iron (III) chloride (4 mL). Once a homogeneous solution was present, the pH was adjusted to 8.0 with 1 N NaOH, then stirred overnight. The samples were lyophilized and the reddish-brown powder stored at 4 °C.

Example 19

Stability of Iron (III) Crosslinked, DOX Loaded Micelles

[00131] Dox loaded micelles (prepared as in Example 17) was dissolved in 20mM Tris, pH 7.5 with varying concentrations (0, 0.1, 1, 5, and 10 mM) of FeCl₃. Samples dissolved such that the final concentration of formulation was 25 mg/mL in the buffer. The pH of the samples was adjusted to 8.0 with 1 N NaOH, followed by overnight incubation at room temperature. Samples were diluted to 0.25 mg/mL polymer concentration with 10 mM pH 8 phosphate buffer, placed in a dialysis bag (Spectra Por 7, 3500 molecular weight cut off) and dialyzed for 6 hours against 10 mM pH 8 phosphate buffer. An aliquot before and after dialysis was analyzed by HPLC to determine the concentration of doxorubicin in each sample. Figure 1 displays the percent of doxorubicin remaining in the dialysis bag compared to pre-dialysis samples as a function of iron (III) chloride concentration. A 10-fold increase in doxorubicin retention is observed for 5 and 10 mM of iron (III) chloride when compared to the sample with no iron added, indicating that crosslinking was achieved.

Example 20

Crosslinking Optimization with Iron (III) Chloride

[00132] Dox loaded micelles (10 mg) (prepared as in Example 17) were dissolved in 20 mM Tris HC1 buffer supplemented with 5 mM iron (III) chloride (0.4 mL). Once homogeneous, the samples were adjusted to various pH values (native, 5.5, 6, 6.5, 7, 7.5, or 8) followed by overnight incubation. Samples were diluted to 0.25 mg/mL polymer concentration with 10 mM pH 8 phosphate buffer, placed in a dialysis bag (Spectra Por 7, 3500 molecular weight cut off) and dialyzed for 6 hours against 10 mM pH 8 phosphate buffer. An aliquot before and after dialysis was analyzed by HPLC to determine the concentration of doxorubicin in each sample. Figure 2 displays the percent of doxorubicin remaining in the dialysis bag compared to pre- dialysis samples as a function of iron (III) chloride concentration. The data demonstrate that adjustment to pH 8.0 yielded the highest crosslinking efficiency.

Example 21

Crosslinking Kinetics with Iron (III) Chloride

[00133] Dox loaded micelles (10 mg) (prepared as in Example 17) were dissolved in 20 mM Tris HCl buffer supplemented with 5 mM iron (III) chloride (0.4 mL). Once homogeneous, the solution was adjusted to pH 8 with 1 N NaOH. Aliquots were removed from the solution at various time points after pH adjustment (2, 4, 6, 8, 12, 16, and 24 h) then diluted to 0.25 mg/mL polymer concentration with 10 mM pH 8 phosphate buffer, placed in a dialysis bag (Spectra Por 7, 3500 molecular weight cut off) and dialyzed for 6 hours against 10 mM pH 8 phosphate buffer. An aliquot before and after dialysis was analyzed by HPLC to determine the

concentration of doxorubicin in each sample. Figure 3 shows the kinetic dependence upon the crosslinking reaction with iron (III) chloride.

Example 22

pH Dependent Release from Iron (III) Crosslinked Micelles

[00134] Dox loaded micelles (prepared as in Example 17) was dissolved in 20mM Tris, pH 7.5 with concentrations of 0, 5 and 10 mM of FeCl₃. Samples dissolved such that the final concentration of formulation was 25 mg/mL in the buffer. The pH of the samples was adjusted to 8.0 with 1 N NaOH, followed by overnight incubation at room temperature. Seven aliquots (50 μί) of each stock solution were then diluted into seven separate 10 mM phosphate buffer (5 mL) at pH 3, 4, 5, 6, 7, 7.4 and 8, giving a final concentration of 0.2 mg formulation per mL buffer. 3 mL of each sample was added to a Spectra-Por 3500 MWCO dialysis bag, then placed in a 400 mL beaker containing a stir bar and 300 mL of the corresponding 10 mM phosphate buffer. The samples were allowed to dialyze for 6 hours, then the samples removed from the dialysis bag. Samples pre and post dialysis were analyzed by HPLC to determine the doxorubicin concentration in each sample. Figure 4 shows the pH dependent release of each sample. The data shows that the samples containing iron demonstrate improved retention of doxorubicin at high pH values, but release of the drug at low pH values.

Example 23

Crosslinking Optimization with Iron (II) and Iron (III) Chloride

[00135] Dox loaded micelles (prepared as in Example 17) was dissolved in 20mM Tris, pH 7.5 with varying concentrations (0, 1, 2, 3, 4, and 5 mM) of FeCl₂ and FeCl₃. Samples dissolved such that the final concentration of formulation was 25 mg/mL in the buffer. The pH of the samples was adjusted to 8.0 with 1 N NaOH, followed by overnight incubation at room temperature. Samples were diluted to 0.25 mg/mL polymer concentration with 10 mM pH 8 phosphate buffer, placed in a dialysis bag (Spectra Por 7, 3500 molecular weight cut off) and dialyzed for 6 hours against 10 mM pH 8 phosphate buffer. An aliquot before and after dialysis was analyzed by HPLC to determine the concentration of doxorubicin in each sample. Figure 5 displays the percent of doxorubicin remaining in the dialysis bag compared to pre-dialysis samples as a function of iron concentration. The data demonstrate effective crosslinking using either FeCl₃ or FeCl₂.

Example 24

pH Dependent Release from Iron (III) Crosslinked Micelles

[00136] Dox loaded micelles (prepared as in Example 17) was dissolved in 20mM Tris, pH 7.5 with concentrations of 1, and 4 mM of FeCl₂ and 4 mM of FeCl₃. Samples dissolved such that the final concentration of formulation was 25 mg/mL in the buffer. The pH of the samples was adjusted to 8.0 with 1 N NaOH, followed by overnight incubation at room temperature. Seven aliquots (50 μί) of each stock solution were then diluted into seven separate 10 mM phosphate buffer (5 mL) at pH 3, 4, 5, 6, 7, 7.4 and 8, giving a final concentration of 0.2 mg formulation per mL buffer. 3 mL of each sample was added to a Spectra-Por 3500 MWCO dialysis bag, then placed in a 400 mL beaker containing a stir bar and 300 mL of the

corresponding 10 mM phosphate buffer. The samples were allowed to dialyze for 6 hours, then the samples removed from the dialysis bag. Samples pre and post dialysis were analyzed by HPLC to determine the doxorubicin concentration in each sample. Figure 6 shows the pH dependent release of each sample. The data demonstrates that the pH dependent release is present for crosslinking performed with iron (II) chloride, and can also be slightly tuned by the varying the addition of iron during the crosslinking step.

Example 25

Pharmacokinetics of Iron Crosslinked DOX loaded micelles

[00137] Fisher rats that possessed a jugular vein catheter were injected with 10 mg/kg of free doxorubicin, uncrosslinked micelles (prepared according to Example 17), and iron (III) crosslinked dox loaded micelles (Example 18) by a fast IV bolus with an injection volume of 1 mL. The delivery vehicle for drug administration was isotonic saline. Rat blood was collected from the catheter into K₂-EDTA tubes by heart puncture at time points of 1, minute, 5 minutes, 15 minutes, 1 hour, 4 hours, 8 hours and 24 hours. Plasma was isolated by centrifugation at 1000 RPM for 5 minutes, and 150 uL of extraction solution (ice cold methanol/100 ng/mL

daunorubicin internal standard) was added to 50 uL of each plasma sample. Samples were then vortexed for 10 minutes, centrifuged at 13,000 RPM for 10 minutes, and 150 uL of the supernatant is transferred to HPLC vials for analysis.

[00138] Samples were analyzed on a Waters Alliance 2695 equiped with a 2475 fluorescence detector (Ex = 470 nm; Em = 580 ). A 5 sample injection was made onto a Waters 4 μιη Nova Pak C18 (3.9 x 150 mm) at 30 °C with a flow rate of 0.750 mL per minute of 10 mM phosphate buffer (pH=1.4), methanol and acetonitrile (gradient from 70/10/20 to 40/10/50 for buffer/methanol/acetonitrile was made over eight minutes). Analyte eluted at 5.9 minutes under these conditions, was normalized to the internal standard, and quantitated using a standard curve comprised of seven standards. The pharmacokinetic parameters are summarized in the table below and the curves are shown in Figure 14. It is important to note that the AUC is nearly 15 times great for the iron (III) crosslinked micelle when compared to the uncrosslinked micelle, again, indicating that the iron crosslinking is stabilizing the micelle when diluted in the bloodstream.

Sample Cmax (ug/mL) AUC (ug*hr/mL)

Free DOX 9.76 2.61

Uncrosslinked Micelles 18. 18 7.05

Iron (III) Crosslinked Micelles 55.77 103.13 Example 26

Preparation of EGFR Targeted, DOX loaded micelles

[00139] N₃-E0270-¾-Poly(Aspio)-¾-Poly(dLeu₂o-co-Tyr₂o)-Ac (97.5 mg) (Example 11) and EGFR-Poly(ethylene oxide)₂7o-¾-Poly(Aspio)-£-Poly(dLeu₂o-co-Tyr₂₀)-Ac (Example 15) was dissolved in water (65 mL) and stirred until homogeneous. Doxorubicin hydrochloride (10 mg) was suspended in a 80% dichloromethane/20% methanol solution (3.9 mL), followed by the addition of triethylamine (11 uL). The resulting doxorubicin suspension was added dropwise to the rapidly stirring aqueous solution. The resulting solution was covered with foil and allowed to stir for an additional eight hours. Over this period of time, a color change from purple to red was noted. The solution was filtered through a 0.22 μιη filter and then lyophilized to give 92 mg (84 % yield) as a red powder.

Example 27

Preparation of uPAR Targeted, DOX loaded micelles

[00140] N₃-E0270-¾-Poly(Aspio)-¾-Poly(dLeu₂o-co-Tyr₂o)-Ac (97.5 mg) (Example 11) and uPAR-Poly(ethylene oxide)₂7o-¾-Poly(Aspio)-£-Poly(dLeu₂o-co-Tyr₂₀)-Ac (Example 13) was dissolved in water (65 mL) and stirred until homogeneous. Doxorubicin hydrochloride (10 mg) was suspended in a 80% dichloromethane/20%) methanol solution (3.9 mL), followed by the addition of triethylamine (11 uL). The resulting doxorubicin suspension was added dropwise to the rapidly stirring aqueous solution. The resulting solution was covered with foil and allowed to stir for an additional eight hours. Over this period of time, a color change from purple to red was noted. The solution was filtered through a 0.22 μιη filter and then lyophilized to give 96 mg (87 % yield) as a red powder.

Example 28

Iron (II) Crosslinking of EGFR Targeted, DOX loaded micelles

[00141] EGFR targeted, Dox loaded micelles (20 mg) (Example 26) was dissolved in 20 mM Tris HC1 buffer supplemented with 1 mM iron (II) chloride (1 mL). Once a homogeneous solution was present, the pH was adjusted to 8.0 with 1 N NaOH, then stirred overnight. The samples were lyophilized and the reddish-brown powder stored at 4 °C. Example 29

Iron (II) Crosslinking of uPAR Targeted, DOX loaded micelles

[00142] uPAR targeted, Dox loaded micelles (20 mg) (Example 27) was dissolved in 20 mM Tris HC1 buffer supplemented with 1 mM iron (II) chloride (1 mL). Once a homogeneous solution was present, the pH was adjusted to 8.0 with 1 N NaOH, then stirred overnight. The samples were lyophilized and the reddish-brown powder stored at 4 °C.

Example 30

pH Dependent Release from Targeted, Iron (II) Crosslinked Micelles

[00143] Dox loaded micelles (prepared as in Example 18, 28, and 29) were dissolved in 20mM Tris, pH 7.5 with 1 mM of FeCl₂. Samples dissolved such that the final concentration of formulation was 25 mg/mL in the buffer. The pH of the samples was adjusted to 8.0 with 1 N NaOH, followed by overnight incubation at room temperature. Seven aliquots (50 μί) of each stock solution were then diluted into seven separate 10 mM phosphate buffer (5 mL) at pH 3, 4, 5, 6, 7, 7.4 and 8, giving a final concentration of 0.2 mg formulation per mL buffer. 3 mL of each sample was added to a Spectra-Por 3500 MWCO dialysis bag, then placed in a 400 mL beaker containing a stir bar and 300 mL of the corresponding 10 mM phosphate buffer. The samples were allowed to dialyze for 6 hours, then the samples removed from the dialysis bag. Samples pre and post dialysis were analyzed by HPLC to determine the doxorubicin

concentration in each sample. Figure 15 shows the pH dependent release of each sample. It is important to note that the pH dependent release of the crosslinked micelles does not change with the addition of targeting groups.

Example 31

Pulse-treatment cytotoxicity Assay

[00144] A549 cells were maintained in F12K medium supplemented with 10%FBS, 2mM L- Glutamine, and lOOunits/mL of penicillin and streptomycin. Cells were plated in 96-well white- walled plates at a concentration of 5.0xl0³ cells per well. The following day, cells were treated with increasing concentrations of free doxorubicin, iron (II) crosslinked micelles (Example 23), EGFR targeted, iron (II) crosslinked micelles (Example 29), or uPAR targeted, iron (II) crosslinked micelles (Example 30) for 1.5 hours. After this time, cells were washed with PBS, fresh growth medium was added and cells were incubated for an additional 72 hours. Cell viability was then determined using the Cell-Titer Glo assay (Promega). Cells were treated in triplicate. Figure 9 shows the in vitro cytotoxicity of the four samples in A549 cells, whereas Figure 10 shows results in 8505C cells. It should be noted that the targeted, crosslinked micelles exhibit a much lower IC₅₀ than the untargted and free doxorubicin.

Example 32

Continuous Cytotoxicity Assay with iron (II) doxorubicin micelles

[00145] Caki-1 cells were maintained in McCoy's 5A media supplemented with 10%FBS, 2mM L-Glutamine, and lOOunits/mL of penicillin and streptomycin. MG-63 cells were maintained in RPMI 1640 media supplemented with 10%FBS, 2mM L-Glutamine, and lOOunits/mL of penicillin and streptomycin. Cells were plated in 96-well white-walled plates at a concentration of l .OxlO⁴ cells (Caki-1) or 7.0xl0³ cells (MG-63) per well. The following day, cells were treated with increasing concentrations of free doxorubicin, or iron (II) crosslinked micelles (Example 23) for 72 hours. Cell viability was then determined using the Cell-Titer Glo assay (Promega). Cells were treated in triplicate. Figure 11 shows the in vitro cytotoxicity of the four samples. It should be noted that the cytotoxicity profile of the iron (II) crosslinked doxorubicin micelle is similar to free doxorubicin.

Example 33

Preparation of Daunorubicin loaded micelles

[00146] N₃-E0270-¾-Poly(Aspio)-¾-Poly(dLeu₂o-co-Tyr₂o)-Ac (3 g) (From Example 11) and water (2 L) was added to a 4 L beaker and stirred until a homogeneous solution was present. Daunorubicin hydrochloride (301 mg) was suspended in 4: 1 dichloromethane:methanol (60 mL), followed by the addition of triethylamine (82 uL). The resulting daunorubicin suspension was added dropwise to the rapidly stirring aqueous solution. The resulting solution was covered with foil and allowed to stir for an additional eight hours. The solution was filtered through a 0.22 μιη filter and then lyophilized to give 2.95 g (89 % yield) as a red powder. Example 34

Crosslinking of daunorubicin micelle

[00147] Daunorubicin loaded micelles (Example 33) were dissolved at 25mg/mL polymer concentration in either water or 20mM Tris, pH 7.5 supplemented with 0, 0.5, 1, 2, 3, 4, or 5mM FeCl₂. Once a homogeneous solution was present, the pH was adjusted to 8.0 with 1 N NaOH, then stirred overnight. Aliquots (50 μί) of each stock solution were then diluted in 10 mM phosphate buffer (5 mL) at pH 8, giving a final concentration of 0.25 mg formulation per mL buffer. 2 mL of each sample was added to a Spectra-Por 3500 MWCO dialysis bag, then dialyzed in 300 mL of 10 mM phosphate buffer pH 8. The samples were allowed to dialyze for 6 hours, then the samples removed from the dialysis bag. Samples pre and post dialysis were analyzed by HPLC to determine the daunorubicin concentration in each sample. Figure 12 demonstrates that crosslinked is attainable using water or 20mM Tris as a buffer. Furthermore, maximal crosslinking is achieved at a concentration of 2mM iron (II) or greater.

Example 35

pH-dependent release of crosslinked daunorubicin micelles

[00148] Daunorubicin loaded micelles (prepared as in Example 33) was dissolved in water with no iron chloride, 2 mM of FeCl₂ or 5 mM of FeCl₂. Once a homogeneous solution was present, the pH was adjusted to 8.0 with 1 N NaOH, then stirred overnight. Six aliquots (50 μί) of each stock solution were then diluted into six separate 10 mM phosphate buffer (5 mL) at pH 3, 4, 5, 6, 7, and 8, giving a final concentration of 0.25 mg formulation per mL buffer. 2 mL of each sample was added to a Spectra-Por 3500 MWCO dialysis bag, then placed in a 400 mL beaker containing a stir bar and 300 mL of the corresponding 10 mM phosphate buffer. The samples were allowed to dialyze for 6 hours, then the samples removed from the dialysis bag. Samples pre and post dialysis were analyzed by HPLC to determine the daunorubicin

concentration in each sample. Figure 13 shows the pH dependent release of each sample. The data demonstrate that the pH dependent release is present for crosslinking performed with either 2mM or 5mM iron (II) chloride. Although the uncrosslinked daunorubicin micelle has some stability and pH-dependent release with this assay, it is less stable than crosslinked daunorubicin micelles. Example 36

Cytotoxicity of daunorubicin micelles compared to free daunorubicin

[00149] All cell lines were obtained from ATCC (Manassas, VA). Media and supplements were obtained from Cell Gro (Manassas, VA). HT-1080 cells were maintained in Eagle's Minimum Essential Medium supplemented with 10%FBS, 2mM L-Glutamine, ImM sodium pyruvate, and lOOunits/mL of penicillin and streptomycin. MG-63 and 786-0 cells were maintained in RPMI 1640 media supplemented with 10%FBS, 2mM L-Glutamine, and lOOunits/mL of penicillin and streptomycin. SKOV-3 cells were maintained in McCoy's 5 A media with 10%FBS, 2mM L-Glutamine, and lOOunits/mL of penicillin and streptomycin. Cells were plated in 96-well white -walled plates at a concentration of 1.5xl0⁴ (HT-1080), 7.0xl0³ (MG-63), l .OxlO⁴ (786-0), or 8.0xl0³ (SKOV-3) cells per well. The following day, cells were treated with increasing concentrations of free daunorubicin, uncrosslinked daunorubicin micelle (Example 33), 2mM iron (II) (Example 34), or 5mM iron (II) (Example 34) crosslinked daunorubicin micelles for 72 hours. Cell viability was then determined using the Cell-Titer Glo assay (Promega). Cells were treated in triplicate. The table below shows lists the IC50 values (in μΜ) of the four samples. It should be noted that the IC50 values of the daunorubicin micelles are very similar to free daunorubicin.

Example 37

Rat pharmacokinetics of daunorubicin micelles compared to free daunorubicin

[00150] Fisher rats that possessed a jugular vein catheter were injected with 10 mg/kg of free daunorubicin, uncrosslinked daunorubicin micelle (prepared according to Example 33), and iron (II) crosslinked daunorubicin loaded micelles (Example 34) by a fast IV bolus with an injection volume of 2 mL on Day 0. The delivery vehicle for drug administration was isotonic saline. Rat blood was collected from the catheter into K₂-EDTA tubes by heart puncture at time points of 1 , minute, 5 minutes, 15 minutes, 1 hour, 4 hours, 8 hours and 24 hours. Plasma was isolated by centrifugation at 1000 RPM for 5 minutes, and 150 uL of extraction solution (ice cold methanol/ 100 ng/mL daunorubicin internal standard) was added to 50 uL of each plasma sample. Samples were then vortexed for 10 minutes, centrifuged at 13,000 RPM for 10 minutes, and 150 uL of the supernatant is transferred to HPLC vials for analysis.

[00151] Samples were analyzed on a Waters Alliance 2695 equiped with a 2475 fluorescence detector (Ex = 470 nm; Em = 580 ). A 5 sample injection was made onto a Waters 4 μιη Nova Pak C18 (3.9 x 150 mm) at 30 °C with a flow rate of 0.750 mL per minute of 10 mM phosphate buffer (pH=1.4), methanol and acetonitrile (gradient from 70/10/20 to 40/10/50 for buffer/methanol/acetonitrile was made over eight minutes). Analyte eluted at 5.9 minutes under these conditions, was normalized to the internal standard, and quantitated using a standard curve comprised of seven standards. The pharmacokinetic parameters are summarized in the table below and the curves are shown in Figure 14.

Sample Cmax (ug/mL) AUC (ug*hr/mL)

Free Dau norubici n 3.29 1.30

Uncrossli nked Daunoru bicin M icelles 2.61 1.48

2m M Iron (II) Crossl in ked M icelles 125.04 27.92

5m M Iron (II) Crossl in ked M icelles 143.50 51.78

[00152] It is important to note that the AUC is nearly 18-35 times (for 2mM and 5mM XL, respectively) greater for the iron (II) crosslinked micelles when compared to the uncrosslinked micelle, again, indicating that the iron crosslinking is stabilizing the micelle when diluted in the bloodstream. This study was repeated, and the micelles were injected in the same rats seven days after the first injection. The pharmacokinetic results are shown in the table below, and graphed in Figure 15. Sample Cmax (ug/mL) AUC (ug*hr/ml_)

Free Dau norubici n 10.64 3.54

Uncrossli nked Daunoru bicin M icelles 7.91 2.95

2m M Iron (II) Crossl in ked M icelles 120.63 35.27

5m M Iron (II) Crossl in ked M icelles 148.60 78.24

[00153] In the repeat experiment, the AUC of the 2mM and 5mM XL micelle was found to be 12-26 times greater than uncrosslinked micelle, and absolute AUC values that were higher than in the first experiment. The Cmax values for the second experiment were similar to the first experiment, with slightly higher Cmax values for free daunorubicin and uncrosslinked micelle. These data demonstrate that similar pharmacokinetic data were obtained following repeat injections of daunorubicin micelles in rats.

Example 38

ELISA assay for Rat IgM induction following multiple administrations of daunorubicin micelles

[00154] Plasma from the experiment described in Example 54 was used in an ELISA assay to determine the concentration of rat IgM following repeated injections of free daunorubicin and daunorubicin micelles. In addition to plasma collected on Day 0, 7, and 14 (days of injection), plasma from the rats was independently obtained on Day 3, 10, and 17. During the study described in Example 37, separate rats were repeatedly injected with the triblock copolymer and plasma was collected throughout the study. The concentration of rat IgM from plasma was determined using an ELISA kit according to the manufacturer's instructions (Bethyl Laboratories, Montgomery, TX). Results from the ELISA are shown in Figure 15. The results demonstrate that none of the groups displayed any significant induction of rat IgM antibody, even after multiple injections.

Claims

Claims We claim:

1. A stabilized, therapeutic agent loaded micelle comprised of a multiblock copolymer, consisting of a polymeric hydrophilic block, a crosslinkable poly(amino acid block) , and a hydrophobic D,L-mixed poly(amino acid) block, and a hydrophobic therapeutic agent wherein the polymer micelle is represented by Formula I:

wherein:

each n is independently 45-450;

each s is independently 1 or 2

each w is independently 0-30;

each x is independently 1-30;

each y is independently 5-80;

each R_y is independently a mixture of 1 or more moieties that represent a natural or unnatural amino acid such that the overall mixture is hydrophobic; and each R l is independently -N₃ ¹

-OCH₃ or ^N—^-N wherein T is a targeting group moiety.

2. A stabilized, therapeutic agent loaded micelle of claim 1, wherein R¹ is -OCH₃.

3. A stabilized, therapeutic agent loaded micelle of claim 1, wherein R_y is selected from a moiety in Table 1 , either singly or in combination.

4. A stabilized, therapeutic agent loaded micelle of claim 1, wherein n is 275 ± 10.

5. A stabilized, therapeutic agent loaded micelle of claim 1, wherein s is 1.

6. A stabilized, therapeutic agent loaded micelle of claim 1, wherein s is 2.

7. A stabilized, therapeutic agent loaded micelle of claim 1, wherein R¹ is -N₃.