# Patent

## Title
Encapsulated contrast agents

## Abstract

The present invention relates to the field of polymer chemistry and more particularly to encapsulated contrast agents and methods for using the same.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. provisional patent application Ser. No. 60/915,014, filed Apr. 30, 2007, and U.S. provisional patent application Ser. No. 61/025,414, filed Feb. 1, 2008, the entirety of each of which is hereby incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to the field of polymer chemistry and more particularly to encapsulated contrast agents and uses thereof.
BACKGROUND OF THE INVENTION
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.
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.
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.
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 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, or other therapeutic agents, in response to the physiological environment encountered in diseased tissues, such as solid tumors.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the results of dynamic light scattering on an exemplary iron oxide nanoparticle-loaded micelle in deionized water.
FIG. 2 depicts the results of dynamic light scattering on an exemplary iron oxide nanoparticle-loaded micelle after lyophilization and reconstitution in deionized water.
FIG. 3 depicts the 100 kV transmission electron microscope image of an exemplary iron oxide nanoparticle-loaded micelle following drying.
FIG. 4 depicts the results of dynamic light scattering on an exemplary iron oxide nanoparticle-loaded micelle in deionized water.
FIG. 5 depicts the results of a cell viability assay performed on an exemplary iron oxide nanoparticle-loaded micelle.
FIG. 6 depicts individual images showing signal increase with repetition time for R1 determination for HS-2-113.
FIG. 7 depicts individual images showing decrease with echo time for R2 determinations for HS-2-113.
FIG. 8 depicts individual images showing signal increase with repetition time for R1 determination for HS-2-165.
FIG. 9 depicts individual images showing signal increase with repetition time for R1 determination for HS-2-166.
FIG. 10 depicts individual images showing decrease with echo time for R2 determinations for HS-2-165.
FIG. 11 depicts individual images showing decrease with echo time for R2 determinations for HS-2-166.
FIG. 12 depicts contrast enhancement in the brains of two animals studied following intravenous administration of HS-2-177.
FIG. 13 depicts contrast enhancement in the brain of one animal studied following intravenous administration of HS-2-177.
FIG. 14 depicts light scattering of iron oxide loaded micelles (HS-2-177) as compared to empty micelles.
FIG. 15 depicts light scattering of iron oxide loaded micelles (HS-2-113) as compared to polymer.
FIG. 16 depicts individual images showing signal increase with repetition time for R1 determination for HS-2-177.
FIG. 17 depicts individual images showing decrease with echo time for R2 determinations for HS-2-177.



DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
1. General Description:
Polymer micelles for use in the present invention are described in detail in International Patent Application publication number WO2006/107903, published Oct. 12, 2006, the entirety of which is incorporated herein by reference.
One embodiment of the present invention provides a micelle having a contrast agent encapsulated therein, comprising a multiblock copolymer which comprises a polymeric hydrophilic block, optionally a poly(amino acid block) that is optionally crosslinkable or crosslinked, and another poly(amino acid) block, characterized in that said micelle has an inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic shell.
2. Definitions:
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, 75th 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”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
As used herein, the term “contrast agent” (also known as “contrast media” and “radiocontrast agents”) refers to a compound used to improve the visibility of internal bodily structures during imaging. Such imaging methods are well known to one of ordinary skill in the art and include MRI, PET, ultrasound, X-ray, computed tomography, or Fluorescence imaging. Such agents include semiconductor materials, such as CdSe, CdS, CdTe, PdSe, CdSe/CdS, CdSe/ZnS, CdS/ZnS, and CdTe/ZnS. Contrast agents also include magnetic materials such as: Fe, Fe2O3, Fe3O4, MnFe2O4, CoFe2O4, NiFe2O4, Co, Ni, FePt, CoPt, CoO, Fe3Pt, Fe2Pt, CO3Pt, CO2Pt, and FeOOH.
As used herein, the term “sequential polymerization”, and variations thereof, refers to the method wherein, after a first monomer (e.g. NCA, lactam, or imide) is incorporated into the polymer, thus forming an amino acid “block”, a second monomer (e.g. NCA, lactam, or imide) is added to the reaction to form a second amino acid block, which process may be continued in a similar fashion to introduce additional amino acid blocks into the resulting multi-block copolymers.
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.
As used herein, the term “triblock copolymer” refers to a polymer comprising one synthetic polymer portion and two poly(amino acid) portions.
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.
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 second (i.e., terminal) 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. It is contemplated that the X′′ block can be a mixed block.
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.
As used herein, the terms “contrast agent-loaded” and “encapsulated”, and derivatives thereof, are used interchangeably. In accordance with the present invention, a “contrast agent-loaded” micelle refers to a micelle having a contrast agent situated within the core of the micelle. This is also referred to as a contrast agent being “encapsulated” within the micelle.
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 polyethylene oxide (also referred to as polyethylene glycol or PEG), and derivatives thereof, poly(N-vinyl-2-pyrolidone), and derivatives thereof, 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.
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 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 other embodiments, an amino acid block comprises one or more monomers such that the overall block is hydrophobic. 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.
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 occurring 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.
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 occurring in proteins, as described above. Such amino acids include the D-isomer of any of the 20 naturally occurring amino acids. Unnatural amino acids also include homoserine, DOPA (also referred to as levodopa or 3,4-dihydroxy phenyl alanine), 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.
As used herein, the phrase “living polymer chain-end” refers to the terminus resulting from a polymerization reaction which maintains the ability to react further with additional monomer or with a polymerization terminator.
As used herein, the term “termination” refers to attaching a terminal group to a polymer chain-end by the reaction of a living polymer with an appropriate compound. Alternatively, the term “termination” may refer to attaching a terminal group to an amine or hydroxyl end, or derivative thereof, of the polymer chain.
As used herein, the term “polymerization terminator” is used interchangeably with the term “polymerization terminating agent” and refers to a compound that reacts with a living polymer chain-end to afford a polymer with a terminal group. Alternatively, the term “polymerization terminator” may refer to a compound that reacts with an amine or hydroxyl end, or derivative thereof, of the polymer chain, to afford a polymer with a terminal group.
As used herein, the term “polymerization initiator” refers to a compound, which reacts with, or whose anion or free base form reacts with, the desired monomer in a manner which results in polymerization of that monomer. In certain embodiments, the polymerization initiator is the compound that reacts with an alkylene oxide to afford a polyalkylene oxide block. In other embodiments, the polymerization initiator is the amine salt described herein.
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.
The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. This includes any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen, or; a substitutable nitrogen of a heterocyclic ring including ═N— as in 3,4-dihydro-2H-pyrrolyl, —NH— as in pyrrolidinyl, or ═N(R†)— as in N-substituted pyrrolidinyl.
The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”.
As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4Ro; —(CH2)0-4ORo; —O—(CH2)0-4C(O)ORo; —(CH2)0-4—CH(ORo)2; —(CH2)0-4SRo; —(CH2)0-4Ph, which may be substituted with Ro; —(CH2)0-4O(CH2)0-1Ph which may be substituted with Ro; —CH═CHPh, which may be substituted with Ro; —NO2; —CN; —N3; —(CH2)0-4N(Ro)2; —(CH2)0-4N(Ro)C(O)Ro; —N(Ro)C(S)Ro; —(CH2)0-4N(Ro)C(O)NRo2; —N(Ro)C(S)NRo2; —(CH2)0-4N(Ro)C(O)ORo; —N(Ro)N(Ro)C(O)Ro; —N(Ro)N(Ro)C(O)NRo2; —N(Ro)N(Ro)C(O)ORo; —(CH2)0-4C(O)Ro; —C(S)Ro; —(CH2)0-4C(O)ORo; —(CH2)0-4C(O)SRo; —(CH2)0-4C(O)OSiRo3; —(CH2)0-4OC(O)Ro; —OC(O)(CH2)0-4SR—, SC(S)SRo; —(CH2)0-4SC(O)Ro; —(CH2)0-4C(O)NRo2; —C(S)NRo2; —C(S)SRo; —SC(S)SRo, —(CH2)0-4OC(O)NRo2; —C(O)N(ORo)Ro; —C(O)C(O)Ro; —C(O)CH2C(O)Ro; —C(NORo)Ro; —(CH2)0-4SSRo; —(CH2)0-4S(O)2Ro; —(CH2)0-4S(O)2ORo; —(CH2)0-4OS(O)2Ro; —S(O)2NRo2; —(CH2)0-4S(O)Ro; —N(Ro)S(O)2NRo2; —N(Ro)S(O)2Ro; —N(ORo)Ro; —C(NH)NRo2; —P(O)2Ro; —P(O)Ro2; —OP(O)Ro2; —OP(O)(ORo)2; SiRo3; —(C1-4 straight or branched alkylene)O—N(Ro)2; or —(C1-4 straight or branched alkylene)C(O)O—N(Ro)2, wherein each Ro may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Ro, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.
Suitable monovalent substituents on Ro (or the ring formed by taking two independent occurrences of Ro together with their intervening atoms), are independently halogen, —(CH2)0-2R•, -(haloR•), —(CH2)0-2OH, —(CH2)0-2OR•, —(CH2)0-2CH(OR•)2; —O(haloR•), —CN, —N3, —(CH2)0-2C(O)R•, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR•, —(CH2)0-2SR•, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR•, —(CH2)0-2NR•2, —NO2, —SiR•3, —OSiR•3, —C(O)SR•, —(C1-4 straight or branched alkylene)C(O)OR•, or —SSR• wherein each R• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of Ro include ═O and ═S.
Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. A suitable tetravalent substituent that is bound to vicinal substitutable methylene carbons of an “optionally substituted” group is the dicobalt hexacarbonyl cluster represented by




when depicted with the methylenes which bear it.

Suitable substituents on the aliphatic group of R* include halogen, —R•, -(haloR•), —OH, —OR•, —O(halo•), —CN, —C(O)OH, —C(O)OR•, —NH2, —NHR•, —NR•2, or —NO2, wherein each R• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R†, —NR†2, —C(O)R†, —C(O)OR†, —C(O)C(O)R†, —C(O)CH2C(O)R†, —S(O)2R†, —S(O)2NR†2, —C(S)NR†2, —C(NH)NR†2, or —N(R†)S(O)2R†; wherein each R† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R† are independently halogen, —R•, -(haloR•), —OH, —ORE, —O(haloR•), —CN, —C(O)OH, —C(O)OR•, —NH2, —NHR•, —NR•2, or —NO2, wherein each R• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Protected hydroxyl groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, proprionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.
Protected amines are well known in the art and include those described in detail in Greene (1999). Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allyl amines, amides, and the like. Examples of suitable mono-protected amino moieties include t-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (—NHCBZ), allylamino, benzylamino (—NHBn), fluorenylmethylcarbonyl (—NHFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like. Suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the like. Suitable di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.
Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl)acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones, and derivatives thereof.
Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Suitable protected carboxylic acids further include, but are not limited to, optionally substituted C1-6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted. Additional suitable protected carboxylic acids include oxazolines and ortho esters.
Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.
A “crown ether moiety” is the radical of a crown ether. A crown ether is a monocyclic polyether comprised of repeating units of —CH2CH2O—. Examples of crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6.
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.
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.
“Primary” labels include radioisotope-containing moieties (e.g., moieties that contain 32P, 33P, 35S, or 14C), mass-tags, and fluorescent labels, and are signal-generating reporter groups which can be detected without further modifications.
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. Fe3O4 and Fe2O3) 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.
“Secondary” labels include moieties such as biotin, or protein antigens, that require the presence of a second compound to produce a detectable signal. For example, in the case of a biotin label, the second compound may include streptavidin-enzyme conjugates. In the case of an antigen label, the second compound may include an antibody-enzyme conjugate. Additionally, certain fluorescent groups can act as secondary labels by transferring energy to another compound or group in a process of nonradiative fluorescent resonance energy transfer (FRET), causing the second compound or group to then generate the signal that is detected.
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.
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.
The term “mass-tag” as used herein refers to any moiety that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques. Examples of mass-tags include electrophore release tags such as N-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecotic Acid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives. The synthesis and utility of these mass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016, 5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270. Other examples of mass-tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition. A large variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds) of an appropriate mass range (100-2000 Daltons) may also be used as mass-tags.
The term “substrate”, as used herein refers to any material or macromolecular complex to which a functionalized end-group of a block copolymer can be attached. Examples of commonly used substrates include, but are not limited to, glass surfaces, silica surfaces, plastic surfaces, metal surfaces, surfaces containing a metallic or chemical coating, membranes (e.g., nylon, polysulfone, silica), micro-beads (e.g., latex, polystyrene, or other polymer), porous polymer matrices (e.g., polyacrylamide gel, polysaccharide, polymethacrylate), macromolecular complexes (e.g., protein, polysaccharide).
3. Description of Exemplary Embodiments:
A. Multiblock Copolymers
As described generally above, one embodiment of the present invention provides a micelle having a contrast agent encapsulated therein, comprising a multiblock copolymer which comprises a polymeric hydrophilic block, optionally a poly(amino acid block) that is optionally crosslinkable or crosslinked, and another poly(amino acid) block, characterized in that said micelle has an inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic shell.
Amphiphilic multiblock copolymers, as described herein, can self-assemble in aqueous solution to form nano- and micron-sized structures. In water, these amphiphilic multiblock copolymers assemble by multi-molecular micellization when present in solution above the critical micelle concentration (CMC). Without wishing to be bound by any particular theory, it is believed that the hydrophobic poly(amino acid) portion or “block” of the copolymer collapses to form the micellar core, while the hydrophilic PEG block forms a peripheral corona and imparts water solubility. In certain embodiments, the multiblock copolymers in accordance with the present invention possess distinct hydrophobic and hydrophilic segments that form micelles. In addition, these multiblock polymers comprise a poly(amino acid) block which contains functionality suitable for crosslinking. It will be appreciated that this functionality is found on the corresponding amino acid side-chain.
Multiblock copolymers of the present invention contain one or more poly(amino acid) blocks and a water-soluble polymer block. Poly(amino acid) (PAA) segments possess a wide range of functionality and are natural building blocks with inherent biocompatibility. In addition, PAA copolymers are hydrolytically stable and can tolerate most chemical transformation conditions yet can be enzymatically degradable.
In certain embodiments, the PEG block possesses a molecular weight of approx. 10,000 Da (225 repeat units) and contains at least one terminal ammonium salt used to initiate the synthesis of poly(amino acid) multi-block copolymers. In some embodiments, the PEG block possesses a molecular weight of approx. 12,000 Da (270 repeat units) and contains at least one terminal ammonium salt used to initiate the synthesis of poly(amino acid) multi-block copolymers. Without wishing to be bound by theory, it is believed that this particular PEG chain length imparts adequate water-solubility to the micelles and provides relatively long in vivo circulation times.
In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer of formula I:




    
    
        wherein:
        
            n is 10-2500;
            m is 0 to 1000;
            m′ is 1 to 1000;
            Rx is a natural or unnatural amino acid side-chain group that is capable of crosslinking;
            Ry is a hydrophobic or ionic, natural or unnatural amino acid side-chain group;
            R1 is -Z(CH2CH2Y)p(CH2)tR3, wherein:
            
                Z is —O—, —S—, —C≡C—, or —CH2—;
                each Y is independently —O— or —S—;
                p is 0-10;
                t is 0-10; and
            
            
            R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
            Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
            each R4 is independently an optionally substituted group selected from hydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
            
                two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
            
            
        
        
    
    


As defined generally above, the n group of formula I is 10-2500. 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 275. In other embodiments, n is about 350. In other embodiments, n is about 10 to about 40. In other embodiments, n is about 40 to about 60. In other embodiments, n is about 60 to about 90. In still other embodiments, n is about 90 to about 150. In other embodiments, n is about 150 to about 200. In still other embodiments, n is about 200 to about 250. In other embodiments, n is about 300 to about 375. In other embodiments, n is about 400 to about 500. In still other embodiments, n is about 650 to about 750. In certain embodiments, n is selected from 50±10. In other embodiments, n is selected from 80±10, 115±10, 180±10, 225±10, 275±10, 315±10, or 340±10.
According to another embodiment, the present invention provides a compound of formula I, as described above, wherein said compound has a polydispersity index (“PDI”) of about 1.0 to about 1.2. According to another embodiment, the present invention provides a compound of formula I, as described above, wherein said compound has a polydispersity index (“PDI”) of about 1.02 to about 1.05. According to yet another embodiment, the present invention provides a compound of formula I, as described above, wherein said compound has a polydispersity index (“PDI”) of about 1.05 to about 1.10. In other embodiments, said compound has a PDI of about 1.01 to about 1.03. In other embodiments, said compound has a PDI of about 1.10 to about 1.15. In still other embodiments, said compound has a PDI of about 1.15 to about 1.20.
In certain embodiments, the m′ group of formula I is about 5 to about 500. In certain embodiments, the m′ group of formula I is about 10 to about 250. In other embodiments, m′ is about 10 to about 50. According to yet another embodiment, m′ is about 15 to about 40. In other embodiments, m′ is about 20 to about 40. According to yet another embodiment, m′ is about 50 to about 75. According to other embodiments, m and m′ are independently about 10 to about 100. In certain embodiments, m is 5-50. In other embodiments, m is 5-25. In certain embodiments, m′ is 5-50. In other embodiments, m′ is 5-10. In other embodiments, m′ is 10-20. In certain embodiments, m and m′ add up to about 30 to about 60. In still other embodiments, m is 1-20 repeat units and m′ is 10-50 repeat units.
In certain embodiments, the m group of formula I is zero, thereby forming a diblock copolymer.
In certain embodiments, the R3 moiety of the R1 group of formula I is —N3.
In certain embodiments, the R3 moiety of the R1 group of formula I is —OCH3.
In other embodiments, the R3 moiety of the R1 group of formula I is —CN.
In still other embodiments, the R3 moiety of the R1 group of formula I is a mono-protected amine or a di-protected amine.
In certain embodiments, R3 is hydrogen.
In certain embodiments, the R3 moiety of the R1 group of formula I is an optionally substituted aliphatic group. Examples include t-butyl, 5-norbornene-2-yl, octane-5-yl, acetylenyl, trimethylsilylacetylenyl, triisopropylsilylacetylenyl, and t-butyldimethylsilylacetylenyl. In some embodiments, said R3 moiety is an optionally substituted alkyl group. In other embodiments, said R3 moiety is an optionally substituted alkynyl or alkenyl group. When said R3 moiety is a substituted aliphatic group, suitable substituents on R3 include CN, N3, trimethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, N-methyl propiolamido, N-methyl-4-acetylenylanilino, N-methyl-4-acetylenylbenzoamido, bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino, di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy, pent-4-ynyloxy, di-but-3-ynyloxy, N-methyl-propargylamino, N-methyl-hex-5-ynyl-amino, N-methyl-pent-4-ynyl-amino, N-methyl-but-3-ynyl-amino, 2-hex-5-ynyldisulfanyl, 2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl, and 2-propargyldisulfanyl. In certain embodiments, the R1 group is 2-(N-methyl-N-(ethynylcarbonyl)amino)ethoxy, 4-ethynylbenzyloxy, or 2-(4-ethynylphenoxy)ethoxy.
In certain embodiments, the R3 moiety of the R1 group of formula I is an optionally substituted aryl group. Examples include optionally substituted phenyl and optionally substituted pyridyl. When said R3 moiety is a substituted aryl group, suitable substituents on R3 include CN, N3, NO2, —CH3, —CH2N3, —CH═CH2, —C≡CH, Br, I, F, bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino, di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy, pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl, 2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl, 2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl, [1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.
In other embodiments, the R3 moiety is an aryl group substituted with a suitably protected amino group. According to another aspect, the R3 moiety is phenyl substituted with a suitably protected amino group.
In other embodiments, the R3 moiety of the R1 group of formula I is a protected hydroxyl group. In certain embodiments the protected hydroxyl of the R3 moiety is an ester, carbonate, sulfonate, allyl ether, ether, silyl ether, alkyl ether, arylalkyl ether, or alkoxyalkyl ether. In certain embodiments, the ester is a formate, acetate, proprionate, pentanoate, crotonate, or benzoate. Exemplary esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benzylbenzoate, 2,4,6-trimethylbenzoate. Exemplary carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers. Exemplary alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof. Exemplary alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether. Exemplary arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.
In certain embodiments, the R3 moiety of the R1 group of formula I is a mono-protected or di-protected amino group. In certain embodiments R3 is a mono-protected amine. In certain embodiments R3 is a mono-protected amine selected from aralkylamines, carbamates, allyl amines, or amides. Exemplary mono-protected amino moieties include t-butyloxycarbonylamino, ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxy-carbonylamino, allyloxycarbonylamino, benzyloxocarbonylamino, allylamino, benzylamino, fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In other embodiments R3 is a di-protected amine. Exemplary di-protected amines include di-benzylamine, di-allylamine, phthalimide, maleimide, succinimide, pyrrole, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, and azide. In certain embodiments, the R3 moiety is phthalimido. In other embodiments, the R3 moiety is mono- or di-benzylamino or mono- or di-allylamino. In certain embodiments, the R1 group is 2-dibenzylaminoethoxy.
In other embodiments, the R3 moiety of the R1 group of formula I is a protected aldehyde group. In certain embodiments the protected aldehydro moiety of R3 is an acyclic acetal, a cyclic acetal, a hydrazone, or an imine. Exemplary R3 groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl)acetal, 1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain embodiments, R3 is an acyclic acetal or a cyclic acetal. In other embodiments, R3 is a dibenzyl acetal.
In yet other embodiments, the R3 moiety of the R1 group of formula I is a protected carboxylic acid group. In certain embodiments, the protected carboxylic acid moiety of R3 is an optionally substituted ester selected from C1-6aliphatic or aryl, or a silyl ester, an activated ester, an amide, or a hydrazide. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester. In other embodiments, the protected carboxylic acid moiety of R3 is an oxazoline or an ortho ester. Examples of such protected carboxylic acid moieties include oxazolin-2-yl and 2-methoxy-[1,3]dioxin-2-yl. In certain embodiments, the R1 group is oxazolin-2-ylmethoxy or 2-oxazolin-2-yl-1-propoxy.
According to another embodiments, the R3 moiety of the R1 group of formula I is a protected thiol group. In certain embodiments, the protected thiol of R3 is a disulfide, thioether, silyl thioether, thioester, thiocarbonate, or a thiocarbamate. Examples of such protected thiols include triisopropylsilyl thioether, t-butyldimethylsilyl thioether, t-butyl thioether, benzyl thioether, p-methylbenzyl thioether, triphenylmethyl thioether, and p-methoxyphenyldiphenylmethyl thioether. In other embodiments, R3 is an optionally substituted thioether selected from alkyl, benzyl, or triphenylmethyl, or trichloroethoxycarbonyl thioester. In certain embodiments, R3 is —S—S-pyridin-2-yl, —S—SBn, —S—SCH3, or —S—S(p-ethynylbenzyl). In other embodiments, R3 is —S—S-pyridin-2-yl. In still other embodiments, the R1 group is 2-triphenylmethylsulfanyl-ethoxy.
In certain embodiments, the R3 moiety of the R1 group of formula I is a crown ether. Examples of such crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6.
In still other embodiments, the R3 moiety of the R1 group of formula I is a detectable moiety. According to one aspect of the invention, the R3 moiety of the R1 group of formula I is a fluorescent moiety. Such fluorescent moieties are well known in the art and include coumarins, quinolones, benzoisoquinolones, hostasol, and Rhodamine dyes, to name but a few. Exemplary fluorescent moieties of the R3 group of R1 include anthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylate of rhodamine B, and the carboxylate of coumarin 343. In certain embodiments, the R3 moiety of the R1 group of formula I is a detectable moiety selected from:







In certain embodiments, the R3 moiety of the R1 group of formula I is a group suitable for Click chemistry. Click reactions tend to involve high-energy (“spring-loaded”) reagents with well-defined reaction coordinates, giving rise to selective bond-forming events of wide scope. Examples include the nucleophilic trapping of strained-ring electrophiles (epoxide, aziridines, aziridinium ions, episulfonium ions), certain forms of carbonyl reactivity (aldehydes and hydrazines or hydroxylamines, for example), and several types of cycloaddition reactions. The azide-alkyne 1,3-dipolar cycloaddition is one such reaction. Click chemistry is known in the art and one of ordinary skill in the art would recognize that certain R3 moieties of the present invention are suitable for Click chemistry.
Compounds of formula I having R3 moieties suitable for Click chemistry are useful for conjugating said compounds to biological systems or macromolecules such as proteins, viruses, and cells, to name but a few. The Click reaction is known to proceed quickly and selectively under physiological conditions. In contrast, most conjugation reactions are carried out using the primary amine functionality on proteins (e.g. lysine or protein end-group). Because most proteins contain a multitude of lysines and arginines, such conjugation occurs uncontrollably at multiple sites on the protein. This is particularly problematic when lysines or arginines are located around the active site of an enzyme or other biomolecule. Thus, another embodiment of the present invention provides a method of conjugating the R1 groups of a compound of formula I to a macromolecule via Click chemistry. Yet another embodiment of the present invention provides a macromolecule conjugated to a compound of formula I via the R1 group.
According to one embodiment, the R3 moiety of the R1 group of formula I is an azide-containing group. According to another embodiment, the R3 moiety of the R1 group of formula I is an alkyne-containing group. In certain embodiments, the R3 moiety of the R1 group of formula I has a terminal alkyne moiety. In other embodiments, R3 moiety of the R1 group of formula I is an alkyne moiety having an electron withdrawing group. Accordingly, in such embodiments, the R3 moiety of the R1 group of formula I is




wherein E is an electron withdrawing group and y is 0-6. Such electron withdrawing groups are known to one of ordinary skill in the art. In certain embodiments, E is an ester. In other embodiments, the R3moiety of the R1 group of formula I is





wherein E is an electron withdrawing group, such as a —C(O)O— group and y is 0-6.

As defined generally above, Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, Q is a valence bond. In other embodiments, Q is a bivalent, saturated C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, or —C(O)—, wherein -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
In certain embodiments, Q is -Cy- (i.e. a C1 alkylene chain wherein the methylene unit is replaced by -Cy-), wherein -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to one aspect of the present invention, -Cy- is an optionally substituted bivalent aryl group. According to another aspect of the present invention, -Cy- is an optionally substituted bivalent phenyl group. In other embodiments, -Cy- is an optionally substituted 5-8 membered bivalent, saturated carbocyclic ring. In still other embodiments, -Cy- is an optionally substituted 5-8 membered bivalent, saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy- groups include bivalent rings selected from phenyl, pyridyl, pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.
In certain embodiments, Rx is a crosslinkable amino acid side-chain group and Ry is a hydrophobic amino acid side-chain group. Such crosslinkable amino acid side-chain groups include tyrosine, serine, cysteine, threonine, aspartic acid (also known as aspartate, when charged), glutamic acid (also known as glutamate, when charged), asparagine, histidine, lysine, arginine, and glutamine. Such hydrophobic amino acid side-chain groups include a suitably protected tyrosine side-chain, a suitably protected serine side-chain, a suitably protected threonine side-chain, phenylalanine, alanine, valine, leucine, tryptophan, proline, benzyl and alkyl glutamates, or benzyl and alkyl aspartates or mixtures thereof. In other embodiments, Ry is an ionic amino acid side-chain group. Such ionic amino acid side chain groups includes a lysine side-chain, arginine side-chain, or a suitably protected lysine or arginine side-chain, an aspartic acid side chain, glutamic acid side-chain, or a suitably protected aspartic acid or glutamic acid side-chain. 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 tyrosine nonpolar and hydrophobic by virtue of protecting the hydroxyl group. Suitable protecting groups for the hydroxyl, amino, and thiol, and carboylate functional groups of Rx and Ry are as described herein.
In other embodiments, Ry comprises a mixture of hydrophobic and hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising Ry is hydrophobic. Such mixtures of amino acid side-chain groups include phenylalanine/tyrosine, phenalanine/serine, leucine/tyrosine, and the like. According to another embodiment, Ry is a hydrophobic amino acid side-chain group selected from phenylalanine, alanine, or leucine, and one or more of tyrosine, serine, or threonine.
As defined above, Rx is a natural or unnatural amino acid side-chain group capable of forming cross-links. It will be appreciated that a variety of amino acid side-chain functional groups are capable of such cross-linking, including, but not limited to, carboxylate, hydroxyl, thiol, and amino groups. Examples of Rx moieties having functional groups capable of forming cross-links include a glutamic acid side-chain, —CH2C(O)CH, an aspartic acid side-chain, —CH2CH2C(O)OH, a cysteine side-chain, —CH2SH, a serine side-chain, —CH2OH, an aldehyde containing side-chain, —CH2C(O)H, a lysine side-chain, —(CH2)4NH2, an arginine side-chain, —(CH2)3NHC(═NH)NH2, a histidine side-chain, —CH2-imidazol-4-yl, or benzimidazolyl.
As defined generally above, the R2a group of formula I is a mono-protected amine, a di-protected amine, —NHR4, —N(R4)2, —NHC(O)R4, —NR4C(O)R4, —NHC(O)NHR4, —NHC(O)N(R4)2, —NR4C(O)NHR4, —NR4C(O)N(R4)2, —NHC(O)OR4, —NR4C(O)OR4, —NHSO2R4, or —NR4SO2R4, wherein each R4 is independently an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10-membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
In certain embodiments, the R2a group of formula I is —NHR4 or —N(R4)2 wherein each R4 is an optionally substituted aliphatic group. One exemplary R4 group is 5-norbornen-2-yl-methyl. According to yet another aspect of the present invention, the R2a group of formula I is —NHR4 wherein R4 is a C1-6 aliphatic group substituted with N3. Examples include —CH2N3. In some embodiments, R4 is an optionally substituted C1-6 alkyl group. Examples include methyl, ethyl, propyl, butyl, pentyl, hexyl, 2-(tetrahydropyran-2-yloxy)ethyl, pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl, (4-acetylenylphenyl)methyl, 3-(methoxycarbonyl)-prop-2-ynyl, methoxycarbonylmethyl, 2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl, 2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl, 4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl, 4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl, 4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl, 2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl, 2-(N-methyl-N-propargylamino)ethyl, and 2-(2-dipropargylaminoethoxy)-ethyl. In other embodiments, R4 is an optionally substituted C2-6 alkenyl group. Examples include vinyl, allyl, crotyl, 2-propenyl, and but-3-enyl. When R4 group is a substituted aliphatic group, suitable substituents on R4 include N3, CN, and halogen. In certain embodiments, R4 is —CH2CN, —CH2CH2CN, —CH2CH(OCH3)2, 4-(bisbenzyloxymethyl)phenylmethyl, and the like.
According to another aspect of the present invention, the R2a group of formula I is —NHR4 wherein R4 is an optionally substituted C2-6 alkynyl group. Examples include —CC≡CH, —CH2C≡CH, —CH2C≡CCH3, and —CH2CH2C≡CH.
In certain embodiments, the R2a group of formula I is —NHR4 wherein R4 is an optionally substituted 5-8-membered aryl ring. In certain embodiments, R4 is optionally substituted phenyl or optionally substituted pyridyl. Examples include phenyl, 4-t-butoxycarbonylaminophenyl, 4-azidomethylphenyl, 4-propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certain embodiments, R2a is 4-t-butoxycarbonylaminophenylamino, 4-azidomethylphenamino, or 4-propargyloxyphenylamino.
In certain embodiments, the R2a group of formula I is —NHR4 wherein R4 is an optionally substituted phenyl ring. Suitable substituents on the R4 phenyl ring include halogen; —(CH2)0-4Ro; —(CH2)0-4ORo; —(CH2)0-4CH(ORo)2; —(CH2)0-4SRo; —(CH2)0-4Ph, which may be substituted with Ro; —(CH2)0-4O(CH2)0-1Ph which may be substituted with Ro; —CH═CHPh, which may be substituted with Ro; —NO2; —CN; —N3; —(CH2)0-4N(Ro)2; —(CH2)0-4N(Ro)C(O)Ro; —N(Ro)C(S)Ro; —(CH2)0-4N(Ro)C(O)NRo2; —N(Ro)C(S)NRo2; —(CH2)0-4N(Ro)C(O)ORo; —N(Ro)N(Ro)C(O)Ro; —N(Ro)N(Ro)C(O)NRo2; —N(Ro)N(Ro)C(O)ORo; —(CH2)0-4C(O)Ro; —C(S)Ro; —(CH2)0-4C(O)ORo; —(CH2)0-4C(O)SRo; —(CH2)0-4C(O)OSiRo3; —(CH2)0-4OC(O)Ro; —(CH2)0-4SC(O)Ro; —(CH2)0-4C(O)NRo2; —C(S)NRo2; —(CH2)0-4OC(O)NRo2; —C(O)N(ORo)Ro; —C(O)C(O)Ro; —C(O)CH2C(O)Ro; —C(NORo)Ro; —(CH2)0-4SSRo; —(CH2)0-4S(O)2Ro; —(CH2)0-4S(O)2ORo; —(CH2)0-4OS(O)2Ro; —S(O)2NRo2; —(CH2)0-4S(O)Ro; —N(Ro)S(O)2NRo2; —N(Ro)S(O)2Ro; —N(ORo)Ro; —C(NH)NRo2; —P(O)2Ro; —P(O)Ro2; —OP(O)Ro2; SiRo3; wherein each independent occurrence of Ro is as defined herein supra. In other embodiments, the R2a group of formula I is —NHR4 wherein R4 is phenyl substituted with one or more optionally substituted C1-6 aliphatic groups. In still other embodiments, R4 is phenyl substituted with vinyl, allyl, acetylenyl, —CH2N3, —CH2CH2N3, —CH2C≡CCH3, or —CH2C≡CH.
In certain embodiments, the R2a group of formula I is —NHR4 wherein R4 is phenyl substituted with N3, N(Ro)2, CO2Ro, or C(O)Ro wherein each Ro is independently as defined herein supra.
In certain embodiments, the R2a group of formula I is —N(R4)2 wherein each R4 is independently an optionally substituted group selected from aliphatic, phenyl, naphthyl, a 5-6 membered aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 8-10 membered bicyclic aryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety.
In other embodiments, the R2a group of formula I is —N(R4)2 wherein the two R4 groups are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. According to another embodiment, the two R4 groups are taken together to form a 5-6-membered saturated or partially unsaturated ring having one nitrogen wherein said ring is substituted with one or two oxo groups. Such R2a groups include, but are not limited to, phthalimide, maleimide and succinimide.
In certain embodiments, the R2a group of formula I is a mono-protected or di-protected amino group. In certain embodiments R2a is a mono-protected amine. In certain embodiments R2a is a mono-protected amine selected from aralkylamines, carbamates, allyl amines, or amides. Exemplary mono-protected amino moieties include t-butyloxycarbonylamino, ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxy-carbonylamino, allyloxycarbonylamino, benzyloxocarbonylamino, allylamino, benzylamino, fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In other embodiments R2a is a di-protected amine. Exemplary di-protected amino moieties include di-benzylamino, di-allylamino, phthalimide, maleimido, succinimido, pyrrolo, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidino, and azido. In certain embodiments, the R2a moiety is phthalimido. In other embodiments, the R2a moiety is mono- or di-benzylamino or mono- or di-allylamino.
In certain embodiments, the R2a group of formula I comprises a group suitable for Click chemistry. One of ordinary skill in the art would recognize that certain R2a groups of the present invention are suitable for Click chemistry.
Compounds of formula I having R2a groups comprising groups suitable for Click chemistry are useful for conjugating said compounds to biological systems such as proteins, viruses, and cells, to name but a few. After conjugation to a biomolecule, drug, cell, substrate, or the like, the other end-group functionality, corresponding to the R1 moiety of formula I, can be used to attach targeting groups for cell specific delivery including, but not limited to, fluorescent dyes, covalent attachment to surfaces, and incorporation into hydrogels. Thus, another embodiment of the present invention provides a method of conjugating the R2a group of a compound of formula I to a fluorescent dye, small molecule drug, or macromolecule via Click chemistry. Yet another embodiment of the present invention provides a macromolecule conjugated to a compound of formula I via the R2a group.
According to one embodiment, the R2a group of formula I is an azide-containing group. According to another embodiment, the R2a group of formula I is an alkyne-containing group.
In certain embodiments, the R2a group of formula I has a terminal alkyne moiety. In other embodiments, the R2a group of formula I is an alkyne-containing moiety having an electron withdrawing group. Accordingly, in such embodiments, the R group of formula I is




wherein E is an electron withdrawing group and y is 0-6. Such electron withdrawing groups are known to one of ordinary skill in the art. In certain embodiments, E is an ester. In other embodiments, the R2a group of formula I is





wherein E is an electron withdrawing group, such as a —C(O)O— group and y is 0-6.

In other embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises multiblock copolymer of formula II:




    
    
        wherein:
        
            n is 10-2500;
            m is 1 to 1000;
            m′ is 1 to 1000;
            Rx is a crosslinked natural or unnatural amino acid side-chain group;
            Ry is a hydrophobic or ionic, natural or unnatural, amino acid side-chain group;
            R1 is -Z(CH2CH2Y)p(CH2)tR3, wherein:
            
                Z is —O—, —S—, —C≡C—, or —CH2—;
                each Y is independently —O— or —S—;
                p is 0-10;
                t is 0-10; and
            
            
            R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
            Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
            each R4 is independently an optionally substituted group selected from hydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
            
                two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
            
            
        
        
    
    


Embodiments for each of R1, Q, n, m, m′, Rx, Ry, and R2a for compounds of formula II are as described herein for compounds of formula I, singly and in combination.
According to another embodiment, the present invention provides compounds of formula II, as described above, wherein said compounds have a polydispersity index (“PDI”) of about 1.0 to about 1.2. According to another embodiment, the present invention provides compounds of formula II, as described above, wherein said compound has a PDI of about 1.03 to about 1.15. According to yet another embodiment, the present invention provides compounds of formula II, as described above, wherein said compound has a PDI of about 1.10 to about 1.20. According to other embodiments, the present invention provides compounds of formula II having a PDI of less than about 1.10.
According to another embodiment, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from those set forth in Tables 1 through 4, below. Table 1 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-50, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.













TABLE 1





Compound
A1
A2
A3
E1
E2




















1






















 


2






















 


3






















 


4






















 


5






















 


6






















 


7






















 


8






















 


9






















 


10






















 


11






















 


12






















 


13






















 


14






















 


15






















 


16






















 


17






















 


18






















 


19






















 


20






















 


21






















 


22






















 


23






















 


24






















 


25






















 


26






















 


27






















 


28






















 


29






















 


30






















 


31






















 


32






















 


33






















 


34






















 


35






















 


36






















 


37






















 


38






















 


39






















 


40






















 


41






















 


42






















 


43






















 


44






















 


45






















 


46






















 


47






















 


48






















 


49






















 


50






















 


51






















 


52






















 


53






















 


54






















 


55






















 


56






















 


57






















 


58






















 


59






















 


60






















 


61






















 


62






















 


63






















 


64






















 


65






















 


66






















 


67






















 


68






















 


69






















 


70






















 


71






















 


72






















 


73






















 


74






















 


75






















 


76






















 


77






















 


78






















 


79






















 


80






















 


81






















 


82






















 


83






















 


84






















 


85






















 


86






















 


87






















 


88






















 


89






















 


90






















 


91






















 


92






















 


93






















 


94






















 


95






















 


96






















 


97






















 


98






















 


99






















 


100






















 


101






















 


102






















 


103






















 


104






















 


105






















 


106






















 


107






















 


108






















 


109






















 


110






















 


111






















 


112






















 


113






















 


114






















 


115






















 


116






















 


117






















 


118






















 


119






















 


120






















 


121






















 


122






















 


123






















 


124






















 


125






















 


126






















 


127






















 


128






















 


129






















 


130






















 


131






















 


132






















 


133






















 


134






















 


135






















 


136






















 


137






















 


138






















 


139






















 


140






















 


141






















 


142






















 


143






















 


144






















 


145






















 


146






















 


147






















 


148






















 


149






















 


150






















 


151






















 


152






















 


153






















 


154






















 


155






















 


156






















 


157






















 


158






















 


159






















 


160






















 


161






















 


162






















 


163






















 


164






















 


165






















 


166






















 


167






















 


168






















 


169






















 


170






















 


171






















 


172






















 


173






















 


174






















 


175






















 


176






















 


177






















 


178






















 


179






















 


180






















 


181






















 


182






















 


183






















 


184






















 


185






















 


186






















 


187






















 


188






















 


189






















 


190






















 


191






















 


192






















 


193






















 


194






















 


195






















 


196






















 


197






















 


198






















 


199






















 


200






















 


201






















 


202






















 


203






















 


204






















 


205






















 


206






















 


207






















 


208






















 


209






















 


210






















 


211






















 


212






















 


213






















 


214






















 


215






















 


216






















 


217






















 


218






















 


219






















 


220






















 


221






















 


222






















 


223






















 


224






















 


225






















 


226






















 


227






















 


228






















 


229






















 


230






















 


231






















 


232






















 


233






















 


234






















 


235






















 


236






















 


237






















 


238






















 


239






















 


240






















 


241






















 


242






















 


243






















 


244






















 


245






















 


246






















 


247






















 


248






















 


249






















 


250






















 


251






















 


252






















 


253






















 


254






















 


255






















 


256






















 


257






















 


258






















 


259






















 


260






















 


261






















 


262






















 


263






















 


264






















 


265






















 


266






















 


267






















 


268






















 


269






















 


270






















 


271






















 


272






















 


273






















 


274






















 


275






















 


276






















 


277






















 


278






















 


279






















 


280






















 


281






















 


282






















 


283






















 


284






















 


285






















 


286






















 


287






















 


288






















 


289






















 


290






















 


291






















 


292






















 


293






















 


294






















 


295






















 


296






















 


297






















 


298






















 


299






















 


300






















 


301






















 


302






















 


303






















 


304






















 


305






















 


306






















 


307






















 


308






















 


309






















 


310






















 


311






















 


312






















 


313






















 


314






















 


315






















 


316






















 


317






















 


318






















 


319






















 


320






















 


321






















 


322






















 


323






















 


324






















 


325






















 


326






















 


327






















 


328






















 


329






















 


330






















 


331






















 


332






















 


333






















 


334






















 


335






















 


336






















 


337






















 


338






















 


339






















 


340






















 


341






















 


342






















 


343






















 


344






















 


345






















 


346






















 


347






















 


348






















 


349






















 


350






















 


351






















 


352






















 


353






















 


354






















 


355






















 


356






















 


357






















 


358






















 


359






















 


360






















 


361






















 


362






















 


363






















 


364






















 


365






















 


366






















 


367






















 


368






















 


369






















 


370






















 


371






















 


372






















 


373






















 


374






















 


375






















 


376






















 


377






















 


378






















 


379






















 


380






















 


381






















 


382






















 


383






















 


384






















 


385






















 


386






















 


387






















 


388






















 


389






















 


390






















 


391






















 


392






















 


393






















 


394






















 


395






















 


396






















 


397






















 


398





























Table 2 sets forth exemplary compounds of the present invention having the formula:




wherein each x is 100-500, each y is 1-50, each z is 1-50, and each dotted bond represents the point of attachment to the rest of the molecule.












TABLE 2





Compound
A1
A2
E1
E2







399


















 


400


















 


401


















 


402


















 


403


















 


404


















 


405


















 


406


















 


407


















 


408


















 


409


















 


410


















 


411


















 


412


















 


413


















 


414


















 


415


















 


416


















 


417


















 


418


















 


419


















 


420


















 


421


















 


422


















 


423


















 


424


















 


425


















 


426


















 


427


















 


428


















 


429


















 


430


















 


431


















 


432


















 


433


















 


434


















 


435


















 


436


















 


437


















 


438


















 


439


















 


440


















 


441


















 


442


















 


443


















 


444


















 


445


















 


446


















 


447


















 


448


















 


449


















 


450


















 


451


















 


452


















 


453


















 


454


















 


455


















 


456


















 


457


















 


458


















 


459


















 


460


















 


461


















 


462


















 


463


















 


464


















 


465


















 


466


















 


467


















 


468


















 


469


















 


470


















 


471


















 


472


















 


473


















 


474


















 


475


















 


476


















 


477


















 


478


















 


479


















 


480


















 


481


















 


482


















 


483


















 


484


















 


485


















 


486


















 


487


















 


488


















 


489


















 


490


















 


491


















 


492

























Table 3 sets forth exemplary compounds of the present invention having the formula:




wherein each v is 100-500, each w is 4-20, x is 4-20, each y is 5-50, each z is 5-50, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.














TABLE 3





Compound
A1
A2
A3
A4
E1
E2







493


























 


494


























 


495


























 


496


























 


497


























 


498


























 


499


























 


500


























 


501


























 


502


























 


503


























 


504


























 


505


























 


506


























 


507


























 


508


























 


509


























 


510


























 


511


























 


512

































Table 4 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, y is 1-50, each z is 1-100, and each dotted bond represents the point of attachment to the rest of the molecule.













TABLE 4





Compound
A1
A2
A3
E1
E2




















513






















 


514






















 


515






















 


516






















 


517






















 


518






















 


519






















 


520






















 


521






















 


522






















 


523






















 


524






















 


525






















 


526






















 


527






















 


528






















 


529






















 


530






















 


531






















 


532






















 


533






















 


534






















 


535






















 


536






















 


537






















 


538






















 


539






















 


540






















 


541






















 


542






















 


543






















 


544






















 


545






















 


546






















 


547






















 


548






















 


549






















 


550






















 


551






















 


552






















 


553






















 


554






















 


555






















 


556






















 


557






















 


558






















 


559






















 


560






















 


561






















 


562






















 


563






















 


564






















 


565






















 


566






















 


567






















 


568






















 


569






















 


570






















 


571






















 


572






















 


573






















 


574






















 


575






















 


576






















 


577






















 


578






















 


579






















 


580






















 


581






















 


582






















 


583






















 


584






















 


585






















 


586






















 


587






















 


588






















 


589






















 


590






















 


591






















 


592






















 


593






















 


594






















 


595






















 


596





























According to another embodiment, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises 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 poly(amino acid) block corresponds to the inner core.
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 α-helix or β-sheet. See α-Aminoacid-N-Caroboxy-Anhydrides and Related Heterocycles, H. R. Kricheldorf, Springer-Verlag, 1987. For example, poly(L-benzyl glutatmate) typically exhibits an α-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.
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 the block copolymers consisting of 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.
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.
Thus, in certain embodiments, the Ry group of any of formulae I, II, and III forms a hydrophobic D,L-mixed poly(amino acid) block. Hydrophobic amino acid side-chain groups are well known in the art and include those described herein. In other embodiments, Ry consists of a mixture of D-hydrophobic and L-hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising Ry is hydrophobic and is a mixture of D- and L-configured amino acids. Such mixtures of amino acid side-chain groups include D-leucine/L-tyrosine, D-leucine/L-aspartic acid, D-leucine/L-glutamic acid, D-phenylalanine/L-tyrosine, D-phenylalanine/L-aspartic acid, D-phenylalanine/L-glutamic acid, D-phenylalanine/L-serine, D-benzyl aspartate/L-tyrosine, D-benzyl aspartate/L-aspartic acid, D-benzyl aspartate/L-glutamic acid, D-benzyl glutamate/L-tyrosine, D-benzyl glutamate/L-aspartic acid and the like. According to another embodiment, Ry is a hydrophobic amino acid side-chain group selected from D-leucine, D-phenylalanine, D-alanine, D-benzyl aspartate, or D-benzyl glutamate, and one or more of L-tyrosine, L-cysteine, L-aspartic acid, L-glutamic acid, L-DOPA, L-histidine, L-lysine, or L-ornithine.
In other embodiments, the Ry group of any of formulae I, II, and III forms a mixture of D-hydrophobic and L-hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising Ry is hydrophobic and is a mixture of D- and L-configured amino acids. Such mixtures of amino acid side-chain groups include D-leucine/L-tyrosine, D-leucine/L-aspartic acid, D-leucine/L-glutamic acid, D-phenylalanine/L-tyrosine, D-phenylalanine/L-aspartic acid, D-phenylalanine/L-glutamic acid, D-phenylalanine/L-cysteine, D-benzyl aspartate/L-tyrosine, D-benzyl aspartate/L-aspartic acid, D-benzyl aspartate/L-glutamic acid, D-benzyl glutamate/L-tyrosine, D-benzyl glutamate/L-aspartic acid and the like. Ratios (D-hydrophobic to L-hydrophilic) of such mixtures include 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, and 1:6.
According to another embodiment, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from those set forth in Tables 5 through 11. Table 5 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 5





Compound
A1
A2
A3







597














 


598














 


599














 


600














 


601














 


602














 


603














 


604














 


605














 


606














 


607














 


608














 


609














 


610














 


611














 


612














 


613














 


614














 


615














 


616














 


617














 


618














 


619














 


620














 


621














 


622














 


623














 


624














 


625














 


626














 


627














 


628














 


629














 


630














 


631














 


632














 


633














 


634














 


635














 


636














 


637














 


638














 


639














 


640














 


641














 


642














 


643














 


644














 


645














 


646














 


647














 


648














 


649














 


650














 


651














 


652














 


653














 


654














 


655














 


656














 


657














 


658














 


659














 


660














 


661














 


662














 


663














 


664














 


665














 


666














 


667














 


668














 


669














 


670














 


671














 


672














 


673














 


674














 


675














 


676














 


677














 


678














 


679














 


680














 


681














 


682














 


683














 


684














 


685














 


686














 


687














 


688














 


689














 


690














 


691














 


692














 


693














 


694














 


695














 


696














 


697














 


698














 


699














 


700














 


701














 


702














 


703














 


704














 


705














 


706














 


707














 


708














 


709














 


710














 


711














 


712














 


713














 


714














 


715














 


716














 


717














 


718














 


719














 


720














 


721














 


722














 


723














 


724














 


725














 


726














 


727














 


728














 


729














 


730














 


731














 


732














 


733














 


734














 


735














 


736














 


737














 


738














 


739














 


740














 


741














 


742














 


743














 


744














 


745














 


746














 


747














 


748














 


749














 


750














 


751














 


752














 


753














 


754














 


755














 


756





















Table 6 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 6





Compound
A1
A2
E1







757














 


758














 


759














 


760














 


761














 


762














 


763














 


764














 


765














 


766














 


767














 


768














 


769














 


770














 


771














 


772














 


773














 


774














 


775














 


776














 


777














 


778














 


779














 


780














 


781














 


782














 


783














 


784














 


785














 


786














 


787














 


788














 


789














 


790














 


791














 


792














 


793














 


794














 


795














 


796














 


797














 


798














 


799














 


800














 


801














 


802














 


803














 


804














 


805














 


806














 


807














 


808














 


809














 


810














 


811














 


812














 


813














 


814














 


815














 


816














 


817














 


818














 


819














 


820














 


821














 


822














 


823














 


824














 


825














 


826














 


827














 


828














 


829














 


830














 


831














 


832














 


833














 


834














 


835














 


836














 


837














 


838














 


839














 


840














 


841














 


842














 


843














 


844














 


845














 


846














 


847














 


848














 


849














 


850














 


851














 


852














 


853














 


854














 


855














 


856














 


857














 


858














 


859














 


860














 


861














 


862














 


863














 


864














 


865














 


866














 


867














 


868














 


869














 


870














 


871














 


872














 


873














 


874














 


875














 


876





















Table 7 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 7





Compound
A1
A2
E1


















877














 


878














 


879














 


880














 


881














 


882














 


883














 


884














 


885














 


886














 


887














 


888














 


889














 


890














 


891














 


892














 


893














 


894














 


895














 


896














 


897














 


898














 


899














 


900














 


901














 


902














 


903














 


904














 


905














 


906














 


907














 


908














 


909














 


910














 


911














 


912














 


913














 


914














 


915














 


916














 


917














 


918














 


919














 


920














 


921














 


922














 


923














 


924














 


925














 


926














 


927














 


928














 


929














 


930














 


931














 


932














 


933














 


934














 


935














 


936














 


937














 


938














 


939














 


940














 


941














 


942














 


943














 


944














 


945














 


946














 


947














 


948














 


949














 


950














 


951














 


952














 


953














 


954














 


955














 


956














 


957














 


958














 


959














 


960














 


961














 


962














 


963














 


964














 


965














 


966














 


967














 


968














 


969














 


970














 


971














 


972














 


973














 


974














 


975














 


976














 


977














 


978














 


979














 


980














 


981














 


982














 


983














 


984














 


985














 


986














 


987














 


988














 


989














 


990














 


991














 


992














 


993














 


994














 


995














 


996





















Table 8 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 8





Compound
A1
A2
E1







997














 


998














 


999














 


1000














 


1001














 


1002














 


1003














 


1004














 


1005














 


1006














 


1007














 


1008














 


1009














 


1010














 


1011














 


1012














 


1013














 


1014














 


1015














 


1016














 


1017














 


1018














 


1019














 


1020














 


1021














 


1022














 


1023














 


1024














 


1025














 


1026














 


1027














 


1028














 


1029














 


1030














 


1031














 


1032














 


1033














 


1034














 


1035














 


1036














 


1037














 


1038














 


1039














 


1040














 


1041














 


1042














 


1043














 


1044














 


1045














 


1046














 


1047














 


1048














 


1049














 


1050














 


1051














 


1052














 


1053














 


1054














 


1055














 


1056














 


1057














 


1058














 


1059














 


1060














 


1061














 


1062














 


1063














 


1064














 


1065














 


1066














 


1067














 


1068














 


1069














 


1070














 


1071














 


1072














 


1073














 


1074














 


1075














 


1076














 


1077














 


1078














 


1079














 


1080














 


1081














 


1082














 


1083














 


1084














 


1085














 


1086














 


1087














 


1088














 


1089














 


1090














 


1091














 


1092














 


1093














 


1094














 


1095














 


1096














 


1097














 


1098














 


1099














 


1100














 


1101














 


1102














 


1103














 


1104














 


1105














 


1106














 


1107














 


1108














 


1109














 


1110














 


1111














 


1112














 


1113














 


1114














 


1115














 


1116





















Table 9 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 9





Compound
A1
A2
E1







1117














 


1118














 


1119














 


1120














 


1121














 


1122














 


1123














 


1124














 


1125














 


1126














 


1127














 


1128














 


1129














 


1130














 


1131














 


1132














 


1133














 


1134














 


1135














 


1136














 


1137














 


1138














 


1139














 


1140














 


1141














 


1142














 


1143














 


1144














 


1145














 


1146














 


1147














 


1148














 


1149














 


1150














 


1151














 


1152














 


1153














 


1154














 


1155














 


1156














 


1157














 


1158














 


1159














 


1160














 


1161














 


1162














 


1163














 


1164














 


1165














 


1166














 


1167














 


1168














 


1169














 


1170














 


1171














 


1172














 


1173














 


1174














 


1175














 


1176














 


1177














 


1178














 


1179














 


1180














 


1181














 


1182














 


1183














 


1184














 


1185














 


1186














 


1187














 


1188














 


1189














 


1190














 


1191














 


1192














 


1193














 


1194














 


1195














 


1196














 


1197














 


1198














 


1199














 


1200














 


1201














 


1202














 


1203














 


1204














 


1205














 


1206














 


1207














 


1208














 


1209














 


1210














 


1211














 


1212














 


1213














 


1214














 


1215














 


1216














 


1217














 


1218














 


1219














 


1220














 


1221














 


1222














 


1223














 


1224














 


1225














 


1226














 


1227














 


1228














 


1229














 


1230














 


1231














 


1232














 


1233














 


1234














 


1235














 


1236





















Table 10 sets forth exemplary compounds of the present invention having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 10





Compound
A1
A2
E2







1237














 


1238














 


1239














 


1240














 


1241














 


1242














 


1243














 


1244














 


1245














 


1246














 


1247














 


1248














 


1249














 


1250














 


1251














 


1252














 


1253














 


1254














 


1255














 


1256














 


1257














 


1258














 


1259














 


1260














 


1261














 


1262














 


1263














 


1264














 


1265














 


1266














 


1267














 


1268














 


1269














 


1270














 


1271














 


1272














 


1273














 


1274














 


1275














 


1276














 


1277














 


1278














 


1279














 


1280














 


1281














 


1282














 


1283














 


1284














 


1285














 


1286














 


1287














 


1288














 


1289














 


1290














 


1291














 


1292














 


1293














 


1294














 


1295














 


1296














 


1297














 


1298














 


1299














 


1300














 


1301














 


1302














 


1303














 


1304














 


1305














 


1306














 


1307














 


1308














 


1309














 


1310














 


1311














 


1312














 


1313














 


1314














 


1315














 


1316














 


1317














 


1318














 


1319














 


1320














 


1321














 


1322














 


1323














 


1324














 


1325














 


1326














 


1327














 


1328














 


1329














 


1330














 


1331














 


1332














 


1333














 


1334














 


1335














 


1336














 


1337














 


1338














 


1339














 


1340














 


1341














 


1342














 


1343














 


1344














 


1345














 


1346














 


1347














 


1348














 


1349














 


1350














 


1351














 


1352














 


1353














 


1354














 


1355














 


1356





















In certain embodiments, the present invention provides a micelle comprising a multiblock copolymer of formula I, wherein m is 0 thus forming a compound of formula I-a:




    
    
        wherein:
        
            n is 10-2500;
            m′ is 2 to 1000;
            Ry forms a hydrophobic D,L-mixed poly(amino acid) block;
            R1 is -Z(CH2CH2Y)p(CH2)tR3, wherein:
            
                Z is —O—, —S—, —C≡C—, or —CH2—;
                each Y is independently —O— or —S—;
                p is 0-10;
                t is 0-10; and
            
            
            R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
            Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
            each R4 is independently hydrogen or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
            
                two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,

wherein each of R1, n, m′, Ry, and R2a, is as described herein singly and in combination.

            
            
        
        
    
    


Exemplary compounds of formula I-a include those set forth in Table 11, below, having the formula:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, p is the sum of y and z, and each dotted bond represents the point of attachment to the rest of the molecule.











TABLE 11





Compound
E1
A1
A2


















1357














 


1358














 


1359














 


1360














 


1361














 


1362














 


1363














 


1364














 


1365














 


1366














 


1367














 


1368














 


1369














 


1370














 


1371














 


1372














 


1373














 


1374














 


1375














 


1376














 


1377














 


1378














 


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In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from:




wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is 1-100, and p is the sum of y and z.

In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from:







wherein each w is 150-350, x is 3-20, y is 3-50, z is 3-50, and p is y+z.

In some embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from:







wherein each of w is 150-350, x is 3-20, y is 3-50, z is 3-50, and p is y+z.

In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from:







wherein each of w is 150-350, x is 3-20, y is 3-50, z is 3-50, and p is y+z.

In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, wherein the micelle comprises a multiblock copolymer selected from:










wherein each of w is 150-350, x is 3-20, y is 3-50, z is 3-50, and p is y+z.

B. Crosslinking Chemistries
As described generally above, in certain embodiments, micelles of the present invention, having a contrast agent encapsulated therein, comprise a crosslinkable or crosslinked “outer core.” The crosslinking of poly(amino acid) groups is known in the art and includes methods described in detail in WO2006/107903.
In certain embodiments, micelles of the present invention, having a contrast agent encapsulated therein, comprise a crosslinked multiblock polymer of formula III:




    
    
        wherein:
        
            n is 10-2500;
            m is 1 to 1000;
            m′ is 1 to 1000;
            L is a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of L are independently replaced by -M-, -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -M- is a suitable bivalent metal;
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            Ry is a hydrophobic or ionic, natural or unnatural amino acid side-chain group;
            R1 is -Z(CH2CH2Y)p(CH2)tR3, wherein:
            
                Z is —O—, —S—, —C≡C—, or —CH2—;
                each Y is independently —O— or —S—;
                p is 0-10;
                t is 0-10; and
            
            
            R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
            Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
            each R4 is independently an optionally substituted group selected from hydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
            
                two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
            
            
        
        
    
    


Embodiments for each of R1, Q, n, m, m′, Rx, Ry, and R2a for compounds of formula III are as described herein for compounds of formula I, singly and in combination.
According to another embodiment, the present invention provides compounds of formula III, as described above, wherein said compounds have a PDI of about 1.0 to about 1.2. According to another embodiment, the present invention provides compounds of formula III, as described above, wherein said compound has a PDI of about 1.03 to about 1.15. According to yet another embodiment, the present invention provides compounds of formula I, as described above, wherein said compound has a PDI of about 1.10 to about 1.20. According to other embodiments, the present invention provides compounds of formula III having a PDI of less than about 1.10.
As defined generally above, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-12 alkylene chain, wherein 0-6 methylene units of L are independently replaced by -M-, Cy, —O—, NH—, —S—, —C(O)—, —SO—, —SO2—,NHC(O)—, C(O)NH—, OC(O)NH—, or —NHC(O)O—, wherein -M- is a suitable bivalent metal, and -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It will be appreciated that the L group of formula III represents crosslinked amino acid side-chain groups. In certain embodiments, the crosslinked amino acid side-chain groups correspond to the Rx moiety of compounds of formulae I and II as described herein. In certain embodiments, the L group of formula III represents a metal crosslinked amino acid side-chain group, a hydrazone crosslinked amino acid side-chain group, an ester crosslinked amino acid side-chain group, an amide crosslinked side-chain group, an imine (e.g. Schiff base) crosslinked side-chain group, or a disulfide crosslinked side-chain group.
In certain embodiments, the L group of formula III comprises -M-. In other embodiments, -M- is zinc, calcium, iron or aluminum. In yet other embodiments, -M- is strontium, manganese, palladium, silver, gold, cadmium, chromium, indium, or lead.
In other embodiments, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-12 alkylene chain wherein 2 methylene units of L are independently replaced by —C(O)—, —C(O)NH—, —NHC(O)—, —S—, —C(O)O—, —OC(O)—, —C(O)NHN—, ═NNHC(O), ═N, N═, -M-OC(O)—, or —C(O)O-M-. According to another embodiment, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-6 alkylene chain, wherein two methylene units of L are replaced by —C(O)— or —C(O)NH—. In other embodiments, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-12 alkylene chain having at least 2 units of unsaturation. According to yet another embodiment, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-12 alkylene chain wherein two methylene units of L are replaced by —NH—. According to yet another embodiment, the L group of formula III is a bivalent, saturated or unsaturated, straight or branched C1-12 alkylene chain wherein two methylene units of L are replaced by —C(O)NHN.
In certain embodiments, the -M- moiety of the L group of formula III is zinc. In other embodiments, L forms a zinc-dicarboxylate crosslinking moiety. In certain embodiments, the crosslinking utilizes zinc-mediated coupling of carboxylic acids, a highly selective and pH-sensitive reaction that is performed in water. This reaction, which is widely used in cough lozenge applications, involves the association of zinc ions with carboxylic acids at basic pH. See Bakar, N. K. A.; Taylor, D. M.; Williams, D. R. Chem. Spec. Bioavail. 1999, 11, 95-101; and Eby, G. A. J. Antimicrob. Chemo. 1997, 40, 483-493. These zinc-carboxylate bonds readily dissociate in the presence of acid.




Scheme 1 above illustrates the reaction of an aqueous zinc ion (e.g. from zinc chloride) with two equivalents of an appropriate carboxylic acid to form the zinc dicarboxylate. This reaction occurs rapidly and irreversibly in a slightly basic pH environment but upon acidification, is reversible within a tunable range of pH 4.0-6.8 to reform ZnX2, where X is the conjugate base. One of ordinary skill in the art will recognize that a variety of natural and unnatural amino acid side-chains have a carboxylic acid moeity that can be crosslinked by zinc or another suitable metal.
In certain embodiments, L represents aspartic acid side-chains crosslinked with zinc. Without wishing to be bound by theory, it is believed that the zinc aspartate crosslinks are stable in the blood compartment (pH 7.4), allowing for effective accumulation of the loaded micelles in solid tumors by passive and active targeting mechanisms. In the presence of lactic acid concentrations commonly encountered in solid tumors or in acidic organelles of cancer cells, rapid degradation of the metal crosslinks leading to micelle dissociation and release of the contrast agent at the tumor site. Preliminary, qualitative studies have shown that crosslinked zinc aspartate segments are reversible in the presence of α-hydroxyacids.
In certain embodiments, the -M- moiety of the L group of formula III is zinc. In some embodiments, L forms a zinc-imidazole crosslinking moiety. In certain embodiments, the crosslinking utilizes zinc-mediated coupling of imidazoles.




Scheme 2, above, illustrates the reaction of an aqueous zinc (II) ion (e.g. from zinc chloride or zinc acetate) with two equivalents of an appropriate imidazole (e.g. histidine) to form a zinc-histidine complex. This reaction occurs rapidly in a slightly basic pH environment and is reversible upon acidification to pH less than 6. (Tezcan, et. al. J. Am. Chem. Soc. 2007, 129, 13347-13375.)
In certain embodiments, Rx is a histidine side-chain crosslinked with zinc. Without wishing to be bound by any particular theory, it is believed that zinc-histidine crosslinks are stable in the blood compartment (pH 7.4), allowing for effective accumulation of therapeutic loaded micelles in solid tumors by passive and/or active targeting mechanisms. In the presence of lactic acid concentrations commonly encountered in solid tumors or hydrochloric acid in acidic organelles of cancer cells, rapid degradation of the metal crosslinks occurs which leads to micelle dissociation and release of the polynucleotide at the tumor site.




Scheme 3 above illustrates the reaction of an aqueous zinc (II) ion (e.g. from zinc chloride or zinc acetate) with two equivalents of an appropriate imidazole (e.g. benzimidazole) to form a zinc-benzimidazole complex.
In certain embodiments, Rx is a benzimidazole side-chain crosslinked with zinc. Without wishing to be bound by any particular theory, it is believed that zinc-benzimidazole crosslinks are stable in the blood compartment (pH 7.4), allowing for effective accumulation of therapeutic loaded micelles in solid tumors by passive and/or active targeting mechanisms. In the presence of lactic acid concentrations commonly encountered in solid tumors or hydrochloric acid in acidic organelles of cancer cells, rapid degradation of the metal crosslinks occurs which leads to micelle dissociation and release of the polynucleotide at the tumor site.
The choice of zinc as a crosslinking metal is advantageous for effective micelle crosslinking. Zinc chloride and the zinc lactate by-product are generally recognized as non-toxic, and other safety concerns are not anticipated. Pharmaceutical grade zinc chloride is commonly used in mouthwash and as a chlorophyll stabilizer in vegetables while zinc lactate is used as an additive in toothpaste and drug preparation. The reaction is reversible within a tunable pH range, selective toward carboxylic acids, and should not alter the encapsulated chemotherapy agents. While zinc has been chosen as an exemplary metal for micelle crosslinking, it should be noted that many other metals undergo acid sensitive coupling with carboxylic acids. These metals include calcium, iron and aluminum, to name but a few. One or more of these metals can be substituted for zinc.
The ultimate goal of metal-mediated crosslinking is to ensure micelle stability when diluted in the blood (pH 7.4) followed by rapid dissolution and contrast agent release in response to a finite pH change such as those found in cancer cells. Previous reports suggest a widely variable and tunable dissociation pH for zinc-acid bonds (from approximately 2.0 to 7.0) depending on the carboxylic acid used and number of bonds formed. See Cannan, R. K.; Kibrick, A. J. Am. Chem. Soc. 1938, 60, 2314-2320. Without wishing to be bound by theory, it is believed that the concentration of zinc chloride and the number of aspartic acid, or other carboxylic acid-containing amino acid, repeat units in the crosslinking block will ultimately control the pH at which complete micelle disassembly occurs. The synthetic versatility of the block copolymer design is advantageous since one or more variables are tuned to achieve the desired pH reversibility. By simple adjustment of zinc chloride/polymer stoichiometry, pH-reversible crosslinking is finely tuned across the pH range of interest. For example, higher zinc concentrations yield more zinc crosslinks which require higher acid concentrations (i.e. lower pH) to dissociate. Adjustments in zinc/polymer stoichiometry will yield the desired pH reversibility, however other variables such as increasing the poly(aspartic acid) block length (i.e. 15-25 repeat units) further tune the reversible crosslinking reaction if necessary.
In other embodiments, L comprises a mixture of crosslinked hydrophilic amino acid side-chain groups. Such mixtures of amino acid side-chain groups include those having a carboxylic acid functionality, a hydroxyl functionality, a thiol functionality, and/or amine functionality. It will be appreciated that when L comprises a mixture of crosslinked hydrophilic amino acid side-chain functionalities, then multiple crosslinking can occur. For example, when L comprises a carboxylic acid-containing side-chain (e.g., aspartic acid or glutamic acid) and a thiol-containing side-chain (e.g., cysteine), then the amino acid block can have both zinc crosslinking and cysteine crosslinking (dithiol). This sort of mixed crosslinked block is advantageous for the delivery of contrast agent to the cytosol of diseased cells because a second stimuli must be present to allow for drug release. For example, micelles possessing both carboxylic acid-zinc crosslinking and cysteine dithiol crosslinking would be required to enter an acidic environment (e.g. a tumor) and enter an environment with a high concentration of glutathione (e.g. in the cell cytoplasm). When L comprises an amine-containing side-chain (e.g., lysine or arginine) and a thiol-containing side-chain (e.g., cysteine), then the amino acid block can have both imine (e.g. Schiff base) crosslinking and cysteine crosslinking (dithiol). The zinc and ester crosslinked carboxylic acid functionality and the imine (e.g. Schiff base) crosslinked amine functionality are reversible in acidic organelles (i.e. endosomes, lysosome) while disulfides are reduced in the cytosol by glutathione or other reducing agents resulting in contrast agent release exclusively in the cytoplasm.
Exemplary R1 groups of any of formulae I, II, and III are set forth in Table 12, below.







TABLE 12





Representative R1 Groups













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















One of ordinary skill in the art would recognize that certain R1 groups depicted in Table 12 are protected groups, e.g. protected amine, protected hydroxyl, protected thiol, protected carboxylic acid, or protected alkyne groups. Each of these protected groups is readily deprotected (see, for example, Green). Accordingly, the deprotected groups corresponding to the protected groups set forth in Table 12 are also contemplated. According to another embodiment, the R1 group of any of formulae I, II, and III is selected from a deprotected group of Table 12.
Additional exemplary R1 groups of any of formulae I, II, and III are set forth in Table 12a, below.







TABLE 12a





Representative R1 Groups













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















In certain embodiments, the R1 group of any of formulae I, II, and III is selected from any of those R1 groups depicted in Table 12, supra. In other embodiments, the R1 group of any of formulae I, II, and III is group k or l. In yet other embodiments, the R1 group of any of formulae I, II, and III is n, o, cc, dd, ee, ff hh, h, ii, jj, ll, or uu. In still other embodiments, the R1 group of any of formulae I, II, and III is h, aa, yy, zz, or aaa.
According to another aspect of the present invention, the R1 group of any of formulae I, II, and III is q, r, s, t, www, xxx, or yyy.
In other embodiments, the R1 group of any of formulae I, II, and III is selected from any of those R1 groups depicted in Tables 1-11, supra.
Exemplary R2a groups of any of formulae I, II, and III are set forth in Table 13, below.







TABLE 13





Representative R2a Groups













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















In certain embodiments, the R2a group of any of formulae I, II, and III is selected from any of those R2a groups depicted in Table 13, supra. In other embodiments, the R2a group of any of formulae I, II, and III is group v, viii, xvi, xix, xxii, xxx, xxxi, xxxii, xxxiii, xxxiv, xxxv, xxxvi, xxxvii, or xlii. In yet other embodiments, the R2a group of any of formulae I, II, and III is xv, xviii, xx, xxi, xxxviii, or xxxix. In certain embodiments, the R2a group of any of formulae I, II, and III is xxxiv.
According to another embodiment, the R2a group of any of formulae I, II, and III is selected from any of those R2a groups depicted in Tables 1-1, supra.
One of ordinary skill in the art would recognize that certain R2a groups depicted in Table 13 are protected groups, e.g. protected amine, protected hydroxyl, protected thiol, protected carboxylic acid, or protected alkyne groups. Each of these protected groups is readily deprotected (see, for example, Green). Accordingly, the deprotected groups corresponding to the protected groups set forth in Table 13 are also contemplated. According to another embodiment, the R2a group of any of formulae I, II, and III is selected from a deprotected group of Table 13.
C. Contrast Agent Encapsulation
As described generally above, in certain embodiments the present invention provides a micelle having a contrast agent encapsulated therein, comprising a multiblock copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly(amino acid block), and a poly(amino acid block), characterized in that said micelle has a contrast agent-loaded inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic shell. In some embodiments the present invention provides a micelle having a contrast agent encapsulated therein, comprising a multiblock copolymer which comprises a polymeric hydrophilic block, crosslinked poly(amino acid block), and a poly(amino acid block), characterized in that said micelle has a contrast agent-loaded inner core, crosslinked outer core, and a hydrophilic shell. As described herein, micelles of the present invention can be loaded with any contrast agent.
The use of various nanoparticles has been demonstrated to be useful in a number of in vitro and in vivo medical fields ranging from the tagging of biological species, in vitro cell separation, immunoassays, immunomagentic array, in vivo contrast agents for MRI, PET, ultrasound, X-ray, computed tomography, fluorescence, disease treatment, targeted therapeutic delivery. See: Jeong, U.; Teng, X.; Wang, Y.; Yang, H.; Xia, Y. “Superparamagnetic Colloids: Controlled Synthesis and Niche Applications” Adv. Mater.; 2007, 19, 33-60.
In order to fully exploit these nanoparticles, delivery systems must be developed that can encapsulate the nanoparticles and impart properties such as long circulation time following injection by reducing RES uptake, accumulation in a targeted region of the body, and functionality for attachment to specific biological molecules. They must be administered in a biocompatible delivery vehicle that does not elicit cytotoxic or immunological responses but can improve their solubility and stability in biological media. The versatility of the triblock copolymer micelle described herein allows for a wide range of nanoparticles to be encapsulated, solubilized, stabilized, and targeted for in vitro and in vivo applications.
Semiconductor nanoparticles, such as CdSe, CdS, CdTe, PdSe, InP, InAs, PbS, CdSe/CdS, CdSe/ZnS, CdS/ZnS, and CdTe/ZnS have been shown to be potentially useful in both in vitro and in vivo as diagnostic agents. Their narrow emission, broad absorption profiles, and resistance to photo-bleaching make them ideal for in vitro and in vivo biological tagging, especially in multiplexing applications. Sizes of such semiconducting nanoparticles typically range from 2 to 50 nm (See Brus, L. “Chemical Approaches to Semiconducting Nanoparticles” J. Phy. Chem. Solids 1998, 59, 459-465. Wang, D.; He, J.; Rosenzweig, N.; Rosenzweig, Z. “Superparamagnetic Fe2O3 Beads-CdSe/ZnS Quantum Dots Core-Shell Nanocomposite Particles for Cell Separation” Nano Letters 2004, 4, 409-413. Yu, W. W.; Falkner, J. C.; Shih, B. S.; Colvin, V. L.; “Preparation and Characterization of Monodisperse PbSe Semiconductor Nanocrystals in a Noncoordinating Solvent” Chem. Mater. 2004, 16, 3318-3322).
Magnetic nanoparticles, such as: Fe, Fe2O3, Fe3O4, MnFe2O4, CoFe2O4, NiFe2O4, Co, Ni, FePt, CoPt, CoO, Fe3Pt, Fe2Pt, CO3Pt, CO2Pt, FeOOH, have also been useful for in vitro and in vivo diagnostics and treatments. Nanoparticles of this type, with sizes ranging from 2 nm-100 nm, have been successfully utilized as contrast agents for magnetic resonance, magnetically-controlled drug delivery vehicles, and in hyperthermia treatments. See: Jeong, U.; Teng, X.; Wang, Y.; Yang, H.; Xia, Y. “Superparamagnetic Colloids: Controlled Synthesis and Niche Applications” Adv. Mater.; 2007, 19, 33-60. Niederberger, M.; Garnweitner, G. “Organic Reaction Pathways in the Nonaqueos Synthesis of Metal Oxide Nanoparticles” 2006, 12, 7282-7302. Sun, S.; Zeng, H.; “Size-controlled Synthesis of Magnetite Nanoparticles” 2002, 124, 8204-8205.
While magnetic nanoparticles have shown a great deal of potential as contrast agents; their application in vivo has been limited in some cases by their limited aqueous solubility, poor biocompatibility, and short circulation lifetime following injection. One approach to address these two problems has been to alter the periphery of the nanoparticles with poly(ethylene glycol) ligands or to encapsulate the nanoparticles in the core of a polymeric micelle. See: Kumagai, M.; Imai, Y.; Nakamura, T.; Yamasaki, Y.; Sekino, M.; Ueno, S.; Hanaoka, K.; Kikuchi, K.; Nagano, T.; Kaneko, E.; Shimokado, K.; Kataoka, K. “Iron Hydroxide Nanoparticles Coated with Poly(ethylene glycol)-poly(aspartic acid) block copolymer as novel magnetic resonance contrast agents for in vivo cancer imaging” Colloids and Surfaces B: Biointerfaces, 2007, 56, 174-181. Kim, D. K.; Mikhaylova, M.; Zhang, Y.; Muhammed, M. “Protective Coating of Superparamagnetic Iron Oxide Nanoparticle” Chem. Mater. 2003, 15, 1617-1627. Ai, H.; Flask, C.; Weinberg, B.; Shuai, X.; Pagel, M.D.; Farrell, D.; Duerk, J.; Gao, J. “Magnetite-Loaded Polymeric Micelles as Ultrasensitive Magnetic-Resonance Probes” Adv. Mater., 2005, 17, 1949-1952. While these approaches have had some limited success in improving nanoparticle solubility, they do not address the inherent instability of the nanoparticle-loaded micelles following administration and dilution in the body. Furthermore, the accumulation of the nanoparticles in a specified area of the body (e.g. solid tumor, lymph nodes, etc.) requires long circulation times which can be improved through crosslinking strategies to improve micelle stability and reduction in uptake by the reticuloendothelial system (RES). Both of these attributes can be imparted using micelles of the present invention which can possess chemical cross-linking in the micelle outer core and stealth properties imparted by the poly(ethylene glycol) corona. Thus, the crosslinked polymer micelles described herein are resistant to dissociation and instability resulting from post-injection dilution and destabilizing blood components.
In addition to the optional crosslinking procedure described above, certain inner core blocks can function as a ligand for the encapsulated nanoparticle. Without wishing to be bound by any particular theory, it is believed that when the inner core block contains certain residues, these residues can function as a multi-dentate ligand for the nanoparticle. For examine, when Ry comprises an aspartic acid or glutamic acid functionality, a tyrosine functionality, or a DOPA functionality, the resulting inner core will function as a multi-dentate ligand for the nanoparticle. Amino-functionalized Ry moieties, such as lysine, ornithine, histidine, and arginine also function as a multi-dentate ligand for the nanoparticle. Thus, in certain embodiments, the present invention provides a micelle having aspartic acid or glutamic acid functionality which functions as a multi-dentate ligand for the encapsulated nanoparticle. In other embodiments, tyrosine containing inner core blocks will function as a multi-dentate ligand for the encapsulated nanoparticle. In yet other embodiments, DOPA containing inner core blocks will function as a multi-dentate ligand for the encapsulated nanoparticle. Without wishing to be bound to any particular theory, in the case where the inner core block contains a moiety capable of functioning as a ligand for the encapsulated nanoparticle, it is believed that the large number of ligand-nanoparticle interactions acts to stabilize the micelle, with the nanoparticle itself acting as the crosslinking agent.
D. Polymer Conjugation
The ability to target the nanoparticles is of importance in allowing for specific imaging of unhealthy cells, e.g. tumors. In order to accomplish this several groups have shown that over expressed receptors can be used as targeting groups. Examples of these targeting groups include Folate, Her-2 peptide, etc. The shortfalls of attaching these targeting units directly to the nanoparticle surface through ligand attachment are that this bonding is not permanent. The ligands have the tendency to debond from the nanoparticle surface, especially as the nanoparticles are diluted. The attachment of targeting moieties directly to the nanoparticle surface in many cases does not advantageously increase the size of the nanoparticle (typically 2-20 nm) to the optimal size range, 50-200 nm, to avoid the endo-reticular system. In contrast, by encapsulating a number of nanoparticles in a given micelle one can deliver a much greater amount of contrast agent with one delivery event. The encapsulated micelles also allow for the generation of a wide range of size ranges from 40 nm to 500 nm. See: Lee, J.; Huh, Y.; Jun, Y. Seo, J.; Jang, J.; Song, H.; Kim, S.; Cho, E.; Yoon, H.; Suh, J.; Cheon, J. “Artificially Engineered Magnetic Nanoparticles for Ultra-sensitive Molecule Imaging” Nature Medicine, 2007, 13, 95-99.
In addition to their core-shell morphology, polymer micelles can be modified to enable passive and active cell-targeting to maximize the benefits of current and future therapeutic agents and contrast agents. Because contrast agent-loaded micelles typically possess diameters greater than 20 nm, they exhibit dramatically increased circulation time when compared to stand-alone contrast agents due to minimized renal clearance. This unique feature of nanovectors and polymeric delivery systems leads to selective accumulation in diseased tissue, especially cancerous tissue due to the enhanced permeation and retention effect (“EPR”). The EPR effect is a consequence of the disorganized nature of the tumor vasculature, which results in increased permeability of polymer therapeutics and contrast agent retention at the tumor site. In addition to passive cell targeting by the EPR effect, micelles are designed to actively target tumor cells through the chemical attachment of targeting groups to the micelle periphery. The incorporation of such groups is most often accomplished through end-group functionalization of the hydrophilic block using chemical conjugation techniques. Like viral particles, micelles functionalized with targeting groups utilize receptor-ligand interactions to control the spatial distribution of the micelles after administration, further enhancing cell-specific delivery of therapeutics. In cancer therapy, targeting groups are designed to interact with receptors that are over-expressed in cancerous tissue relative to normal tissue such as folic acid, oligopeptides, sugars, and monoclonal antibodies. See Pan, D.; Turner, J. L.; Wooley, K. L. Chem. Commun. 2003, 2400-2401; Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. Adv. Drug Deliv. Rev. 2004, 56, 1177-1202; Reynolds, P. N.; Dmitriev, I.; Curiel, D. T. Vector. Gene Ther. 1999, 6, 1336-1339; Derycke, A. S. L.; Kamuhabwa, A.; Gijsens, A.; Roskams, T.; De Vos, D.; Kasran, A.; Huwyler, J.; Missiaen, L.; de Witte, P. A. M. T J. Nat. Cancer Inst. 2004, 96, 1620-30; Nasongkla, N., Shuai, X., Ai, H.,; Weinberg, B. D. P., J.; Boothman, D. A.; Gao, J. Angew. Chem. Int. Ed. 2004, 43, 6323-6327; Jule, E.; Nagasaki, Y.; Kataoka, K. Bioconj. Chem. 2003, 14, 177-186; Stubenrauch, K.; Gleiter, S.; Brinkmann, U.; Rudolph, R.; Lilie, H. Biochem. J. 2001, 356, 867-873; Kurschus, F. C.; Kleinschmidt, M.; Fellows, E.; Dommair, K.; Rudolph, R.; Lilie, H.; Jenne, D. E. FEBS Lett. 2004, 562, 87-92; and Jones, S. D.; Marasco, W. A. Adv. Drug Del. Rev. 1998, 31, 153-170.
Compounds of any of formulae I, II, and III having R3 moieties suitable for Click chemistry are useful for conjugating said compounds to biological systems or macromolecules such as proteins, viruses, and cells, to name but a few. The Click reaction is known to proceed quickly and selectively under physiological conditions. In contrast, most conjugation reactions are carried out using the primary amine functionality on proteins (e.g. lysine or protein end-group). Because most proteins contain a multitude of lysines and arginines, such conjugation occurs uncontrollably at multiple sites on the protein. This is particularly problematic when lysines or arginines are located around the active site of an enzyme or other biomolecule. Thus, another embodiment of the present invention provides a method of conjugating the R1 groups of a compound of any of formulae I, II, and III to a macromolecule via Click chemistry. Yet another embodiment of the present invention provides a macromolecule conjugated to a compound of any of formulae I, II, and III via the R1 group.
After incorporating the poly(amino acid) block portions into the multi-block copolymer of the present invention resulting in a multi-block copolymer of the form W—X—X′, the other end-group functionality, corresponding to the R1 moiety of any of formulae I, II, and III, can be used to attach targeting groups for cell specific delivery including, but not limited to, attach targeting groups for cell specific delivery including, but not limited to, proteins, oliogopeptides, antibodies, monosaccarides, oligosaccharides, vitamins, or other small biomolecules. Such targeting groups include, but or not limited to monoclonal and polyclonal antibodies (e.g. IgG, IgA, IgM, IgD, IgE antibodies), sugars (e.g. mannose, mannose-6-phosphate, galactose), proteins (e.g. Transferrin), oligopeptides (e.g. cyclic and acylic RGD-containing oligopeptides), and vitamins (e.g. folate). Alternatively, the R1 moiety of any of formulae I, II, and III is bonded to a biomolecule, drug, cell, or other suitable substrate.
In other embodiments, the R1 moiety of any of formulae I, II, and III is bonded to biomolecules which promote cell entry and/or endosomal escape. Such biomolecules include, but are not limited to, oligopeptides containing protein transduction domains such as the HIV Tat peptide sequence (GRKKRRQRRR) or oligoarginine (RRRRRRRRR). Oligopeptides which undergo conformational changes in varying pH environments such oligohistidine (HHHHH) also promote cell entry and endosomal escape.
In other embodiments, the R1 moiety of any of formulae I, II, and III is bonded to detectable moieties, such as fluorescent dyes or labels for positron emission tomography including molecules containing radioisotopes (e.g. 18F) or ligands with bound radioactive metals (e.g. 62Cu). In other embodiments, the R1 moiety of any of formulae I, II, and III is bonded to a contrast agents for magnetic resonance imaging such as gadolinium, gadolinium chelates, or iron oxide (e.g. Fe3O4 and Fe2O3) particles. In other embodiments, the R1 moiety of any of formulae I, II, and III is bonded to a semiconducting nanoparticle such as cadmium selenide, cadmium sulfide, or cadmium telluride or bonded to other metal nanoparticles such as colloidal gold. In other embodiments, the R1 moiety of any of formulae I, II, and III is bonded to natural or synthetic surfaces, cells, viruses, dyes, drugs, chelating agents, or used for incorporation into hydrogels or other tissue scaffolds.
In one embodiment, the R1 moiety of any of formulae I, II, and III is an acetylene or an acetylene derivative which is capable of undergoing [3+2] cycloaddition reactions with complementary azide-bearing molecules and biomolecules. In another embodiment, the R1 moiety of any of formulae I, II, and III is an azide or an azide derivative which is capable of undergoing [3+2] cycloaddition reactions with complementary alkyne-bearing molecules and biomolecules (i.e. click chemistry).
Click chemistry has become a popular method of bioconjugation due to its high reactivity and selectivity, even in biological media. See Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004-2021; and Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. In addition, currently available recombinant techniques permit the introduction of azides and alkyne-bearing non-canonical amino acids into proteins, cells, viruses, bacteria, and other biological entities that consist of or display proteins. See Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598-10602; Deiters, A.; Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, C.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 11782-11783.
In another embodiment, the [3+2] cycloaddition reaction of azide or acetylene-bearing nanovectors and complimentary azide or acetylene-bearing biomolecules are transition metal catalyzed. Copper-containing molecules which catalyze the “click” reaction include, but are not limited to, copper bromide (CuBr), copper chloride (CuCl), copper sulfate (CuSO4), copper iodide (CuI), [Cu(MeCN)4](OTf), and [Cu(MeCN)4](PF6). Organic and inorganic metal-binding ligands can be used in conjunction with metal catalysts and include, but are not limited to, sodium ascorbate, tris(triazolyl)amine ligands, tris(carboxyethyl)phosphine (TCEP), and sulfonated bathophenanthroline ligands.
In another embodiment, the R1 moiety of any of formulae I, II, and III is an hydrazine or hydrazide derivative which is capable of undergoing reaction with biomolecules containing aldehydes or ketones to form hydrazone linkages. In another embodiment, the R1 moiety of any of formulae I, II, and III is an aldehyde or ketone derivative which is capable of undergoing reaction with biomolecules containing a hydrazine or hydrazide derivative to form hydrazone linkages.
In another embodiment, the R1 moiety of any of formulae I, II, and III is a hydroxylamine derivative which is capable of undergoing reaction with biomolecules containing aldehydes or ketones. In another embodiment, the R1 moiety of any of formulae I, II, and III is an aldehyde or ketone which is capable of undergoing reaction with biomolecules containing a hydroxylamine, or a hydroxylamine derivative.
In yet another embodiment, the R1 moiety of any of formulae I, II, and III is an aldehyde or ketone derivative which is capable of undergoing reaction with biomolecules containing primary or secondary amines to form imine linkages. In another embodiment, the R1 moiety of any of formulae I, II, and III is a primary or secondary amine which is capable of undergoing reaction with biomolecules containing an aldehyde or ketone functionality to form imine linkages. It will be appreciated that imine linkages can be further converted to stable amine linkages by treatment with a suitable reducing agent (e.g. lithium aluminum hydride, sodium borohydride, sodium cyanoborohydride, etc.)
In yet another embodiment, the R1 moiety of any of formulae I, II, and III is an amine (primary or secondary) or alcohol which is capable of undergoing reaction with biomolecules containing activated esters (e.g. 4-nitrophenol ester, N-hydroxysuccinimide, pentafluorophenyl ester, ortho-pyridylthioester), to form amide or ester linkages. In still other embodiments, the R1 moiety of any of formulae I, II, and III is an activated ester which is capable of undergoing reaction with biomolecules possessing amine (primary or secondary) or alcohols to form amide or ester linkages.
In still other embodiments, the R1 moiety of any of formulae I, II, and III is an amine or alcohol which is bound to biomolecules with carboxylic acid functionality using a suitable coupling agent. In still other embodiments, the R1 moiety of any of formulae I, II, and III is a carboxylic acid functionality which is bound to biomolecules containing amine or alcohol functionality using a suitable coupling agent. Such coupling agents include, but are not limited to, carbodiimides (e.g. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), diisopropyl carbodiimide (DIC), dicyclohexyl carbodiimide (DCC)), aminium or phosphonium derivatives (e.g. PyBOP, PyAOP, TBTU, HATU, HBTU), or a combination of 1-hydroxybenzotriazole (HOBt) and a aminium or phosphonium derivative.
In another embodiment, the R1 moiety of any of formulae I, II, and III is an electrophile such as maleimide, a maleimide derivative, or a bromoacetamide derivative, which is capable of reaction with biomolecules containing thiols or amines. In another embodiment, the R1 moiety of any of formulae I, II, and III is a nucleophile such as an amine or thiol which is capable or reaction with biomolecules containing electrophilic functionality such as maleimide, a maleimide derivative, or a bromoacetamide derivative.
In still other embodiments, the R1 moiety of any of formulae I, II, and III is a ortho-pyridyl disulfide moiety which undergoes disulfide exchange with biomolecules containing thiol functionality. In still other embodiments, the R1 moiety of any of formulae I, II, and III is a thiol or thiol derivative which undergoes disulfide exchange with biomolecules containing ortho-pyridyl disulfide functionality. It will be appreciated that such exchange reactions result in a disulfide linkage which is reversible in the presence of a suitable reducing agent (e.g. glutathione, dithiothreitol (DTT), etc.).
In certain embodiments, micelles of the present invention are mixed micelles comprising one or more compounds of formula I, II, or III. It will be appreciated that mixed micelles having different R1 groups, as described herein, can be conjugated to multiple other compounds and/or macromolecules. For example, a mixed micelle of the present invention can have one R1 group suitable for Click chemistry and another R1 group suitable for covalent attachment via a variety of coupling reactions. Such a mixed micelle can be conjugated to different compounds and/or macromolecules via these different R1 groups. Such conjugation reactions are well known to one of ordinary skill in the art and include those described herein.
4. General Methods for Providing Compounds of the Present Invention
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 U.S. patent application Ser. No. 11/325,020 filed Jan. 4, 2006 and published as US 20060172914 on Aug. 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.




Scheme 4 above depicts a general method for preparing multiblock polymers of the present invention. A macroinitiator of formula A is treated with a first amino acid NCA to form a compound of formula B having a first amino acid block. The second amino acid NCA is added to the living polymer of formula B to form a compound of formula I′ having two differing amino acid blocks. Each of the R1, A, n, Q, Rx, Ry, m, and m′ groups depicted in Scheme 4 are as defined and described in classes and subclasses, singly and in combination, herein.
One step in the preparation of a compound of formula I comprises terminating the living polymer chain-end of the compound of formula I′ with a suitable polymerization terminator to afford a compound of formula I. One of ordinary skill in the art would recognize that the polymerization terminator provides the R group of formula I. Accordingly, embodiments directed to the R2a group of formula I as set forth above and herein, are also directed to the suitable polymerization terminator itself, and similarly, embodiments directed to the suitable polymerization terminator, as set forth above and herein, are also directed to the R2a group of formula I.
As described above, compounds of formula I are prepared from compounds of formula I′ by treatment with a suitable terminating agent. One of ordinary skill in the art would recognize that compounds of formula I are also readily prepared directly from compounds of formula I′. In such cases, and in certain embodiments, the compound of formula I′ is treated with a base to form the freebase compound prior to, or concurrent with, treatment with the suitable terminating agent. For example, it is contemplated that a compound of formula I′ is treated with a base and suitable terminating agent in the same reaction to form a freebase of that compound. In such cases, it is also contemplated that the base may also serve as the reaction medium.
One of ordinary skill in the art would also recognize that the above method for preparing a compound of formula I may be performed as a “one-pot” synthesis of compounds of formula I that utilizes the living polymer chain-end to incorporate the R2a group of formula I. Alternatively, compounds of formula I may also be prepared in a multi-step fashion. For example, the living polymer chain-end of a compound of formula I′ may be quenched to afford an amino group which may then be further derivatized, according to known methods, to afford a compound of formula I.
One of ordinary skill in the art will recognize that a variety of polymerization terminating agents are suitable for the present invention. Such polymerization terminating agents include any R2a-containing group capable of reacting with the living polymer chain-end of a compound of formula I′, or the free-based amino group of formula I′, to afford a compound of formula I. Thus, polymerization terminating agents include anhydrides, and other acylating agents, and groups that contain a suitable leaving group LG that is subject to nucleophilic displacement.
Alternatively, compounds of formula I′ may be coupled to carboxylic acid-containing groups to form an amide thereof. Thus, it is contemplated that the amine group of formula I′ or freease thereof, may be coupled with a carboxylic acid moiety to afford compounds of formula I wherein R2a is —NHC(O)R4. Such coupling reactions are well known in the art. In certain embodiments, the coupling is achieved with a suitable coupling reagent. Such reagents are well known in the art and include, for example, DCC and EDC, among others. In other embodiments, the carboxylic acid moiety is activated for use in the coupling reaction. Such activation includes formation of an acyl halide, use of a Mukaiyama reagent, and the like. These methods, and others, are known to one of ordinary skill in the art, e.g., see, “Advanced Organic Chemistry,” Jerry March, 5th Ed., pp. 351-357, John Wiley and Sons, N.Y.
A “suitable leaving group that is subject to nucleophilic displacement” is a chemical group that is readily displaced by a desired incoming chemical moiety. Suitable leaving groups are well known in the art, e.g., see, March. Such leaving groups include, but are not limited to, halogen, alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, and diazonium moieties. Examples of suitable leaving groups include chloro, iodo, bromo, fluoro, methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy, nitro-phenylsulfonyloxy (nosyloxy), and bromo-phenylsulfonyloxy (brosyloxy).
According to an alternate embodiment, the suitable leaving group may be generated in situ within the reaction medium. For example, a leaving group may be generated in situ from a precursor of that compound wherein said precursor contains a group readily replaced by said leaving group in situ.
Alternatively, when the R2a group of formula I is a mono- or di-protected amine, the protecting group(s) is removed and that functional group may be derivatized or protected with a different protecting group. It will be appreciated that the removal of any protecting group of the R2a group of formula I is performed by methods suitable for that protecting group. Such methods are described in detail in Green.
In other embodiments, the R2a group of formula I is incorporated by derivatization of the amino group of formula I′, or freebase thereof, via anhydride coupling, optionally in the presence of base as appropriate. One of ordinary skill in the art would recognize that anhydride polymerization terminating agents containing an azide, an aldehyde, a hydroxyl, an alkyne, and other groups, or protected forms thereof, may be used to incorporate said azide, said aldehyde, said protected hydroxyl, said alkyne, and other groups into the R2a group of compounds of formula I. It will also be appreciated that such anhydride polymerization terminating agents are also suitable for terminating the living polymer chain-end of a compound of formula I′, or freebase thereof. Such anhydride polymerization terminating agents include, but are not limited to, those set forth in Table 14, below.







TABLE 14





Representative Anhydride Polymerization Terminating Agents













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















In other embodiments, the R4 moiety of the R2a group of formula III is incorporated by derivatization of the amino group of formula I′, or freebase thereof, via reaction with a polymerization terminating agent having a suitable leaving group. It will also be appreciated that such polymerization terminating agents are also suitable for terminating the living polymer chain-end of a compound of formula I′, or freebase thereof. Examples of these polymerization terminating agents include, but are not limited to, those set forth in Table 15, below.







TABLE 15





Representative Polymerization Terminating Agents













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















wherein each L is a suitable leaving group as defined above and in classes and subclasses as described above and herein.

In certain embodiments, the hydrophilic polymer block is poly(ethylene glycol) (PEG) having a terminal amine salt (“PEG macroinitiator”). This PEG macroinitiator initiates the polymerization of NCAs to provide the multiblock copolymers of the present invention. Such polymers having a terminal amine salt may be prepared from synthetic polymers having a terminal amine. Such synthetic polymers having a terminal amine group are known in the art and include PEG-amines. PEG-amines may be obtained by the deprotection of a suitably protected PEG-amine. Preparation of such suitably protected PEG-amines, and methods of deprotecting the same, is described in detail in U.S. patent application Ser. No. 11/256,735, filed Oct. 24, 2005 and published as US 20060142506 on Jun. 29, 2006, the entirety of which is hereby incorporated herein by reference.
As described in US 20060142506, suitably protected PEG-amines may be formed by terminating the living polymer chain end of a PEG with a terminating agent that contains a suitably protected amine. The suitably protected amine may then be deprotected to generate a PEG that is terminated with a free amine that may subsequently be converted into the corresponding PEG-amine salt macroinitiator. In certain embodiments, the PEG-amine salt macroinitiator of the present invention is prepared directly from a suitably protected PEG-amine by deprotecting said protected amine with an acid. Accordingly, in other embodiments, the terminating agent has suitably protected amino group wherein the protecting group is acid-labile.
Alternatively, suitable synthetic polymers having a terminal amine salt may be prepared from synthetic polymers that contain terminal functional groups that may be converted to amine salts by known synthetic routes. In certain embodiments, the conversion of the terminal functional groups to the amine salts is conducted in a single synthetic step. In other embodiments, the conversion of the terminal functional groups to the amine salts is achieved by way of a multi-step sequence. Functional group transformations that afford amines, amine salts, or protected amines are well known in the art and include those described in Larock, R. C., “Comprehensive Organic Transformations,” John Wiley & Sons, New York, 1999.




Scheme 5 above shows one exemplary method for preparing the bifunctional PEGs used to prepare the multiblock copolymers of the present invention. At step (a), the polymerization initiator is treated with a suitable base to form D. A variety of bases are suitable for the reaction at step (a). Such bases include, but are not limited to, potassium naphthalenide, diphenylmethyl potassium, triphenylmethyl potassium, and potassium hydride. At step (b), the resulting anion is treated with ethylene oxide to form the polymer E. Polymer E can be transformed at step (d) to a compound of formula A directly by terminating the living polymer chain-end of E with a suitable polymerization terminator to afford a compound of formula A. Alternatively, polymer E may be quenched at step (c) to form the hydroxyl compound F. Compound F is then derivatized to afford a compound of formula A by methods known in the art, including those described herein. Each of the R1, A, n, and Q groups depicted in Scheme 5 are as defined and described in classes and subclasses, singly and in combination, herein.
Although certain exemplary embodiments are depicted and described above and herein, it will be appreciated that compounds of the invention can be prepared according to the methods described generally above using appropriate starting materials by methods generally available to one of ordinary skill in the art. Additional embodiments are exemplified in more detail herein.
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-methylpyrollidinone, 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 a water miscible organic solvent such as N-methylpyrollidinone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dimethylacetamide and added dropwise to water or another aqueous medium. Micelles prepared by these methods can be sterilized by sterile filtration and then isolated by lyophilization.
In one embodiment, inventive micelles having a contrast agent encapsulated therein and possessing carboxylic acid functionality in the outer core are optionally crosslinked by addition of zinc chloride to the micelle solution along with a small amount of sodium bicarbonate to neutralize any hydrochloric acid by-product. In this basic pH environment, the reaction of zinc chloride with the poly(aspartic acid) crosslinking block is rapid and irreversible.
In another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing amine functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multi-functional aldehyde-containing molecule which forms pH-reversible imine crosslinks. In another embodiment, micelles, having a contrast agent encapsulated therein, possessing aldehyde functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multi-functional amine-containing molecule which forms pH-reversible imine crosslinks.
In another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing alcohol or amine functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multi-functional carboxylic acid-containing molecules and a coupling agent to form amide or ester crosslinks. In yet another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing carboxylic acid functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multi-functional amine or alcohol-containing molecules and a coupling agent to form amide or ester crosslinks. Such coupling agents include, but are not limited to, carbodiimides (e.g. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), diisopropyl carbodiimide (DIC), dicyclohexyl carbodiimide (DCC)), aminium or phosphonium derivatives (e.g. PyBOP, PyAOP, TBTU, HATU, HBTU), or a combination of 1-hydroxybenzotriazole (HOBt) and a aminium or phosphonium derivative.
In another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing aldehyde or ketone functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multifunctional hydrazine or hydrazide-containing molecule to form pH-reversible hydrazone crosslinks. In still other embodiments, inventive micelles having a contrast agent encapsulated therein and hydrazine or hydrazide-functionality in the outer core are optionally crosslinked by the addition of a bifunctional, or multifunctional aldehyde or ketone-containing molecule to form pH-reversible hydrazone crosslinks.
In another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing thiol functionality in the outer core are optionally crosslinked by the addition of an oxidizing agent (e.g. metal oxides, halogens, oxygen, peroxides, ozone, peroxyacids, etc.) to form disulfide crosslinks. It will be appreciated that disulfide crosslinks are reversible in the presence of a suitable reducing agent (e.g. glutathione, dithiothreitol (DTT), etc.).
In yet another embodiment, inventive micelles having a contrast agent encapsulated therein and possessing both carboxylic acid and thiol functionality in the outer core can be dual crosslinked by the addition of an oxidizing agent (e.g. metal oxides, halogens, oxygen, peroxides, ozone, peroxyacids, etc.) to form disulfide crosslinks followed by the addition of zinc chloride to the micelle solution along with a small amount of sodium bicarbonate to neutralize any hydrochloric acid by-product. It will be appreciated that such a dual-crosslinked micelle is reversible only in the presence of acid and a reducing agent (e.g. glutathione, dithiothreitol (DTT), etc.).
According to another aspect, the present invention provides a method for preparing a micelle having a contrast agent encapsulated therein, comprising a multiblock copolymer which comprises a polymeric hydrophilic block, a crosslinked poly(amino acid block), and a poly(amino acid) block, characterized in that said micelle has an inner core, a crosslinked outer core, and a hydrophilic shell, said method comprising the steps of:
(a) providing a multiblock copolymer of formula I:




    
    
        wherein:
        
            n is 10-2500;
            m is 1 to 1000;
            m′ is 1 to 1000;
            Rx is a natural or unnatural amino acid side-chain group that is capable of crosslinking;
            Ry is a hydrophobic or ionic, natural or unnatural amino acid side-chain group;
            R1 is -Z(CH2CH2Y)p(CH2)tR3, wherein:
            
                Z is —O—, —S—, —C≡C—, or —CH2—;
                each Y is independently —O— or —S—;
                p is 0-10;
                t is 0-10; and
            
            
            R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
            Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
            
                -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
            
            
            R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
            each R4 is independently an optionally substituted group selected from hydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
            
                two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,

(b) combining said compound of formula I with a contrast agent; and

(c) treating the resulting micelle with a crosslinking reagent to crosslink Rx.

            
            
        
        
    
    


In one embodiment, contrast agents are loaded into the micelle inner core by adding an aliquot of a copolymer solution in water to the contrast agent to be incorporated. For example, a stock solution of the contrast agent in a polar organic solvent is made and the solvent is allowed to evaporate. The copolymer/water solution is subsequently added and loading can be achieved through stirring, vortex mixing, sonication, and the like. In another embodiment, the contrast agent is incorporated using an oil in water emulsion technique. In this case, the contrast agent is dissolved in a water immiscible organic solvent and added dropwise to the agitated micelle solution in water, and the contrast agent is incorporated into the micelle during solvent evaporation. In another embodiment, the contrast agent and polymer can be dissolved in a water immiscible organic solvent and added dropwise to water while stirring, vortex mixing, or other methods of agitation. The contrast agent is incorporated into the micelle during solvent evaporation. In another embodiment, the contrast agent is dissolved with the copolymer in a common polar organic solvent and dialyzed against water or another aqueous medium. See Allen, C.; Maysinger, D.; Eisenberg A. Colloid Surface B 1999, 16, 3-27.
In still another embodiment, the loading and crosslinking of contrast agent-filled micelles is carried out by dissolving the contrast agent and the block copolymer in a polar solvent such as acetone or ethanol, followed by slow addition to water or buffer solution. Due to the limited solubility of the contrast agent in water, the contrast agent is forced into the core of the micelle, effectively encapsulating the contrast agent.
5. Uses, Methods, and Compositions
Although bones are easily visualized using x-ray imaging, many other organs and tissues cannot be easily imaged without contrast enhancement. Contrast agents, also known as contrast media or diagnostic agents, are often used during medical imaging examinations to highlight specific parts of the body (e.g. tissues and organs) and make them easier to visualize and improve disease diagnosis. Contrast agents can be used with many types of imaging examinations, including x-ray exams, computed tomography scans, magnetic resonance imaging, and positron emission tomography to name but a few.
As described herein, micelles of the present invention can encapsulate a wide variety of contrast agents. In certain embodiments, the present invention provides a micelle having a contrast agent encapsulated therein, as described herein, wherein said micelle is useful for enhancing the visualization of tissues and organs. Such visualization is useful for diagnosing various diseases and injuries.
In certain embodiments, the present invention provides a method for imaging at least one tissue in a patient said method comprising administering to said patient a provided micelle having a contrast agent encapsulated therein, or composition thereof, and detecting the contrast agent. One of ordinary skill in the art will recognize that various imaging methods are useful for the detecting step. Exemplary imaging methods include x-ray, magnetic resonance, ultrasound, optical imaging, sonoluminescence, photoacoustic imaging, nuclear imaging, positron emission tomography, absorption, light scattering, and computed tomography.
In certain embodiments, the present invention provides a diagnostic imaging method comprising the steps of: (a) administering to a patient a provided micelle having a contrast agent encapsulated therein, or composition thereof, and (b) imaging the contrast agent after administration of the micelle to the patient. In some embodiments, the present invention provides a diagnostic imaging method comprising the steps of: (a) administering to a patient a provided micelle having a contrast agent encapsulated therein wherein the micelle is conjugated to a targeting group, or composition thereof, and (b) imaging the contrast agent after administration of the micelle to the patient.
In certain embodiments, the imaging step is selected from magnetic resonance imaging, ultrasound imaging, optical imaging, sonoluminescence imaging, photoacoustic imaging, or nuclear imaging.
In certain embodiments, the present invention provides a method of imaging at least one tissue in a patient comprising administering a provided micelle, or composition thereof, and performing an imaging procedure.
Compositions
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. In some embodiments, compositions of the present invention are formulated for parenteral administration.
The term “patient”, as used herein, means an animal, preferably a mammal, and most preferably a human.
The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a non-toxic 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.
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.
Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and N+(C1-4 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.
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, intra-articular, 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 non-toxic 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.
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.
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.
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 agent. Such materials include cocoa butter, beeswax and polyethylene glycols.
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.
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.
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.
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.
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.
In certain embodiments, the pharmaceutically acceptable compositions of this invention are formulated for oral administration.
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 contrast agent can be administered to a patient receiving these compositions.
It will be appreciated that dosages typically employed for the encapsulated contrast agent are contemplated by the present invention. In certain embodiments, a patient is administered a contrast agent-loaded micelle of the present invention wherein the dosage of the contrast agent is equivalent to what is typically administered for that contrast agent. In other embodiments, a patient is administered a contrast agent-loaded micelle of the present invention wherein the dosage of the drug is lower than is typically administered for that contrast agent.
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.
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.
EXAMPLES
Preparation of Bifunctional Pegs and Multiblock Copolymers of the Present Invention
As described generally above, multiblock copolymers of the present invention are prepared using the heterobifunctional PEGs described herein and in U.S. patent application Ser. No. 11/256,735, filed Oct. 24, 2005 and published as US20060142506 on Jun. 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 U.S. patent application Ser. No. 11/325,020, filed Jan. 4, 2006 and published as US 20060172914 on Aug. 3, 2006, the entirety of which is hereby incorporated herein by reference.
Example 1




mPEG-hydrochloride

To a 500 mL 2-neck round bottom flask was added mPEG (40 g, 5 mmol), phthalimide (4.41 g, 30 mmol) and triphenyl phosphine (6.55 g, 25 mmol). The reagents were dissolved in anhydrous THF (300 mL) and stirred at room temperature. Once a homogeneous solution was present, DIAD (4.04 g, 20 mmol) was added and the solution stirred for 16 h. The solvent was evaporated and the residue purified by solid phase extraction (3% MeOH in CHCl3 (1 L) followed by 10% MeOH in CHCl3 (1 L) which contained the polymer product). The solvent was removed and the resulting liquid dissolved in ethanol (200 mL) and hydrazine hydrate (10 mL). The solution was stirred at reflux for 14 h, allowed to cool, then concentrated HCl (15 mL) was added dropwise to the solution. The solution was filtered and the solvent evaporated. The residue was dissolved in water and the polymer product extracted with CHCl3 (4×500 mL). The combined organic layers were dried over MgSO4, filtered and the solvent evaporated. The resulting liquid was diluted with a minimal amount of methanol and precipitated in to diethyl ether. A white powder (28.2 g, 71%) was isolated following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 7.79 br-s, 3.7-3.3 br-m, 2.96 t. GPC (DMF, PEG standards) Mn=7,800; PDI=1.03.
Example 2




mPEG-PAsp-(PAsp-co-PBLG)-Ac

To a 100 mL round bottom flask was added mPEG-hydrochloride (1.04 g, 0.14 mmol) and t-butyl aspartic acid NCA (0.3 g, 1.4 mmol). The reagents were dried under vacuum for 1 hour, then NMP (15 mL) added. The solution was degassed under vacuum the backfilled with N2, and stirred at 80° C. After 48 h, benzyl glutamate NCA (0.54 g, 2.1 mmol) and t-butyl aspartic acid NCA (0.15 g, 0.70 mmol) was dissolved in NMP (10 mL) and added to the reaction. After an additional 48 h, the solution was allowed to cool, then DMAP (0.17 g, 1.4 mmol) and acetic anhydride (0.14 g, 1.4 mmol) added to the stirred solution. After 1 hour, the solution was precipitated into diethyl ether/hexanes (3:2, 300 mL). A white solid was recovered after filtration, which was dissolved in TFA/H2O (95:5, 40 mL) and stirred for 4 hours at room temperature. The solvent was evaporated and the residue precipitated into ether (300 mL). A white powder (1.15 g, 68% yield) was recovered following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 12.21, 8.19, 7.36, 5.08, 4.50, 3.7-3.3, 3.16, 2.67, 2.38, 2.01, 1.80.
Example 3




mPEG-PAsp-(PAsp-co-DBzGlu)-Ac

To a 100 mL round bottom flask was added mPEG-hydrochloride (1.0 g, 0.13 mmol) and t-butyl aspartic acid NCA (0.28 g, 1.3 mmol). The reagents were dried under vacuum for 1 hour, then NMP (10 mL) added. The solution was degassed under vacuum the backfilled with N2, and stirred at 80° C. After 48 h, D-benzyl glutamate NCA (0.34 g, 1.3 mmol) and t-butyl aspartic acid NCA (0.28 g, 1.3 mmol) was dissolved in NMP (5 mL) and added to the reaction. After an additional 48 h, the solution was allowed to cool, then DMAP (0.16 g, 1.3 mmol) and acetic anhydride (0.13 g, 1.3 mmol) added to the stirred solution. After 1 hour, the solution was precipitated into diethyl ether/hexanes (3:2, 300 mL). A white solid was recovered after filtration, which was dissolved in TFA/H2O (95:5, 40 mL) and stirred for 4 hours at room temperature. The solvent was evaporated and the residue precipitated into ether (300 mL). A white powder (0.7 g, 52% yield) was recovered following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 12.37, 8.23, 7.97, 7.55, 7.34, 6.97, 5.06, 4.51, 4.27, 3.7-3.3, 3.19, 2.67, 2.35, 2.01, 1.83.
Example 4
4 nm Fe3O4 nanoparticles (2.5 mg) (prepared according to Sun, S.; Zeng, H. “Size-Controlled Synthesis of Magnetite Nanoparticles” J. Am. Chem. Soc. 2002, 124, 8204-8205.) dissolved in CH2Cl2 (1 mL) was added to PEG-b-PAsp-b-P(Phe-co-Tyr) (50 mg) dissolved in water. The biphasic mixture was agitated in an incubated shaker for 3 hours. The homogenous solution was lyophilized and the dry powder was stored at 4° C.
Example 5
A solution of 4 nm Fe3O4 nanoparticles (2.5 mg) and mPEG-PAsp-(PAsp-co-DBzGlu)-Ac (25 mg) dissolved in CHCl3 (1 mL) was added dropwise to a vortexing flask containing water (20 mL). The mixture was vortexed until a homogenous solution was formed. The homogenous solution was analyzed by dynamic light scattering (FIG. 1) then lyophilized and the dry powder was stored at 4° C. A small portion of the powder (5 mg) was reconstituted in water and again analyzed by dynamic light scattering (FIG. 2). Diameter=60+/−8.8 nm pre-lyophilization, 65+/−9.7 nm post-lyophilization.
Example 6
A portion of the iron oxide encapsulated nanoparticles (Example 5, 5 mg) were reconstituted in water (0.5 mL). One drop was added to a carbon-coated, 400 Mesh copper TEM grid. The encapusalted nanoparticles were then imaged on a 100 kV transmission electron microscope (FIG. 3).
Example 7
A solution consisting of 4 nm Fe3O4 nanoparticles (6.35 mg) and mPEG-PAsp-(PAsp-co-PBLG)-Ac (63.5 mg) dissolved in CHCl3 (1 mL) was added dropwise to a vortexing flask containing water (12.5 mL). The mixture was vortexed until a homogenous solution was formed. An aqueous solution of zinc chloride (0.1 M, 12.5 μL) was added and vortexing was continued for 5 minutes, followed by 30 minutes of sonication. The pH of the solution was then adjusted to 7.8 with dilute aqueous sodium hydroxide. The solution was lyophilized and the dry powder was stored at 4° C. A small portion of the powder (5 mg) was reconstituted in water (1 mL) and analyzed by dynamic light scattering (FIG. 4). Diameter=64+/−13 nm.
Example 8
Iron Oxide micelle cytotoxicity assay: 1.5×104 MCF-7, 1.5×104 MDA-MB-231, or 3×103 MCF10A cells were plated in 96 well-plates. Twenty-four hours later, growth media was replaced with 0, 50, 250, 500, 1000, 2500, or 5000 μg/mL of iron oxide-loaded micelles. After incubation at 37 degrees Celsius for 72 h, cell viability was determined using Cell-Titer Glo reagent. Experiments were performed in triplicate and are shown in FIG. 5.
Example 9




To a 2 L 2-neck round bottom flask was added mPEG (180 g, 15 mmol) and anhydrous methylene chloride (1 L). Once a homogeneous solution was present, methane sulfonyl chloride (2.3 mL, 30 mmol) was added, followed by triethyl amine (4.2 mL, 30 mmol) and the solution stirred for 16 h at room temperature. The solvent was evaporated and the residue purified by solid phase extraction (3% MeOH in CHCl3 (2 L) followed by 10% MeOH in CHCl3 (3 L) which contained the polymer product). The solvent was removed and the resulting liquid dissolved in ammonia in methanol (7M, 600 mL). The solution was stirred at 60° C. for 48 h and allowed to cool. The solution was concentrated and redissolved in brine/0.1N HCl solution. The aqueous phase was washed with ether (1 L), then the polymer product extracted with CHCl3 (4×1 L). The combined organic layers were dried over MgSO4, filtered and the solvent evaporated. The resulting liquid was diluted with a minimal amount of methanol and precipitated in to diethyl ether. A white powder (110 g, 69%) was isolated following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 7.84 br-s, 3.7-3.3 br-m, 2.97 t. GPC (DMF, PEG standards) Mn=10,600; PDI=1.06.
Example 10




mPEG227-b-(PAsp)10-b-(PAsp15-co-PLBzAsp15)-Ac To a 100 mL round bottom flask was added mPEG-hydrochloride (1.0 g, 0.13 mmol) and t-butyl aspartic acid NCA (0.28 g, 1.3 mmol). The reagents were dried under vacuum for 1 hour, then NMP (10 mL) added. The solution was degassed under vacuum the backfilled with N2, and stirred at 80° C. After 48 h, d-benzyl aspartate NCA (0.48 g, 1.95 mmol) and t-butyl aspartic acid NCA (0.41 g, 1.95 mmol) was dissolved in NMP (5 mL) and added to the reaction. After an additional 48 h, the solution was allowed to cool, then DMAP (0.16 g, 1.3 mmol) and acetic anhydride (0.13 g, 1.3 mmol) added to the stirred solution. After 1 hour, the solution was precipitated into diethyl ether/hexanes (3:2, 300 mL). A white solid was recovered after filtration, which was dissolved in TFA/H2O (95:5, 40 mL) and stirred for 4 hours at room temperature. The solvent was evaporated and the residue precipitated into ether (300 mL). A white powder (0.75 g, 54% yield) was recovered following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 12.38, 8.26, 7.97, 7.54, 7.34, 6.92, 5.08, 4.53, 4.28, 3.7-3.3, 3.19, 2.32, 2.05, 1.84.
Example 11




mPEG227-b-(PAsp)10-b-(PAsp15-co-PBzGlu15)-Ac To a 100 mL round bottom flask was added mPEG-hydrochloride (1.0 g, 0.13 mmol) and t-butyl aspartic acid NCA (0.28 g, 1.3 mmol). The reagents were dried under vacuum for 1 hour, then NMP (10 mL) added. The solution was degassed under vacuum the backfilled with N2, and stirred at 80° C. After 48 h, benzyl glutamate NCA (0.52 g, 1.95 mmol) and t-butyl aspartic acid NCA (0.41 g, 1.95 mmol) was dissolved in NMP (5 mL) and added to the reaction. After an additional 48 h, the solution was allowed to cool, then DMAP (0.16 g, 1.3 mmol) and acetic anhydride (0.13 g, 1.3 mmol) added to the stirred solution. After 1 hour, the solution was precipitated into diethyl ether/hexanes (3:2, 300 mL). A white solid was recovered after filtration, which was dissolved in TFA/H2O (95:5, 40 mL) and stirred for 4 hours at room temperature. The solvent was evaporated and the residue precipitated into ether (300 mL). A white powder (0.9 g, 58% yield) was recovered following filtration. 1H NMR (400 MHz, DMSO-d6, δ) 12.38, 8.25, 7.97, 7.54, 7.34, 6.93, 5.11, 4.52, 4.27, 3.7-3.3, 3.19, 2.66, 2.32, 1.99, 1.80.
Example 12
HS-2-165
mPEG227-b-(PAsp)10-b-(PAsp15-co-PLBzAsp15)-Ac (0.5 g) and 11 nm MnFe2O4 nanoparticles (56.4 mg in 2 mL hexanes) were dissolved in 5 mL CH2Cl2. The homogenous CH2Cl2 solution was added dropwise to a vortexing flask filled with 100 mL of water. The flask was vortexed for 10 minutes until no odor could be detected and the solution became homogenous. The solution was filtered through a 0.22 micron filter. A brown/black solid was obtained after the sample was lyophilzed.
Example 13
HS-2-166
mPEG227-b-(PAsp)10-b-(PAsp15-co-PLBzAsp15)-Ac (0.5 g) and 15 nm MnFe2O4 nanoparticles (56.4 mg in 2 mL hexanes) were dissolved in 5 mL CH2Cl2. The homogenous CH2Cl2 solution was added dropwise to a vortexing flask filled with 100 mL of water. The flask was vortexed for 10 minutes until no odor could be detected and the solution became homogenous. The solution was filtered through a 0.22 micron filter. A brown/black solid was obtained after the sample was lyophilzed.
Example 14
HS-2-113
mPEG227-b-(PAsp)10-b-(PAsp15-co-PBzGlu15)-Ac (0.5 g) and 4 nm Fe3O4 nanoparticles (50.0 mg) were dissolved in 8.78 mL CH2Cl2. The homogenous CH2Cl2 solution was added dropwise to a vortexing flask filled with 100 mL of water. The flask was vortexed for 10 minutes until no odor could be detected and the solution became homogenous. The solution was filtered through a 0.22 micron filter. A brown/black solid was obtained after the sample was lyophilzed.
Example 15
HS-2-177
mPEG227-b-(PAsp)10-b-(PAsp15-co-PLBzAsp15)-Ac (3.5 g) and 15 nm MnFe2O4 nanoparticles (875 mg in 20 mL of a 2/1 solution of hexanes/CH2Cl2) were dissolved in 30 mL CH2Cl2. The homogenous CH2Cl2 solution was added dropwise to a stirring beaker filled with 700 mL of water. The beaker was stirred at 1700 rpm for 60 minutes until no odor could be detected and the solution became homogenous. The solution was filtered through a 0.22 micron filter. A brown/black solid was obtained after the sample was lyophilzed.
Example 16
The focus of this study was to measure the R1 and R2 relativities of HS-2-113 as a function of concentration using MRI. HS-2-113 was determined to have R1 relaxivity of 0.012 s−1 mmol Fe-1, and an R2 relaxivity of 14.1 s−1 mmol Fe-1. The R1, and R2 relaxivities are lower than that of Feridex (0.68 s−1 mmol Fe-1, and 100 s−1 mol Fe-1 respectively), based on MIR historical data.
HS-2-113 was serially diluted with sterile saline for relaxivity imaging in 1/10, 1/50, 1/200, 1/1000, and 1/5000, dilutions with water. Test dilutions were contained in 0.2 ml Eppendorf tubes.
Imaging
Each set of diluted sample tubes was arranged such that all the tubes could be imaged simultaneously. A tube of sterile water was also included to use as a benchmark for minimal relaxivity. The tubes were imaged using the following scans and scan parameters:










MRI scans used, with associated variable parameters









Relaxivity
Image



Type
Scan Type
Variable Parameters





R1
Spin-echo
Repetition Time (TR) = 0.1, 0.2, 0.4, 0.7, 1,




2, 5, 7.5, 10, 15, 20 s


R2
Spin-echo
Echo time (TE) = 8.4, 10, 12.5, 15, 20, 25,




30, 40, 50, 75, 150, 300, 600 ms









For the R1 measurements, an echo time of 8 ms was used, and for the R2 measurements, a repetition time of 2 s was used. All images were acquired using three 1.5 mm thick transaxial slices through the tubes. The images were manually segmented by defining regions-of-interest (ROIs) for each tube in each slice of each image, and calculating the signal intensity time courses for each tube, averaged over all three slices.
The R2 data were then fitted to a decaying exponential to calculate R2:

S=S0*exp(−R2*t)+C 

where S=signal intensity, t=time, S0=signal intensity for t=0, C=constant (offset).

The R1 data were fitted to an exponential recovery:

S=Seq*(1−exp(−R1*t))+C 

where S=signal intensity, Seq=asymptotic signal value, t=time, C=constant (offset).

All fitting was performed using the Sigmaplot mathematical package. R1 and R2 relaxivity were then calculated as a function of Fe concentration by performing a linear regression over the relaxivity (R1 or R2) vs. concentration curve, and expressed in the standard units of s-1 mmol Fe−1. Historical data for the FDA approved Fe-based contrast agent, Feridex, was used as a relaxivity benchmark.
Individual images showing signal decrease with echo time (for R2 determinations) and signal increase with repetition time (for R1 determination) are displayed in FIG. 6 and FIG. 7. The calculated relaxivities for the dilutions used are shown in Table 9 below.







TABLE 9







Calculated relaxivities for each dilution of HS-2-113 and water











Dilution
R1 (s−1)
R2 (s−1)















1/10
0.244
77.3



1/50
0.244
17.8



1/200
0.202
5.9



1/500
0.179
2.9



1/1000
0.187
1.1



1/5000
0.192
3.6



Water
0.152
0.9










Table 10, below, shows the linear correlations between relaxivity and concentration for HS-2-113 and Feridex, for R1 and R2. Linear regressions on these curves provided quantification.







TABLE 10







Concentration-dependent relaxivity comparisons


between HS-2-113 and Feridex











Relaxivity Parameter
HS-2-113
Feridex















R1 (s−1 mmol Fe−1)
0.012
0.68



R2 (s−1 mmol Fe−1)
14.1
100










The measured relaxivity values show that R1 for HS-2-113 was significantly less than that of Feridex, while R2 was about one seventh that of Feridex. While HS-2-113 has lower relaxivity than Feridex, the R2 relaxivity is estimated to be in the range required to obtain in vivo contrast. The lower R1 relaxivity compared with that of Feridex is favorable for a negative contrast agent as it reduces the potential for R1-mediated positive contrast effects which could offset and reduce the overall negative contrast induced by the agent.
Example 17
The focus of this study was to measure the R1 and R2 relaxivities of HS-2-165 and HS-2-166 as a function of concentration using MRI.
HS-2-165 was determined to have R1 relaxivity of 0.038 s−1 mmol Fe−1, and R2 relaxivity of 31 s−1 mmol Fe−1. The R1 and R2 relaxivities are lower than that of Feridex (0.68 s−1 mmol Fe−1, and 100 s−1 mmol Fe−1 respectively), based on MIR historical data.
HS-2-166 was determined to have R1 relaxivity of 0.017s−1 mmol Fe−1, and an R2 relaxivity of 156 s−1 mmol Fe−1. For HS-2-166, the R1 relaxivity was found to be lower than that of Feridex (0.68 s−1 mmol Fe−1), and the R2 relaxivity was found to be higher than that of Feridex (100 s−1 mmol Fe−1).
HS-2-165 and HS-2-166 were serially diluted with sterile saline for relaxivity imaging, beginning with a 15 mg/ml solution (top concentration, TC), then diluting to obtain additional 0.1×TC, 0.067×TC, 0.04×TC, and 0.02×TC dilutions. Test dilutions were contained in 0.2 ml Eppendorf tubes.
Imaging
Each set of diluted sample tubes was arranged such that all the tubes could be imaged simultaneously. A tube of sterile water was also included to use as a benchmark for minimal relaxivity. The tubes were imaged using the following scans and scan parameters set forth in Table 11, below:







TABLE 11







MRI Scans Used with Associated Variable Parameters









Relaxivity
Image



Type
Scan Type
Variable Parameters





R1
Spin-echo
Repetition Time (TR) = 0.1, 0.2, 0.4, 0.7, 1, 2, 5,




7.5, 10, 15, 20 s


R2
Spin-echo
Echo time (TE) = 8.4, 10, 12.5, 15, 20, 25,




30, 40, 50, 75, 150, 300, 600 ms









For the R1 measurements, an echo time of 8 ms was used, and for the R2 measurements, a repetition time of 2s was used. All images were acquired using three 1.5 mm thick transaxial slices through the tubes. The images were manually segmented by defining regions-of-interest (ROIs) for each tube in each slice of each image, and calculating the signal intensity time courses for each tube, averaged over all three slices.
The R2 data were then fitted to a decaying exponential to calculate R2:

S=S0*exp(−R2*t)+C 

where S=signal intensity, t=time, S0=signal intensity for t=0, C=constant (offset).

The R1 data were fitted to an exponential recovery:

S=Seq*(1−exp(−R1*t))+C 

where S=signal intensity, Seq=asymptotic signal value, t=time, C=constant (offset).
All fitting was performed using the Sigmaplot mathematical package. R1 and R2 relaxivity were then calculated as a function of Fe concentration by performing a linear regression over the relaxivity (R1 or R2) vs. concentration curve, and expressed in the standard units of s-1 mmol Fe−1. Historical data for the FDA approved Fe-based contrast agent, Feridex, was used as a relaxivity benchmark.
Results
Individual images showing signal decrease with echo time (for R2 determinations) and signal increase with repetition time (for R1 determination) for HS-2-165 and HS-2-166 are displayed in FIGS. 8-11. The calculated relaxivities for the dilutions used are shown in Table 12, below.







TABLE 12







Calculated Relaxivities for Each Dilution of HS-2-165 and HS-2-166











Concentration






Relative to Top


Concentration
R1 (s−1)
R2 (s−1)
R1 (s−1)
R2 (s−1)


(15 mg/ml)
HS-2-165
HS-2-165
HS-2-166
HS-2-166














0.01x
0.267
31
0.349
258


0.02x
0.267
63
0.373
442


0.04x
0.346
120
0.414
698


0.067x
0.420
190
0.453
1011


0.1x
0.592
308
0.462
1294









Table 13, below, shows the linear correlations between relaxivity and concentration for HS-2-165 and HS-2-166 and Feridex, for R1 and R2. Linear regressions on these curves provided quantification of the relaxivity for HS-2-165 and HS-2-166, compared with Feridex in the standard units of s−1 mmol Fe−1.







TABLE 13







Concentration-Dependent Relaxivity Comparisons:


HS-2-165, HS-2-166, and Feridex












Relaxivity Parameter
HS-2-165
HS-2-166
Feridex
















R1 (s−1 mmol Fe−1)
0.038
0.017
0.68



R2 (s−1 mmol Fe−1)
31
156
100










The measured relaxivity values show that R1 for HS-2-165 was significantly less than that of Feridex, while R2 was about one third that of Feridex. R1 for HS-2-166 was significantly less than that of Feridex, while the R2 value was about 50% higher than that of Feridex. While HS-2-165 has lower R2 than Feridex, the R2 relaxivity is estimated to be in the range required to obtain in vivo contrast given a tolerated dosage of approximately 400 mg/kg. The R2 relaxivity for HS-2-166 suggests that it would provide greater in vivo contrast than HS-2-165 at a similar dosage level. The lower R1 relaxivity for both particles compared with that of Feridex is favorable for a negative contrast agent as this reduces the potential for R1-mediated positive contrast effects which could offset and reduce the overall negative contrast induced by the agent.
Example 18
The purpose of this study was to determine the maximum tolerated dose for the contrast agent HS-2-113. HS-2-113 was well tolerated at the single, maximal dosage tested in Fischer 344 rats.
The test compound, HS-2-113, was obtained as a powder and stored at room temperature in a cool dry cabinet protected from light. It was first formulated in saline at a concentration of 100 mg/ml. The concentration was slowly increased to a final concentration of 164.6 mg/ml. It was observed that the powder did not dissolve immediately when added to saline, but did dissolve after several hours, without vortexing, sonication or heat. The dosing solution was made on Day 1 of treatment. All dosage levels in this report are expressed in terms of parent compound.
Animals and Husbandry
Female Fischer 344 rats were obtained from Harlan. They were 6 weeks old on Day 1 of the experiment. The animals were fed irradiated Rodent Diet 5053 (LabDietTM) and water ad libitum. Rats were housed in ThorenTM microisolator caging with Bed-O'CobsTM bedding. All treatments, body weight determinations, and tumor measurements were carried out in laminar down flow cabinets. All procedures carried out in this experiment were conducted in compliance with all the laws, regulations and guidelines of the National Institutes of Health (NIH).
Treatment was administered on Day 1, after the animals were housed and their body weights measured. All animals weighed ≧134.8 g at the initiation of therapy. Mean group body weights at treatment were well matched (range, 135.6-140.2 g). All animals were dosed according to individual body weight on the day of treatment (0.2 ml/20g). The HS-2-113 solution was administered via slow injection (over 3 minutes).
Assessment of Side Effects
All animals were observed for one hour following the dose for signs of acute toxicity. Throughout the study, all animals were observed for clinical signs at least once daily. Animals were weighed on the day of treatment and twice weekly thereafter. Individual body weights were recorded twice weekly. Treatment-related weight loss in excess of 20% is generally considered unacceptably toxic. In this report, a dosage level is described as tolerated if treatment-related weight loss (during and two weeks after treatment) is <20% and mortality during this period in the absence of potentially lethal tumor burdens is ≦10%.
Dosing and Toxicity
The treated animals in this study showed no signs of toxicity from the treatments. The animals in the Treatment Group experienced no weight loss; however, their weight gain was slightly slower than that of the Untreated Group. Given the small group sizes (n=3), this difference is not thought to be significant. Animal 2 of the Treatment Group displayed slight discoloration of the tail. This occurred one week after treatment and remained until the end of treatment, and was likely due to leakage of the agent from the tail vein into the sub-dermal space.
HS-2-113 was well tolerated at the dosage level tested. The only unusual occurrence was discoloration of the tail in one treated animal. This discoloration was closely monitored and although it did not disappear, it also did not get any worse. This animal survived through the duration of the experiment and did not show any other adverse clinical signs.
Example 19
HS-2-177 was well tolerated at 150 mg/ml and showed significant in vivo contrast enhancement in the 9L intracranial glioma rat model for all three animals studied.
HS-2-177 is an iron-oxide based nanoparticle that is under development as an MRI contrast agent. The current study was designed to verify a tolerated dose in vivo, and to test in vivo contrast enhancement of a newly synthesized nanoparticle, HS-2-177, in the 9L orthotopic rat brain tumor model.
Animals and Husbandry
Intracranial 9L tumors were implanted in male Fischer 344 rats weighing between 125 and 150 g. 9L cells (105) were implanted in the right forebrain at a depth of 3 mm through a 1 mm burr hole on Day 0. The surgical field was cleaned with 70% ethanol and the burr hole was filled with bone wax to prevent extracranial extension of the tumor. Animals were imaged using MRI beginning at 15 days post cell implantation to select tumors for in vivo nanoparticle (NP) studies. A total of 3 animals were selected for the study from 5 that were implanted. The animals imaged in this study had tumors in the range 40-89 μl in volume.
Nanoparticle Preparation and Administration
HS-2-177 was supplied as a powder, and formulated in saline at a concentration of 150 mg/ml to create a fine, opaque black suspension ready for injection. Tolerance of IV injection of HS-2-177 at 10 ml/kg using the 150 mg/ml formulation was initially tested in a triage rat (Fischer 344) from the tumor pool, but further testing could not be done as the remaining triage animals had succumbed to tumor burden at the time of injection. HS-2-177 was therefore tested in one additional Female Dark Agouti rat that was available. For both animals, HS-2-177 was dosed intravenously over approximately 180 seconds. Animals were observed for acute signs of toxicity for 30 minutes, and then monitored every 15 minutes for a further hour. Additional monitoring of the animals was done over the next 4 days while the imaging was done. Nanoparticle administration began on Day 21. All animals weighed >240 g at the time of administration. HS-2-177 was dosed (10 ml/kg) by tail vein injection of the formulated material. An AngiocathTM Teflon catheter was placed in the tail vein of the animal and flushed with 0.2 ml heparin in saline (10 units/ml). A pre-primed infusion line was connected to the AngiocathTM. The HS-2-177 was injected over 30 seconds using a Harvard syringe pump during dynamic MR scanning (see below).
In vivo MRI
After animal preparation and anesthesia induction using 1.5% isoflurane, an anatomical T2-weighted scout image was obtained to locate the tumor using a multi-slice fast spinecho protocol with a 25×25 mm field of view (FOV), 128×128 image matrix, TR=4s, echo spacing=15 ms, and 8 echoes with k-space centered on the 4th echo. To determine the distribution and preliminary pharmacokinetic behavior of HS-2-177, MR images were obtained using T2*-weighted gradient echo MRI. For imaging nanoparticle uptake, gradient echo images were acquired using a 25×25 mm field of view over a 64×64 matrix, in contiguous 1 mm axial slices which covered the entire region of the tumor. Pre-IVbolus scans were obtained with TR=2s/TE=15 ms (referred to as TR2k protocol). During IV injection of the nanoparticle suspension, a dynamic gradient-echo sequence with a time resolution of 10.24 sec (TR=80 ms/TE=7.5 ms) was used to characterize the uptake of the particle into normal tissue and the tumor over 7 minutes. Post-IV-injection TR2k gradient-echo scans were then acquired at approximately 3 hours to examine the rate clearance of HS-2-177.
Measurements and Endpoints
Images were analyzed by measuring signal intensity time courses within manually drawn regions of interest (ROIs) in vessel, normal brain, and tumor. Relative concentration of the contrast agents is a measure of the relative change in tissue contrast agent concentration over time and can be derived from the tissue signal intensity:

Relative concentration∝ΔR2*∝log(S/S0)/TE 

where R2*(i.e. inverse of T2*) is the transverse relaxation rate or relaxivity and ΔR2* is the change in R2* after injection of the contrast agent, S is signal intensity following administration of the contrast agent, S0 is the initial signal intensity and TE is the echo time. Exponential nanoparticle clearance was assumed and therefore, the extended time courses of relative concentration (nanoparticle washout curves), were fitted to decaying exponentials and half-lives derived for clearance of the nanoparticles from vasculature, contralateral normal brain tissue and tumor tissue.

The contrast-to-noise ratio (CNR) was also determined from the uptake data according to the relation:

CNR=[S(brain)−S(tumor)]/N, 

where S(brain) and S(tumor) are the average signal intensities of normal brain and tumor, respectively, and N is the average noise signal level. The CNR between normal brain and tumor were calculated at baseline (pre-injection) and maximum (postinjection).

A second parameter used to quantify the level of contrast is the Brain/Tumor Signal ratio defined as:

Brain/Tumor Signal ratio=[signal intensity in contralateral brain tissue]/[signal intensity in tumor].

Results

The 9L rat glioma model was chosen because it provides an excellent model for showing contrast enhancement (through contrast between tumor and normal brain tissue) using MRI, and is especially suited toward proof of principle studies. This study was preceded by the following studies:
1. Tested tolerance of HS-2-113 and showed that a 164.6 mg/ml formulation was well tolerated at the maximal dose volume of 10 ml/kg.
2. Tested in vitro relaxivity of HS-2-113 and determined that the relaxivity of HS-2-113 was approximately 7 times lower than that of Feridex.
3. Tested in vitro relaxivity of HS-2-165 and HS-2-166. It was determined that HS-2-166 had R2 relaxivity approximately 1.5 times greater than that of Feridex, while HS-2-165 R2 relaxivity was approximately 3 times lower than that of Feridex.
Significant contrast enhancement was detected in the brains of three animals studied following intravenous administration of HS-2-177. See FIG. 12 and FIG. 13. The tumor signal was approximately decreased to the noise level 1-2 minutes after the injection. Heterogeneity was observed across the tumor in all cases, which is typical due to normal tumor heterogeneity. The mean tumor CNR value obtained was 10 (averaged over the entire tumor in each case), with two animals showing CNR values of 13 (range=4-13). The commercial standard in this agent class has shown peak CNR of 50-60. The mean brain/tumor signal ratio for HS-2-177 was calculated to be 2.1 (range=1.9-2.5). In previous work, Feridex produced a brain/tumor signal ratio of approximately 3.
Based on a comparison of a single image acquired at approximately 90 minutes after the HS-2-177 administration with the images acquired immediately after administration, it was estimated by relative concentration exponential decay analyses that the half life for HS-2-177 was approximately 26 minutes. This is less then the half life that has been measured for Feridex (46 minutes). In summary, this study demonstrated that HS-2-177 was well tolerated and had favorable in vivo contrast enhancement properties that approximate those of Feridex in the 9L tumor model.
Example 20
As depicted in FIGS. 14 and 15, empty micelles and iron oxide loaded micelles were dissolved at various concentrations in an aqueous solution of sodium dodecyl sulfate (5 mg/mL) and the scattering intensity recorded for each solution. FIG. 14 depicts HS-2-177 compared to mPEG227-b-(PAsp)10-b-(PAsp15-co-PLBzAsp15)-Ac). FIG. 15 depicts HS-2-113 compared to (mPEG227-b-(PAsp)10-b-(PAsp15-co-PBzGlu15)-Ac). The significantly higher scattering intensity for the iron oxide loaded micelles suggests the persistence of the micellar structure throughout dilution. Dynamic light scattering of the iron oxide loaded micelle solutions exhibited a size distribution centered around 60 nm, confirming the presence of micelles throughout the dilution. DLS sizing of the empty micelle solution exhibited a size distribution of less than ten nanometers, indicating free polymer and the disruption of the micelle structure.
Example 21
The focus of this study was to measure the R1 and R2 relaxivities of HS-2-177 as a function of concentration using MRI, and to benchmark the measured relaxivity values against those measured for Feridex in a previous study.
HS-2-177 was obtained as a brown powder and was stored in a dark dry cabinet. It was serially diluted with sterile saline for relaxivity imaging, resulting in suspensions that varied from an opaque black appearance (highest concentration) to a transparent appearance (lowest concentration). The intermediate concentrations were transparent in appearance with varying shades of brown. The various concentrations of the test agent were contained in 0.2 ml Eppendorf tubes. The concentrations formulated were:

    
    
        Tube 1: 0.300 mg/ml; Tube 2: 0.200 mg/ml
        Tube 3: 0.120 mg/ml
        Tube 4: 0.060 mg/ml
        Tube 5: 0.030 mg/ml

Imaging

    
    


The sample tubes containing the concentrations listed above were arranged such that all the tubes could be imaged simultaneously. The tubes were imaged using the following scans and scan parameters:







TABLE 14







MRI Scans Used with Associated Variable Parameters









Relaxivity
Image



Type
Scan Type
Variable Parameters





R1
Spin-echo
Repetition Time (TR) = 0.1, 0.2, 0.4, 0.7, 1, 2, 5,




7.5, 10, 15, 20 s


R2
Spin-echo
Echo time (TE) = 8.4, 10, 12.5, 15, 20, 25, 30,




40, 50, 75, 150, 300, 600 ms









For the R1 measurements, an echo time of 8 ms was used, and for the R2 measurements, a repetition time of 2 s was used. All images were acquired using three 1.5 mm thick transaxial slices through the tubes. The images were manually segmented by defining regions-of-interest (ROIs) for each tube in each slice of each image, and calculating the signal intensity time courses for each tube, averaged over all three slices.
The R2 data were then fitted to a decaying exponential to calculate R2:

S=S0*exp(−R2*t)+C 

where S=signal intensity, t=time, S0=signal intensity for t=0, C=constant (offset).

The R1 data were fitted to an exponential recovery:

S=Seq*(1−exp(−R1*t))+C 

where S=signal intensity, Seq=asymptotic signal value, t=time, C=constant (offset).

All fitting was performed using the Sigmaplot mathematical package. R1 and R2 relaxivity were then calculated as a function of Fe concentration by performing a linear regression over the relaxivity (R1 or R2) vs. concentration curve, and expressed in the standard units of s−1 mmol Fe−1. Historical data for the FDA approved Fe-based contrast agent, Feridex, was used as a relaxivity benchmark.
Results
Individual images showing signal decrease with echo time (for R2 determinations) and signal increase with repetition time (for R1 determination) are displayed in FIGS. 16 and 17.







TABLE 15







Calculated Relaxivities for Each Dilution of HS-2-177









Concentration
R1 (s−1)
R2 (s−1)


(mg/ml)
HS-2-177
HS-2-177












0.300
1.49
159.07


0.200
2.52
104.48


0.120
0.98
51.94


0.060
0.57
28.46


0.03
0.36
4.34









Table 16, below, shows the linear correlations between relaxivity and concentration for HS-2-177 and Feridex, for R1 and R2.







TABLE 16







Concentration-Dependent Relaxivity Comparisons


Between HS-2-177 and Feridex











Relaxivity





Parameter
HS-2-17
Feridex















R1 (s−1 mmol Fe−1)
3.3
0.68



R2 (s−1 mmol Fe−1)
237
100










As reported in Table 16, above, the measured relaxivity values show that R1 and R2 relaxivities for HS-2-177 were higher than those of Feridex.
Example 22
The purpose of this study was to determine the effect of particle size and weight loading on R1 and R2 relaxivity of MnFe2O4 loaded micelles. These nanoparticle-loaded micelles were prepared with 7 nm, 11 nm, and 15 nm MnFe2O4 particles with feed ratios of 20%, 50%, 60% or 80% by mass with respect to the polymer. MnFe2O4 loaded micelles were prepared according to Example 14. The actual Fe concentration was determined by fully dissolving the micelles in 10% HNO3 then subjecting this solution to ICP-MS analysis.
Samples for relaxivity measurements were prepared by dissolving ca. 2 mg of micelle in 10% D2O in H2O, then serially diluting the samples four times to give a range of Fe concentrations. R1 and R2 values were calculated for each Fe concentration from measurements performed on a 400 MHz NMR under the following conditions:
T1 measurements performed using an inversion recovery pulse sequence with recovery times of 0.1000, 0.1479, 0.2169, 0.3234, 0.4782, 0.7071, 1.0456, 1.5463, 2.2865, 3.3812, and 5.0000 seconds. The T1 value was calculated by the VNMRJ 2.2C software package. The R1 value for each sample was calculated by the following equation:




  
    R
    ⁢
    
      
    
    ⁢
    1
  
  =
  
    1
    
      T
      1
    
  




The R1 values were plotted against the iron concentration in mmol/L and a linear regression (y=mx) fit to the data, where the slope of the line is equal to the R1 relaxivity.
R2 measurements were performed using a Carr-Purcell pulse sequence with echo times of 0.00500, 0.00793, 0.01257, 0.01994, 0.03162, 0.05014, 0.07952, 0.1261, and 0.2000 seconds. The amplitude of the H2O resonance was recorded for each echo time and plotted against the echo time. The data was fitted to the following equation:

y=a*e−R2x 

The R2 values for each sample were plotted against Fe concentration in mmol/L and the data fit to a linear regression (y=mx), where the slope of the line is equal to the R2 relaxivity.
A summary of the results is found in Table 17.












TABLE 17





Micelle
Target wt %
ICP wt %
R1
R2
Contrast (R2/R1)















7 nm particles












HS-2-235-20
20
4.13
3.00
85.0
28


HS-2-235-50
50
4.76
3.07
108.0
35







11 nm particles












HS-2-236-20
20
7.27
2.66
146.6
55


HS-2-236-50
50
11.84
2.23
381.7
171







15 nm particles












HS-2-237-20
20
15.69
1.07
177.2
166


HS-2-237-50
50
22.07
0.87
711.7
818


HS-2-237-60
60
25.96
0.68
605.0
890


HS-2-237-80
80
24.88
0.51
491.1
963









While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

## Claims

1. A micelle, having a contrast agent encapsulated therein, comprising a multiblock copolymer of formula I:



wherein:
n is 10-2500;
m is 5-50;
m′ is 5-50;

Rx is a natural or unnatural amino acid side-chain group that is capable of crosslinking;
Ry is a hydrophobic or ionic, natural or unnatural amino acid side-chain group;
R1 is —Z(CH2CH2Y)p(CH2)tR3, wherein:
Z is —O—, —S—, —C≡C—, or —CH2—;
each Y is independently —O— or —S—;
p is 0-10;
t is 0-10; and

R3 is hydrogen, —N3, —CN, a mono-protected amine, a di-protected amine, a protected aldehyde, a protected hydroxyl, a protected carboxylic acid, a protected thiol, a 9-30 membered crown ether, or an optionally substituted group selected from aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;
Q is a valence bond or a bivalent, saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO2—, —NHSO2—, —SO2NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:
-Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R2a is a mono-protected amine, a di-protected amine, —N(R4)2, —NR4C(O)R4, —NR4C(O)N(R4)2, —NR4C(O)OR4, or —NR4SO2R4; and
each R4 is independently an optionally substituted group selected from hydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety, or:
two R4 on the same nitrogen atom are taken together with said nitrogen atom to form an optionally substituted 4-7 membered saturated, partially unsaturated, or aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

2. The micelle according to claim 1, wherein R3 is an optionally substituted aliphatic group.

3. The micelle according to claim 1, wherein R3 is an azide-containing group or an alkyne-containing group.

4. The micelle according to claim 1, wherein Q is a valence bond.

5. The micelle according to claim 1, wherein Q is a bivalent, saturated C1-12 alkylene chain, wherein 0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, or —C(O)—, wherein -Cy- is an optionally substituted 5-8 membered bivalent, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered bivalent saturated, partially unsaturated, or aryl bicyclic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

6. The micelle according to claim 1, wherein Rx is a natural or unnatural amino acid side-chain group selected from a glutamic acid side-chain, an aspartic acid side-chain, a cysteine side-chain, a serine side-chain, an aldehyde containing side-chain, an imidazole containing side-chain, a benzimidazole containing side-chain, a lysine side-chain, an arginine side-chain, or a histidine side-chain.

7. The micelle according to claim 1, wherein Ry is a hydrophobic amino acid side-chain selected from a suitably protected aspartic acid, a suitably protected glutamic acid, a suitably protected tyrosine side-chain, a suitably protected serine side-chain, a suitably protected threonine side-chain, a phenylalanine side-chain, an alanine side-chain, a valine side-chain, a leucine side-chain, a tryptophan side-chain, a proline side-chain, or a mixture thereof.

8. The micelle according to claim 1, wherein Ry comprises a mixture of hydrophobic and hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising Ry is hydrophobic.

9. The micelle according to claim 1, wherein Ry consists of a mixture of D-hydrophobic and L-hydrophilic amino acid side-chain groups such that the overall poly(amino acid) block comprising Ry is hydrophobic and is a mixture of D- and L-configured amino acids.

10. The micelle according to claim 1, wherein Ry consists of a mixture of hydrophobic amino acid side-chain groups selected from D-leucine, D-phenylalanine, D-alanine, D-benzyl aspartate, or D-benzyl glutamate, and one or more of L-tyrosine, L-cysteine, L-aspartic acid, L-glutamic acid, L-DOPA, L-histidine, L-lysine, or L-ornithine.

11. The micelle according to claim 9, wherein the mixture of D-hydrophobic and L-hydrophilic amino acid side-chain groups is selected from D-leucine/L-tyrosine, D-leucine/L-aspartic acid, D-leucine/L-glutamic acid, D-phenylalanine/L-tyrosine, D- phenylalanine/L-aspartic acid, D-phenylalanine/L-glutamic acid, D-phenylalanine/L-serine, D-benzyl aspartate/L-tyrosine, D-benzyl aspartate/L-aspartic acid, D-benzyl aspartate/L-glutamic acid, D-benzyl glutamate/L-tyrosine, D-benzyl glutamate/L-aspartic acid.

12. The micelle according to claim 2, wherein R2a is —NHC(O)R4, —NHR4 or —N(R4)2, wherein each R4 is an optionally substituted aliphatic group.

13. The micelle according to claim 1, wherein R2a is —NHR4 or —N(R4)2, wherein each R4 is independently methyl, ethyl, propyl, butyl, pentyl, hexyl, 2-(tetrahydropyran-2-yloxy)ethyl, pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl, (4-acetylenylphenyl)methyl, 3-(methoxycarbonyl)-prop-2-ynyl, methoxycarbonylmethyl, 2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl, 2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl, 4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl, 4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl, 4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl, 2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl, 2-(N-methyl-N-propargylamino)ethyl, 2-(2-dipropargylaminoethoxy)-ethyl, vinyl, allyl, crotyl, 2-propenyl, but-3-enyl, —CH2CN, —CH2CH2CN, —CH2CH(OCH3)2, 4-(bisbenzyloxymethyl)phenylmethyl, —CC≡CH, —CH2C≡CH, —CH2C≡CCH3, or —CH2CH2C≡CH.

14. The micelle according to claim 1, wherein the contrast agent is selected from a magnetic nanoparticle or a semiconductor nanoparticle.

15. The micelle according to claim 1, wherein the contrast agent is selected from Fe, Fe2O3, Fe3O4, MnFe2O4, CoFe2O4, NiFe2O4, Co, Ni, FePt, CoPt, CoO, Fe3Pt, Fe2Pt, CO3Pt, CO2Pt, or FeOOH.