Nanoparticles containing a taxane and their use

A OP ARTICLES CONTAINING A TA A E AND THEIR USE

FIELD

[0001 ] The present disclosure reiates to a surface-modified branched polymer (MBP) or a linear polymer, which can either be a surface-modified symmetrically branched polymer (SBP); a surface-modified asymmetrically branched polymer (ABP); or a linear polymer with at least one chain end modified with a hydrophobic group, which on exposure to a water insoluble or poorly water soluble taxane forms a composite nanoparticle or nanoaggregate, wherei the drug is dispersed or deposited at or near hydrophobic domains, such as, at the surface or at structures where hydrophobic portions, segments or sites are located. The particles or aggregates of interest are stable, for example, can be desiccated and rehydrated. The nanoparticl.es or nanoaggregaies can range from about 20 tun to about 500 am in diameter.

Hydrophobic, electrostatic, metai-ligand interactions, hydrogen bonding and other molecular interactions may be involved in the spontaneous interactions between the water insoluble or poorly water soluble iaxane and the homopoiymer to form aggregates. The particles or aggregates of interest have a controlled release profile and thus .find utility, for example, as a carrier for the controlled release of a iaxane in a host for treating a suitable disorder; and the like. For example, the present

disclosure relates to the use of such polymers for the in vivo delivery of a taxane, such as, paclitaxel and derivatives thereof with lower toxicity, improved solubility, greater bioavailability and enhanced efficacy in treating cancers.

BACKGROUND

Symmetrically Branched Polymers

[0002 j A new class of polymers called dendritic polymers, including Starbwst dendrimers (or Dense Star polymers) and Combburst dendrigrafts (or hyper comb-branched polymers), recently was developed and studied for various industrial applications. Those polymers often possess: (a) a well -defined core molecule, (b) at least two concentric dendritic layers (generations.) with symmetrical (equal length) branches and branch junctures and (c ) exterior surface groups, such as.

poiyamidoauiine (PAMAM)-based branched polymers and dendrimers described in U.S. Pat Nos. 4,435,548: 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524; and i 5,714 J 66. Other examples include polyethyleneuufne (PE.I) dendrimers, such as those disclosed in U.S. Pat. No. 4,631,337; poljpropyleuei me (PPi) dendrimers, such as those disclosed in U.S. Pat. Nos. 5,530,092; 5,610,268; and 5,698.662;

Frechet-lype polyether and polyester dendrimers. core shell tectodendrimers and others, as described, for example, in, "Dendritic Molecules," edited by Newkome et a ., VCH Weinheim, 1 96, " eiidrimers and Other Dendritic Polymers/' edited by Frechet <& Tomalia, John Wiley & Sons, Ltd., 2001; and U.S. Pat No. 7,754,500.

[0003} Combburst dendri grafts are constructed with a core molecule and concentric layers with symmetrical branches through a stepwise synthetic method. In contrast to dendrimers, Combburst dendrigraf s or polymers are generated with monodisperse linear polymeric building blocks (U.S. Pat. Nos. 5,773,527; 5,631,329 and 5, 19,442). Moreover, the branch pattern is different from that of dendrimers. For example, Combbnrst dendrigrafts form branch junctures along the polymeric backbones (chain branches), while Starburst dendrimers often branch at the termini (terminal branches). Due to the living polymerization techniques osed, the molecular weight distributions (Μ_¾/Μ„) of those polymers (core and branches) often are narrow. Thus, Combbnrst dendrigrafts produced through a graft-on-graft process are welt defined with M_wM_n ratios often approaching 1.

[0004] SBPs, such as dendrimers, are produced predominantly by repetitive protecting and dep.rotecii.ng procedures through either a divergent or a. convergent synthetic approach. Since dendrimers utilize small molecules as building blocks for the cores and the branches, the molecular weight distribution of the dendrimers often is defined. In the ease of lower generations, a single molecular weight dendrimer often is obtained While dendrimers often utilize small molecule monomers as building blocks, dendrigrafts use linear polymers as building blocks.

[0005] In addition io dendrimers and dendrigrafts, other SBP's include symmetrical star-shaped or comb-shaped polymers, such as, symmetrical star-shaped or comb-shaped polyethyleneoxide (PEG), polyethyJeneglyco! (PEG), PEL PPL polyosazoline (POX), poiyniethyloxazo.ii.ue (PMO.X). polyethyloxazo!ine (PEOX), polystyrene, polymethylmethacrylate, poiydiraethylsiloxane or a combination ihereof. Asymmetrically Branched Polymers

[000 j Unlike SBPs, asymmetrically branched polymers (ABP), particularly asymmetricall branched dendrimers or regular ABP (reg-ABP), often possess a core, controlled and well-defined asymmetrical ( unequal length) branches and

asymmetrical branch junctures as described in U.S. Pat. Nos. 4,289,872; 4,360,646; and 4,410,688.

[0007] On the other hand, a random ABP (ran- ABP) possesses: a) no core, b) functional groups both at the exterior and in the interior, c) random/variable branch lengths and patterns (i.e., termini and chain branches), and d) unevenly distributed interior void spaces.

1000 1 The synthesis and mechanisms ofran-ABPs, such as those made from PEL were reported by Jones et a!., J. Org. Chem. 9, 125 (1 44), Jones et al,, J. Org. Chera. 30, 1994 (.1965) and Dick et al., J. Macromoi. Sci. Chem., A4 (6), 1301-1314, ( 1 70)). Ran-ABP, such as those made of POX, i.e., po!y(2-methy Soxa .oline) and poly(2 -ethyl ox azoline), was repotted by Lid (J, Macromoi. Sci, Chem, A9(5),

703-727 (1 75)) and Warakomski (J. Polym. Sci. Poiyra. Chem. 28, 3551 (1990)). The synthesis of ran-ABP's often can involve a one-pot divergent or a one-pot convergent method.

Homepoiymers

[0009] A homopolymer can relate to a polymer or to a polymer backbone composed of the same repeat unit, that is, the homopolymer is generated from the same monomer (e.g., PEI linear polymers, POX linear polymers, PEI dendrimers, po^'lyaniidoamine (PAA) dendrimers or POX dendrigrafts and randomly ranched polymers). The monomer can be a simple compound or a complex or an assemblage of compounds where the assemblage or complex is the repeat uni in the

homopolymer. Thus, if an assemblage is composed of three compounds, A, B and C; the complex can be depicted as ABC. On the other band, a polymer composed of (ABC)~(ABC)~(ABC).. J.s a homopolymer for the purposes of the instant disclosure. The homopolymer may be linear or branched. Thus, in the case of a randomly branched PEI, although there are branches of different length and branches occur randomly, that molecule is a homopolymer for the purposes of the instant disclosure because that branched polymer is composed, of a single monomer, the ethylenei.rmne or aziridine repeat unit. Also, one or more of the monomer or complex monomer components can be modified, substituted, derivatized and. so on, for example, modified to carry a junctional group. Such molecules are horaopoiyraers for the purposes of the insiani disclosure as the backbone is composed of a single simple or complex monomer.

Poorly Water Soluble Drugs: Tavanes

[001 j Paclitaxel is a water insoluble drug soid as Taxof by Bristol-Myers Squibb. Paclitaxel is derived from Che Pacific Yew tree, Taxus brevifolta (Wan et al., J, Am, Chem. Soc. 93:2325, 1 971). Taxanes, including paclitaxel and docetaxcl (also sold as Taxotere*^'),, are used to treat various cancers, including, breast, ovarian and lung cancers, as well as colon, and head and neck carreers, etc.

[001 1 ] However, the poor aqueous solubility of paclitaxel has hampered the widespread use thereof. Currently, Taxol * and generics thereof are formulated using a 1 : 1 solution of ethanohCremaphor* (polyethyoxylated castor oil) to solubili e the drug. The presenc of Cremaphor* has been linked to severe hypersensitivity reactions and consequently requires medication of patients with corticosteroids (e.g., dexarnethasone) and antihistamines.

[0012] Alternatively, conjugated paclitaxel, for example, Abraxane^, which is produced by mixing paclitaxel with human serum albumin, has eliminated the need for corticosteroids and antihistamine injections. However, Abraxane*^' generates undesirable side effects, such as, severe cardiovascular events, including chest pain, cardiac arrest, supraventricular tachycardia, edema, thrombos s, pulmonary thromboembolism, pulmonary emboli, hypertension -etc., which prevents patients with high cardiovascular risk from using the drug.

Delivery of Poorly Water Soluble Drugs

[0013] Although, branched polymers, including SBPs and ABPs, have been used for drug delivery, those attempts are focused, primarily on the chemical, attachment of the drug to the polymer, or physical encapsulation of such drugs in the interior through unimolecular encapsulation (U.S. Pat. Nos. 5,773,527; 5,631,329; 5. 19,442; and 6,716,450). 0014} For example, dendriniers and. dendrigrafts are believed to entrap physically bioactive molecules using unimoiecular encapsulation approaches, as described in U.S. Pat os. 5338,532; 5,527,524; and 5,714, 166 for dense star polymers, and U.S. Pat, No, 5,91.9,442 for hyper comb-branched polymers. Similarly, the unimolecutar encapsulation of various drugs using SBPs to form a, "dendrimer box," was reported in Toma!ia et al, Angew. Chem, Int. Ed. Engl, 1990, 29, 138, and in, "Dendrimers and Other Dendritic Polymers," edited by Frechet & Tomaiia, John Wiley & Sons, Lid., 2001 , pp. 387-424.

[0 1 ] Branched core shell polymers with a hydrophobic core and a

hydrophUic shell may be used to entrap a poorly water soluble drag through

molecular encapsulation. Randomly branched and hyperbranched core shell, structures with a hydrophihc core and a hydrophobic shell have also been used to carry a drag through unimoiecular encapsulatio and pre-formed nanomicelles (U.S. Pat. No. 6,716,450 and Liu et al., Biomateriais 2010, 10, 1334-1341), However, those unimoiecular and pre-forraed micelle structures are generated in the absence of a drug,

[0016] In embodiments, block copolymers, such as, miktoarm polymers (i.e., Y shaped/ABiHype star polymers) and linear (A)-deridri«c (8) block copolymers, were observed to form stereocomplexes with paclitaxel. (Nederberg et al.,

Biomacromolecules 2009, 10, 1460-1468 and Ltio et al., Bioconjugate Chem. 20.10, 21, 1216). Those block copolymers closely resemble traditional lipid or AB-type linear block copolymers., which are well, known surfactants used for the generation of micelles.

[001 ] However, such branched block copolymers are difficult to make and thus, are not suitable for mass production.

[001 ] There is no description of modi fying branched, or li near homopo!ymers with a hydrophobic group, which on exposure to a poori soluble or water insoluble drug, spontaneously form stable aggregates which are suitable for controlled dru delivery. SUMMARY

[00 j In one aspect., the present: disclosure is directed to use of modified branched polymers ( BP) or linear polymers to increase the solubility of taxanes, such as, pac!itaxei, and derivatives thereof.

0020] in another aspect of the disclosure, the asymmetrically branched polymer (ABF) has either random or regular, asymmetrical branches. The random ABP can also have a mixture of terminal and chain branching patterns.

[0021 j In another aspect of the disclosure, both ABPs and SBPs can be modified further with at. least one molecule or group capable of formi ng additional branches at a given time so that new material properties can be achieved, wherein additional functional groups further ma be attached. AH of the modified polymers cart be defined as modified SBP or ABP's.

[0022] In another aspect of the disclosure, the unmodified and modified branched polymers either can be produced by a divergent or a convergent method, and either a stepwise or a one-step synthetic process can be used.

[0023 j In another aspect of the disclosure, the SBP includes, but is not limited to, PAA dendrimers; PEI dendrimers; PPI dendrimers; polyether dendrimers;

polyester dendrimers; eomb-bmnched star-branched. polymers, such as, PAA, poSyethyleneoxide (PEO), pofyethyleneglycol (PEG), PMOX, PEOX,

polymethylmethacrylate (PMA), polystyrene, poiybutadiene, polyisoprene and polydimethylsiloxane; comb-branched dendri grafts, such as, PEOX, PMOX, polypropyloxazoline (PPOX), polybutyloxazolme, PEL PAA: and so on.

[0024] In a further aspect of the disclosure, the SBP can have an interior void space, while the ABP can have unevenly distributed void spaces.

[0025] in another aspect of the disclosure, a hybrid branched polymer comprising the aforementioned SBPs, such as, dendrimers or dendrigrafis, and ABPs, such as, regular and randoml branched, polymers, as well as star-branched and comb-branched polymers, or combination thereof, also can be used for the generation of said drug-induced aggregates or nanopardcles of .interest.

[0026] In another aspect of the disclosure, the branched polymers are modified with functional groups, such as, but not limited to, NH_¾ NHR, NR_¾ NR_S^! , COOR, COOH, COO^', OH, C(0) , C(0}N¾ C(0)NHR or C(0)NR₂, wherein R can be any aliphatic group, aromatic group or combination thereof; an aliphatic group (e.g., a hydrocarbon chain), which can be branched, cart contain one or more double and/or triple bonds and/or may be substituted; an aromatic group, which may contain a plurality of rings, which may be fused or separated, the rings may be of varying size and/or may contain subsiituents; perOuorocarbon chains; saccharides and/or

polysaccharides, which may be of varying ring sizes, the rings may contain a

heteroatom, such as a sulfur or a nitrogen atom, may be substituted, may contain more than one species of saccharide, may be branched and or may be substituted;

•polyethylene glycols; and (he like.

[0027] The molecular weight of the MBPs can range from abottt 500 to over 5,000,000; from about 500 to about i ,000,000; from about 1 ,000 to about 500,000; from about 2,000 to about 100,000.

[0028] In another aspect of the disclosure, the surface of the SBP's and ABP's is modified so that the physical properties of the surface groups- will be more

compatible with a taxane, thus making taxane more raise ible with the surface group region, domain, portion or segment of the BP's.

[0029] In an embodiment, the modification of a branched polymer or a linear polymer at a chain end is with a hydrophobic functional group, such as, aliphatic chains (e.g., hydrocarbon chains comprising I to about 22 carbons, whether linear or branched), aromatic structures (e.g. containing one or more aromatic rings, which may be fused) or combinations thereof.

[0030] In contrast to known drag carriers, the branched or linear polymer structures of the instant invention do not physically entrap taxane within each polymer molecule, instead, a taxane either can be located, at or dispersed in the

domains/regions containing functional groups of each branched or linear polymer.

[0031 ] The resulting structures of interest optionally can be preserved, for example, by ly philizatiou or other form of desiccation, which may further stabilize the structures of interest. Once redissolved. in water or a buffer, nanopardcles with sizes ranging from about 50 to about 500 nm in diameter can be obtained.

[0032] The presence of .multiple, often funciionalixed branches enables the formation of intramolecular and iniermoieciiiar crosslinks, which may stabilize the taxane-containmg nanoparticles. On dilution, said physical aggregate or nanopartic-le deconstructs releasing drug at a controlled rate. [0033 j In. another aspect of the disclosure, a mixture of linear and branched polymers also can be utilized to encapsulate a iaxane. At leas one end group of said linear and/or bmnched polymer is modified with a hydrophobic moiet or functional group. A hydrophobic moiety or functional group can include, hot is not limited to, hydrocarbon chains (e.g., containing 1 -22 carbons wit either saturated or non-saturated chemical bonds) and hydrophobic groups containing ara!kyL aromatic- rings, fluorocarbons etc,

0034} In another aspec t of the disclosure, the branched or linear polymer can comprise targeting moieti.es/groiips including, but not limited to, an antibody or antigen-binding portion thereof antigen, cognate carbohydrates (e.g., sialic acid), a eel! surface receptor ligand, a moiety bound by a cell surface receptor, such as, a prostate-specific membrane antigen (PSMA), a moiety that binds a cell surface saccharide, an extracellular matrix ligand, a cytosolic receptor ligand, a growth factor, a cytokine, an incretin, a hormone, a lectin, a lectin ligand, such as, a galactose, a galactose derivative, an N-acetySga!actosamine, a mannose, a mannose derivative and the like, a vitamin, such as, a folate or a biotin; avidin, streptavidin, neutravidra, DNA, RNA etc. Such targeted nanoparticles release drug at the preferred treatment locations, and therefore,, enhance local effective concentrations and c-an minimize undesired side effects.

[0035] In another aspect of the disclosure, a targeting moiety/group and a functional group, including, hydrophobic, hydrpphtlic and/or ionic functional groups, are attached to the branched polymer prior to the formation of the composite naooparticle for targeted drag delivery.

[0036] In another aspect of (he disclosure-, specific ranges of

monomer -initiator and polymer: taxane ratios result in drag nanoparticles of appropriate size to facilitate large scale manufacturing of the drug nanoparticles, sterilization of drag nanoparticles, and result in improved drag efficacy as compared to other monomer; . initiator and/or polymentaxane ratios.

[0037] Additional features and advantages of the present disclosure are described in, and will be apparent from, the following Detailed Description and the attached Figures. BRIEF DESCRIPTION OF THE FIGURES

[0038} The following description, of the figures and. the respective drawings are non-limiting examples that depict various embodiments that exemplify the present disclosure.

0039] Figure .1 depicts SBPs including a dendrimer. a star-shaped polymer, a dendrigraft and a comb-shaped polymer. Ail have a core, whether globular or linear,

[0040} Figure 2 depicts a chemical structure of symmetrically branched PPi dendriiners.

[0041] Figure 3 depicts chemical modification reactions of symmetrically branched PPI. dendri aers. The numbers, 8, 16, 32 and so on. indicate the number of reactive groups at the surface of the dendrimer.

10042] Figures 4A and 48 depict random (A) and regular (B) ABPs with asymmetric branch junctures and patterns.

[0043} Figure 5 depicts a chemical structure of a random asymmetrically branched PEI homopolymer.

[0044} Figures 6A and 6B depict synthetic schemes. Figure 6A presents chemical modification reactions of random asymmetrically branched ΡΕΪ

ho.mopoiy.mers. Figure 6B depicts a one-pot synthesis of hydrophobically modified, randomly branched. poly(2-ethyioxazoline) with a primary amino group at the focal point of the polymer. The initiator/surface group 0.) is the brominated hydrocarbon. The reaction opens the oxazoline .ring.

[ 0045} Figure ? illustrates a drug loaded in or at the surface domain, or region of the branched polymer (S.BP's and AB 's.h in the and other figures, R indicates a surface group and a solid circle depicts a drug of interest

[0046] Figure 8 illustrates one ty pe of composite-based nanopartieks containing both drug molecules and branched polymers,

[0047] Figure 9 illustrates an insoluble or poorly water soluble drug that is loaded at hydrophobic surface groups of branched poiyiners (SBP's and ABP's). In the and other .figures, a thin, wavy Sine depicts a hydrophobic surface group.

[ 0048] Figure 10 illustrates various drug-containing nanoparticles also carrying at least one targeting group or moiety, such as, an antibody, depicfed herein and in other figures as a, ^**Y." 0049} Figure 11 shows the size comparison of polymer-only and

•polymer-drag aggregates with the polymer concentration at 25 mg/mL and the drug concentration at 5 mg/mL in saline. The polymer is a hydropfaobically-modified, randomly-branched PEOX and die drug is paciitaxei,

0050] Figure .12 shows the size comparison of polymer-only and

polymer-drug aggregates with the polymer concentration at 2.5 mg/mL and the drug concentration at 0.5 mg/mL in saline. The polymer is a ydrophobically-modified, randomly-branched PEOX and the drug is paclHaxel.

[00 1] Figure 1.3 shows the size comparison of polymer-only and

polymer-drag aggregates with the polymer concentration at 2.50 g/mL and the drug concentration at 50 i saline. The polymer is a hydtophobically-modified, randomly-branched PEOX and the drug is paclitaxel.

10052 ] Figure 4 shows the size comparison of polymer-only and

polymer-drug aggregates with the polymer concentration at 25 tg/raL and the drug concentration at 5 mL in saline. The polymer is a hydrophobically-modified, randomly-branched PEOX and the drug is paclitaxel.

[0053] Figure 15 depicts normal ceil survival on exposure to three taxane formulations.

10054] Figure .16 depicts A.549 lung cancer cell cytotoxicity on exposure to three different taxane formulations.

[0055] Figure 17 depicts MDA-MB-231 triple negative breast cancer cytotoxicity on exposure to three different taxane form lations.

[0056] Figure 1.8 depicts OV-90 ovarian cancer cytotoxicity on exposure to three different taxane formulations.

[0057] Figure 1 depicts phannacokinetk (P .) profiles of three different taxane fo.nmilaiio.ns depicting plasma concentration, over time.

[ 0058] Figure 20 depicts 49 long cancer tumor volume in a mouse xenograft model with two control treatments and expostsi'e to three different taxane for ulations.

[ 0059] Figure 21 presents images of excised lung cancer cell tumors grown, as xenografts in a mouse and treatment of the mice with two controls and two forms of taxane. 0060} Figure 22 depicts impact of two negative controls and three formulations of taxane on ovarian cancer tumor size in a mouse xenograft model.

[0061] Figure 23 presents images of excised ovary cancer ceil tumors grown as xenografts in a moose and treatment of the mice with two controls and three forms of taxane.

DETAILED DESCRIPTION OF THE DISCLOSU E

[0062 } The drug solubility in the instant disclosure is defined as, relative to parts of solvent required to solubilke one part of drug, < 30 (soluble), 30- 1 0 (poorly soluble) and > 100 {insoluble).

100631 For the purposes of the instant disclosure, the wards, such as, "about," "substantially," and the like are defined as a range of values no greater than 1 % from the stated value or figure. "Horaopo!ymer," is as described hereinabove.

Drug of Interest

[ 0064} The drug of interest described is a taxane and comprises paclitaxel and other taxane derivatives, such as, doceiaxel. Paclitaxel is water-insoluble and has well-defined performance characteristics, such as, a low maximum tolerated dose (MTD), PK profile and limited efficacies in treating various types of cancer. The present disclosure covers the use of A^'BPs, as previously described, in improving those performance characteristics.

Nanocomposite, Naitoparticle or Nanoaggregate

[0065] A nanocomposite is a physical mixture of two or more materials or components (e.g., polymer and a taxane). in the instant disclosure, such a mixture could contain different nanoscopic phases or domains formed between a taxane and a branched homopolymer molecule in either solid or liquid state, Nanoeomposiies can include a combination of a bulk matrix (e.g., branched homopoiymers and a. taxane) and nanodiniensional phase(s), which may exhibit different properties due to dissimilarities of structure and chemistry (e.g., the domain formed by a taxane and the surface groups of branched 'polymer, as well as the domains formed, by the interior of the branched polymers). Since the solubility of the domains/phases may be different, on dissolving the nanocomposite in an aqueous solution, one of the phases may

I I dissolve faster than (he other or others, resulting in a gradual breakdown of the composite aggregate resulting in a graded and controlled release of the composite components and optionally, reformation of one or more of the components into a novel form, such as, a new aggregate. The terms, "nanocomposite," "nanopatli e " and "nanoaggregate," are equivalent and are used interchangeably herein .

[0066] The size of the aggregates described m the disclosure ranges from between about 10 to about 500 mn in diameter, from about 30 mn to about 300 nm in diameter. Aggregates may exhibit size-related properties that differ significantly from those observed, for micropariicles or bulk materials.

[0067] SBP's are depicted in Figure i, with symmetric branches, wherein, all the homopolymers of interest possess a cote and exhibit symmetric branch junctures consisting either of terminal or chain, branches throughout the homopolymer. The functional groups are present predominantly at the exterior.

[0068] The modified SBP's can be obtained, for example, through chemically linking functional groups on, for example, symmetrically branched PAMA or PPI dendri triers, commercially a vailable from Aklrich, polyether dendrimers, polyester de.adri.mers, comb-branched/star-branched polymers, such as, those containing PBO, PEG, P GX or PEOX, polystyrene, and comb-branched dendrigrafts, such as, those containing PEOX, PMOX or PEL

[0069] The synthetic procedures for making such SBPVdendrimers are known (see, for example, "Dendrimers and Other Dendritic Polymers," Frechet & Tomah^'a, eds., John Wile & Sons, Ltd., 2001 ) using commercially available reagents (for example, various generations of PPI dendrimers, Figure 2} or a number of SBP's are commercially available. The synthesis of comb-branched and combburst polymers is know (see, for example, U.S. Pat. Nos. 5,773,527; 5,631,329; and

5, 19,442).

[0070] The higher branching densities of SBP's render the polymers molecularly compact with a well-defined interior void space, which makes such molecules suitable as a carrier for a iaxane entrapped or encased, therein..

[0071] The surface modifications can enhance the properties and uses of the resulting modified SBP's. For example, with suitable modification, a water insoluble SBP can become water soluble, while an SBP with a high charge density can be modified to carry very low or no charge on the polymer or at the polymer surface. On the other hand, a water sol able SBP cm be modified with hydrophobic surface groups to enhance the ability to soiubilize water insoluble or pooriy water soluble drugs at the surface thereof. Modification can occur at any site of a polymer, for example, at a terminus, a branch, a backbone residue and so on,

0072] in one embodiment of the instant disclosure, the SBP (for example, either a symmetrically branched PE1 den.drirrj.er, a PP1 dendrimer, a PAMAM dendrimer or a symmetrically branched ΡΕΪ dendrigraft, for example) can be modified with different kinds of, for example, primary amine groups through, for example, Michael addition or an. addition of acrylic esters onto amine groups of the

homopoJymer. Thus, for example, through a Michael addition reaction, methyl aery late can be introduced onto the primary and or secondary amino groups of PHI, PPi and poSyl.ysi.ne (FIX) homopolymers. The ester groups then can be derivatized further, for example, by an amidation reaction. Thus, lor example, such an amidation reaction with, for example, ethy!enediam ne (EDA), can ield the addition of an amino group at the terminus of the newly formed branch. Other modifications to the homopoJymer can be made using known chemistries, for example, as provided in, "Poly(amines) and Poly(anunontum salts)," in, "Handbook of Polymer Synthesis," (Part A), Kricheldorf, ed.. New York, Marcel Dekker, 1 94; and, "Dendrimers and Other Dendritic Polymers " Frechet & Tomalia, eds., John Wiley & Sons, Lid., 200 J. Derivatives of ED A also can be used and include any molecular entity that comprises a reactive EDA, a substituted EDA or, for example, other members of the

polyethylene amine family, such as, diethylenetriaraine, triethyienetetrarnine, teiraethylenepentamlne, pen.taethyS.enehexamine, and so on including polyeth lene amine, tetranietliylethylenediamine and so on.

[0073] in embodiments, a modification can compose a moiety that contributes to or enhances hydrophobicity of a polymer or a portion, of a polymer. For example, hydrophobic functional groups, such as, aliphatic chains (e.g., hydrocarbon chains comprising 1 to about 22 carbons, whether saturated or unsaturated, linear, cyclic or branched), aromatic structures (e.g. containing one or more aromatic rings, which, may be fused) or combinations thereof, can be used as a modifying agent and added to a polymer as taught herein practicing chemistries as provided herein.

[0074] On such addition, a modified SBP. such as, a modified PEI, PPl, PAMAM dendrimer or PEl dendrigraft, is formed. As an extension of the SBP, such as PPI and PEI, (he resulting modified SBP also is symmetrically branched.

Depending on the solvent environment (i.e. pH or polarity), the surface functional groups can carry different charge and/or charge density, and/or hydrophobic groups. The molecular shape and surface functional group locations (i.e., surface functional group back folding) then can be tuned further, based on those characteristic properties.

100731 in another embodiment of the disclosure, the modified SBP's can be produced using any of a variety of synthetic schemes that, for example, are known to be amenable to reac tion with a suitable site on the homopolymer. Moreover, an of a variety of reagents can be used in a synthetic scheme of choice to y ield any of a variety of modifications or additions to the homopolymer backbone. Thus, for example, in the case of the Michael addition reaction to an amine described above, the addition of any of a variety of substituents can be used, for example, at the aikylation stage, using for example, any of a variety of acrylate reagents, such as, a acrylate comprising a hydrocarbon substiluent, such as saturated or unsaturated hydrocarbon comprising 1 to about 22 carbons, which may be substituted, aliphatic, aromatic, ringed, saturated at one or more bonds or a combination thereof. Thus, suitable reactants include, methyl aerylate, ethyl acrylate, propyl acrylate, butyl acrylate, peniyl acrylate, hexyl acrylate, heptyi acrylate, octyl acrylate, nony! acrylate, deeyl acrylate, undecyi acrylate, dodecyl acrylate and so on, and mixtures thereof.

Similarly, at the amidation stage in the example exemplified above, any of a variety of amines can be used. For example, EDA, monoetbano!arnine,

tris(hydroxymet^:hyl amjnometharie, alky! amine, ailyl amine or any ammo-modified polymer, including those comprising PEG, PEO, perfiaoropoiymers, polystyrene, polyethylene, poiydimeUiy!si!oxane, polyacrylate, polymethyirnethacrylate and the like, and mixtures thereof, can be used.

10076] Such a synthetic strategy would allow not only symmetric growth of the molecule, where more branches with different chemical compositions can be introduced, but also the addition of multiple functional groups at the exterior of the structure. The precursor homopolymer can be modified, and continuously, using the same or a different synthetic process until the desired SBPs with appropriate molecular weight and functional groups are attained. In addition, the hydrophobic and hydrophilic properties, as well as charge densities of such polymers, can be tailored to fit specific application needs using appropriate monomers for constructing the homopolymer and suitable modification reactions.

[0077] in another embodiment of the disclosure, if a divergent synthetic procedure is used, the chain end of symmetrically star-branched or comb-branched homopolymer, such as, a po!y(2~substituted oxazaline), including, for example, poiy(2-metliyloxazoliiie)_s poiy(2-ethyloxazoline), poly(2-propyloxazoline) and ^•poly(2-b tyloxazoline_t etc.), PE1, PEO/glycol, polyvinylpyrrolidone (PVP),

•polyphosphate, polyvinyl alcohol (PVA) or polystyrene, can be niodified with another smal l molecule or polymer to generate various functional groups at the

homopolymeric chain ends including a primary, secondary or tertiary amine, carbox late, hydroxy!, aliphatic (e.g., hydrocarbon chain), aromatic, flaoroalkyL aryl. PEG, PEO, acetate, amide and/or ester groups. Alternatively, various initiators also can be utilized so that the same type of functional groups can. be introduced at die chain end if a convergent synthetic approach is utilized ("Dendritic Molecules," e konie et al, eds., VCH, Weiriheiro, 1.996; "Dendrimers and Other Dendritic Polymers," Frechet & Toma!ia, eds., John Wiley & Sons, Ltd., 2001 ; and J.

Macromol. Set. Cheni. A9(5), pp. 703-727 (1975)).

[0078] The initiator can he a hydrophobic electrophiltc .molecule, .including hydrocarbons, aliphatic hydrocarbons, aromatic hydrocarbons or a combination of both, along with a halide functional group, such as, aikyl liatid.es, aralkyl hahdes, acy! halides or combinations thereof. Examples of such compounds are monoiiinetional. initiators such as hydrocarbons containing from 1 to about 22 hydrocarbons with either saturated, or unsaturated chemical bonds, such as, methyl

iodide/bromide/chloride, ethyl iodide/bromide/chloride, ί -iodo bromo/chloro butane, 1 -iodobromo/chloro hexane, .1 -iodo/brorao/chloro dodeeane, 1 -iodo bromo/chloro octadodecane, benzyl iodide/broniide/ch!oride and so on. Other initiators include ally! bromides chlorides. Acy^'l halides, such as, acy^'l bromide/chloride, benzoyl bromide/chloride and tosyl group-cotiiaining compounds, such as, p-toiuenesulfonk acid, methyl tosylate and other tosylate esters can also be used. Any one or more initiators can be used in combination.

[0079] During polymerization, an initiator can be used to start polymerization. When used, various molar ratios of monomer to initiator can be used to obtain particular polymers. The particular polymers cart have differing properties, such as, molecular size. Hence, suitable monomer to initiator molar ratios can be 20; I to 80; 1, such as, 25:1, 30; 1, 35; 1,40:1, 45:1, 50:1 , 55: 1 , 60:1 , 65: 1 , 70:1 or 75:1 including 21:1,22:1, 23:1, 24:1, 26:1, 27:1,28:1,29:1, 31:1, 32:1, 33:1, 34:1, 36:1, 37: L 38:1, 39:1 , 41 :L 42:1, 43:1,44:1 ,46:1 ,47:1, 48:1, 49:1, 5! ;.i, 52:1, 53:1,54:1 , 56:1,57:1, 58:1, 59:1, 61:1, 62:1, 63:1, 64:1, 66: 1,67:1, 68:1 ,69:1,71:1,72:1, 73: 1 , 74: 1 , 76: 1 , 77:1, 78:1, 79:1 and so on.

[0080) ABP's are depicted in Figures 4A and 4B with asymmetric branches, wherein some of the polymers of i nterest possess no core and exhibit asymmetrical branch junctures consisting of both chain and terminal branches throughout the entire homopolymer. The functional groups often are present both at the exterior and in the interior. However, when a larger functional group (e.g., a large hydrophobic or hydrophiJic group) is used, the functional groups often can be attached preferentiall and perhaps necessarily at the exterior of the ABP, for example, possibly due to steric effects. Therefore, such surface MBP's can be utilized for solubilization of or aggregate formation with an insoluble or poorly soluble drug,

[0081 ) The modified ABP's can he obtained, for example, through chemically linking functional groups on regular ABP's, such as, polylysine (e.g., branched PLL), on random ABP's, such as, PEFs (commercially available from Akirich,

PoJysciences, or BASF under the trade name, .Luposal™) or poiyoxazoliiies, which can be prepared according to the procedure of Li (j. MacromoL Sci. Chem. A9(5), pp.703-727 (1975)). Other ABP's can include, hut are not limited to,

polyacrylamides, polyphosphates, PVP's, PVA^'s etc.

[0082) A variety of known starting materials can be used. For making such modified ABP's. Such monomers and polymers are available commercially in large quantities at modest cost. For example, one such precursor monomer that can be used to synthesize a homopolymer of interest Is FBI. The synthesis of random

asymmetrically branched PEFs is known (Jones et ah, J. Org. Chem.9, 125 (1 44)). PEFs with various molecular weights are available commercially from different sources, such as, Akirich, Polyscienees and BASF (under the trade name Luposaf^'"). The random asymmetrically branched PE Fs are produced primarily through eatiome ring opening polymerization of ring-strained cyclic imine monomers, such as, aziridines (ethyl eneiniine) and axetidines (propylenei ine), with Lewis or Bronsted acids as initiators (Dernier et al., "Ethylenediamine and Other Aztridines," Academic Press, New York, ( 1969); and Pel.!, J. Chem. Sac, 7.1 (1959)). Since many of the methods are essentially one-pot processes, large quantities of random ABP's can be produced readily. Randomly branched poiy{2-substimted oxazoline) polymers can be prepared using the procedure of Li it (J. Macromol Sci. Chem, A (5). pp. 703-727 (1975)).

[0083] The synthetic processes for making ABP's often generate various branch junctures within the macromolecule. in other words, a mixture of terminal and chain branch junc tures is distributed throughout the molecular structure. The

branching densities of the random ABP's can be lower, and the molecular structure can be more open when compared with dendrimers and dendrigrafts. Although the branch pattern is random, the average ratio of primary, secondary and tertiary amine groups can be relatively consistent with a ratio of about 1 -.2: 1 , as described by Dick et at, I. Macromol. Sci. Chem., A4 (6), 1301 -1314 ( 1970) and Lukovkin, Eur. Polyrti. J. 9, 559 (1973).

[0084 j The presence of the branch junctures can make the random ABP^'s. such as, asymmetrically branched FBI's, form, macromolecules with a possible spherical ovoid or similar configuration.. Within the globular structure, there are various sizes of pockets formed from the imperfect branch junctures at the interior of the macromolecule. Unlike dendrimers and dendrigrafts where interior pockets are always located around the center core of the molecule, the pockets of random ABP's are spread unevenly throughout the entire molecule. As a result, random ABP's possess both exterior and unevenly distributed interior functional groups that can be reacted further with, a variety of molecules, thus forming new macromolecular architectures, a modified random ABP of interest.

[0085] Although having a core, the functional groups of the regular ABP are also distributed both at the exterior and in the interior, which is vers¹ similar to the random ABP. One such homopolymer is PLL, which can be made as described in U.S. Pat. Nos. 4,289,872; 4,360,646; and 4,410,688. Such homopoiyraers also can be modified in. a manner similar as that for random ABP's, as taught herein, and as known in the art.

[0086] In a embodiment of the disclosure, the ABP (for example, either a random asymmetrically branched PEI or a regular asymmetrically branched PLL) is modified with differen kinds of primary amine groups through, for example, Michael addition or an addition of acrylic esters onto amines of the polymer. Tims, for example, through a Michael addition reaction, methyl acrylate or other acrylates as provided herein can be introduced onto the primary and/or secondary amino groups of, for example, PHI and PLL hornopolymers. The ester groups then can be farther deriva&ed, for example, by an amidation reaction. Thus, for example, such an amidation reaction with, for example, EDA, can yield the addition of an amino group at the terminus of the newly formed branch. Other modifications to the polymer can be made using known chemistries, for example, as provided in. ^*'Poly(amines) and Po.ly(aiumonium salts)," in, "Handbook of Polymer Synthesis" f Part A), richeldorf, ed.. New York, Marcel ekker, 1 94.

[0087] On such addition, a modified ABP, such as, a modified ΡΕΪ or PLL homopolymer, is formed. As an extension of the ABP, such, as PE1 and PLL, die resulting modified ABP also is branched, asymmetric-ally. Depending on the solvent environment (i.e. pli or polarity), fee surface functional groups can carry different charge and charge density. The molecular shape and functional group locations (i.e., functional group back folding) then can be further tuned, based on those characteristic- properties.

[0088] In another embodiment, the modified ABP's can be- produced using any of a variety of synthetic schemes that, for example, are known to be amenable to reaction with a suitable site on the homopolymer. Moreover, any of a variety of reagents ca be used in a synthetic scheme of choice to yield any of a variety of modific tions or additions to the polymer backbone. Thus, for example, in the case of the Michael addition reaction to an amine described above, the addition of any of a variety of substituems can be used at the alk lation stage, as provided hereinabove, for example, with an acrylate, which can comprise a saturated or unsaturated hydrocarbon, such a one comprising one carbon to about 22 carbons, which may be aliphatic, branched, saturated, aromatic, ringed or combination thereof Suitable reactants include methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, peniyi acrylate, hexyl acrylate, hepiyl acrylate. octyl acrylate, nonyl acrylate, decy! acrylate, imdecyl acrylate, dodeey! acrylate and the like, and mixtures thereof. Similarly, at the amidation stage in the example exemplified above, any of a variety of amines can be used in the methods provided herein and known in the art. For example, EDA, monoethanolamine, tris(hydroxymethyl)aminomethane, alky! amine, allyl amine or any aiwiaomodified polymers, including PEG, perOuoropolyraers.. polystyrene, polyethylene, polydiraethylsil.ixaiie, polyacrylate, polyme&ylmethacrylate and the like, and mixtures thereof; can be used, in addition, the linking of the hydrophobic groups, including aliphatic (e.g., hydrocarbons from Cj to about (^';-) groups, aromatic groups, polyethylene polymers, polystyrene polymers, periluoropolytners, poiydirnethylsiloxanes, polyacrylates, polymethylmethacrylates, as well as, hydrophilic groups, including a OH group, hydrophilic polymers, such as, PEOX, PEG, PEG etc. to a modified ABP can be achieved by using, for example, epoxy reactions, ami dation reactions, Michael addi tion reactions, including using a -SH or an - Hj group reacted with maleimide, aldehyde/ketone-amine hydraxide coupling reactions, iodo/iodoacet l-SH coupling reactions, hydroxyiamine-aldehyde ketone coupling reactions etc. Such synthetic strategies allow not. only asymmetric growth of the molecule, where more pockets are introduced., but also the addition of multiple functional groups at both the .interior and the exterior of the structure. The homopoiymer can be modified further using the same or a different synthetic process until the desired ABP's with appropriate molecular weight and functional groups are attained, in addition, the hydrophobic and hydrophilic properties, as well as charge density of such hoTOopolyrae s, cars be tailored to fit specific application needs using appropriate monomers .for constructing the homopoiymer and suitable modification reactions.

[0089] In another embodiment of the disclosure, a focal point (merged from various reacti ve chain ends during a convergent synthesis) of a random ABP, such as, POX, can be terminated or reacted with, another small molecule to generate various functional groups at the homopolymeric chain ends, including primary, secondary or tertiary amines, carbox late, hydroxy!, alkyl, tlaoroalkyl, aryi, PEG, acetate, amide and/or ester groups. Alternatively, various initiators also can be utilized so that the same type of functional group can be introduced at the surface groups where a polymerization begins during a convergent synthesis (J. Macromof ScL Chem. A9 (5), pp. 703-727 (1975)).

[0090] An. alkyl surface-modified, randomly branched poly(2-eihy!oxazoline} with a primary amine group at the focal point of the branched polymer can be prepared using the Litt and Warakomski procedures, supra. For example,

CHj(C!¾)j?-Br can be utilized as an initiator for 2-ethyloxazoline polymerization through a calionic ring opening process to generate a randomly branched polymer, followed by quenching with N-iert-buiyloxycarbonylpiperazine (N~Boc-piperazine) or EDA. The teramiation with a large excess of EDA allows the hydrophobically modified branched poly(2-ed¾y!oxa oike) polymer to be fimctionalized with a primary amine group at the focal point (Figure 6B). Alternatively,

N-Boe-piperazine-termiriated hydrophobically-modified branched

poly(2-eihyioxazoline) polymer also can be deprotected to generate a free amino group at the focal point. If not terminated, the focal point of the polymer can be hydrolyzed to, for example, a hydroxy! group on dissolving in water (e.g., containing, for example, IN N¾CO; .

100 1 J Whi !e the introduction of a primary amine group to a

hydropbobically-modified branched poly l-oxazoline) homopo!ymer enhances drug solubility and produces taxane-induced aggregates, the primary amine group also allows the attachment of various targeting groups, such as, an antibody,

antigen-binding portion thereof, an antigen or a member of a binding pair, such as, to the hydrophohically modified branched poiy(2-oxazoline) polymer (Figure 10). Such aggregates or nanopartides containing such targeting groups and modifications thereto can provide a targeting ability on the aggregate with a taxane and enable taxane to be released preferentially or solely a the desired treatment location.

[ 0092] As taught herein, the MBP's, such as, a hydrophobically-modified homopolymers, including both SBP's and ABP's, can be used to generate an encapsulating polymer or nanocapsule for solubilking water insoluble or poorly water soluble taxanes, or for forming taxane-induced nanopartides with water insoluble or poorly water soluble taxanes, such as, paditaxe^'l. in an organic sol vent environment, the hydrophi!ic or amphiphiik interior can be poly(2-oxazoiine), poiy(2-substii:uted oxazolines), including poly(2-methy!oxazoli«e, poly(2-ethyIoxazohne),

poly(2-propy1oxazoline) and poly(2-butyloxa¾olirte) etc., PEG, PEO,

poiyphosphonafe and the like. The hydrophobic exterior can comprise aliphatic hydrocarbons (such as, from Cj to about C22), aromatic hydrocarbons, polyethylene polymers, polystyrene polymers, perfluoropolymers, polydimethyisiloxanes, po!yaerylates, polymethylmethacrylates and the like, in an aqueous environment, the reverse is true. In the drug-induced aggregates in an aqueous environment, the drug molecules such as taxanes are associated with the hydrophobic groups/domains of the MBP's (.Figure 9). The branching density (e.g., from low generation., such as, star and comb homopoiymers, to high generation of dendriraers and dendrigrafts), as well as the amount of hydrophobic surface group coverage (e.g., from 0% to 100% coverage) of the branched homopoiymers can affect significantly homopoiymer solubility, which in turn, also affects the ability to dissolve or to adsorb/absorb a iaxane. For example, the increase in branching density and the amount of hydrophobic group coverage will make the homopoiymer more compatible with a iaxane.

[0093 j In some cases, the ABP's and SBP's with from about 0.1 to about 30 % or more surface ^'hydrophobic component by weight are effective at solubilmng or dispersing poorly water soluble or water insoluble taxanes, such as, paclitaxel. In. addition, the branched homopoiymers utilized, for example, a POX, a PEOX, a

PMQ.X, PEO/PEG, polyacryiamides, polyphosphates, PVP's and PVA's are soluble in both water and in various organic solvents, thereby facilitating forming various taxane-contaming nanopartic!es or aggregates. The good water solubility along with good hydrophobic drug miscibility in an aqueous solution, with or without other organic solvents, makes such homopoiymers useful for enhancing the solubility of poorly water soluble taxanes. For example, the homopoiymers of interest simplify manufacturing processes and decrease production cost by reducing formulation steps, processing time, as well as the need to use complex and expensive equipment currently used in the pharmaceutical industry. If additional branching densities are needed, the SBP's or ABP's first can be modified with additional groups as described herein, and then, for example, attached with additional hydrophobic functional groups for enhancing taxane solubility.

[0094] On mixing hydrophobically-modified SBP's or ABP's with a water insoluble or poorly water soluble taxane, such as, paclitaxel,. a distinct physical aggregate is formed of size distinct from aggregates formed only of polymer

(Figures 1 1 -13), When the homopoiymer and iaxane concentrations decrease, the size and distribution of the polymer taxane aggregates become much more similar to that of polymer only aggregates suggesting iaxane is released from the induced aggregates or nanoparticles. The broad size distribution of polymer-only aggregates is similar to that observed for other structures composed of lipid, whether or not associated with a taxane. On the other hand, the taxane-induced aggregates of interest are of a

particular size of narrower distribution, tha is, unique aggregates of certain size are produced. As taxane concentration in the aggregate decreases, homopolymer concentration in the aggregate decreases, aggregate concentration decreases or any combination thereof, the aggregates of interest release paclitaxel, as evidenced by a. reduction of aggregate ske and/or a broader distribution of aggregate size. The broader distribution may result from a mixture ofhomopolymer-only aggregates and polymer/taxane aggregates of varying size due to taxane release, until the only aggregates observed are those which have the characteristics of those which are homopolymer only. In other words, taxane is released gradually after introduced into a host, such as, in the circulatory system. That mechanism is important for various drug delivery applications including, intravenous (TV), oral, transdermal, ocular, intramuscular and the like modes of administration, and where a delayed release or sustained release profile may be desirable.

10095] Suitable weight ratios of polymer to taxane are 6 to 8, such as, 6.5, 7 or 7,5, including 6.1 , 6.2, 6.3, 6.4, 6.6, 6.7, 6.8, 6.9, 7.1 , 7.2, 7.3, 7.4, 7.6, 7.7, 7.8, 7.9 and so on.

[0096 j The combination of the molar ratio of monomer to i nitiator in the polymerization and the we ight ratio of polymer to taxane in the nanoparticles determines large scale nranufacturability of the drug nanoparticles, nanoparticle size, and efficacy as a tumor-reducing treatment. As an example, taxane-induced aggregates prepared with a polymer: taxane weight ratio of 5:1, using a polymer synthesized with 100:1 monomer: initiator molar ratio results in larger nanoparticles, for example, in the 120-140 nm range before lyophilization. Such large nanoparticles are difficult to pass through a 0.2 pm filter (a required sterilization step for injectables) when manufactured in large quantities.

[0097] in comparison, when a polymer synthesized using a monome mtiator molar ratio of 60: 1 was mixed with taxane with, a poiyraer.iaxane weight ratio of 7: 1 , the nanoparticles formed were 70-90 am in size before lyophilixaiioa, which allows the particles to pass through a 0.2 pm filter with little difficulty.

[0098] Smaller nanoparticles at about 100 ura or less in size before lyophilization reduced tumors at Sower dose concentrations than did larger particles. For example, smaller nanoparticles achieve the same cancer treatment efficacy with only 1/5 of the taxane content when compared to larger nanoparticles. Thus, lower doses of drug can be used and the risk of side effects is minimized. 0099} The taxane-induced aggregates also can be linked with a targeting moiety or group including, but not limited to, an antibody (or antigen-binding portion thereof), antigen, cognate carbohydrates (e.g., sialic acid), a ceil surface receptor ligand, a moiety that binds a cell surface receptor, such as. prostate-specific

membrane antigen (PSMA), a moiety that, binds a cell surface saccharide, an extracellular matrix ligand, a cyiosoiic receptor ligand, a growth factor, a cytokine, an meretin, a hormone, a lectio, a lectin target, such as, a galactose, a galactose derivative, an N-acetylga!actosamioe, a mannose, a mannose derivative and the like, a vitamin, such as, a folate, a bioiin. and the like, an avidin, a streplavidin, a neutravidin, a D A, an RNA etc. to form a conjugate so that the targeting groupfs) are

incorporated with, nanocomposite particle of interest (Figure .10).

Drug Formulation and anopartick' Preparation

100100 } Taxane and modi fied homopolymer can be suspended

individually in suitable buffers and/or solvents, such as, a buffer, methanol, acetone, ethanol and the like, at suitable concentrations, such as those which are established for in vivo use, generally in milligram or nanogram quantities. Then, the two solutions are mixed at a suitable temperature, such as, room temperature or at another temperature known, to be acceptable for maintaining integrity of the taxane and homo-polymer, for a suitabl e period of time, such as, one hour, two hows and so on. Other incubation toes can vary from minutes to hours as the aggregates of interest are stable once formed. The aggregates can be concentrated or collected practicing methods known, in the art, for example, fay filtration, centriJftigatton, evaporation, lyophi!izaticm, dialysis and the like. The aggregates caa be desiccated for extended_, shelf life.

[001.01 J For example, a taxane, such as, paclftaxel, was dissolved in methanol or ethanol in various amounts of up to 40 mg mL. A hydrocarbon

(CH3(CHj)r?)-modified randomly branched ΡΒΟΧ6Θ (monomer to initiator ratio ~ 60: i ) was prepared as taught herein and dissolved at varying concentrations of op 1 0 mg/mL in methanol or ethanol.

[00102] The two solutions then were mixed in various volumes to result in final homopolymer to taxane weight ratios in the mixtures ranging from 2; 1 to 10: 1 and rotary evaporated to dryness. The mixtures then were redissolved in water or saline, followed by sterile filtration by a 0.2 μΜ filter and iyophiiixaiion for 20 to 72 hours depending on volatile to yield a dry powder.

[00103] The size of the aggregates or nanoparttc!es, as measured by light scattering, can range from about 50 to about 100 am, from about 60 to about 95 tin from about 70 to about 90 am (e.g., at 3 mg paclitaxel per uiL) before lyophilization to about 1.10 to about 150 ran, from about 115 to about 145 urn, from about 120 to about 1 0 nm (e.g., at 5 m paclitaxel per ml,) m diameter after l ophilization.

[00104] A pharmaceutical composition of die disclosure for use as disclosed herein is formulated to be compatible with, the intended route of

administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal and recta! administration.. Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include a sterile diluent, such as, water for injection, saline, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as, benzyl alcohol or methyl parabens; antioxidants, such as, ascorbic acid or sodium bisulfite; chelating agents, such as, EDTA; buffers, such as, acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as, sodium chloride or dextrose. pH can be adjusted with acids or bases, such as HQ or NaOH. The parenteral preparation can. be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic as an article of manufacture. Generally, an in vivo diagnostic agent will be administered orally, recialiy, intravenously, intrapemoneally and so on.

[00105] ^'Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous

administration, suitable carriers include physiological saline, bacteriostatic water or phosphate-buffered saline (PBS). The composition generally is sterile and is fluid to the extent that syrmgability exists. ^'The composition must be stable under the conditions of manufacture and storage and must be preserved, against the

contaminating aciion of microorganisms, such as, bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyoi (for example, glycerol, propylene glycol, liquid PEG, polysorbat.es and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such, as lecithin, by the maintenance of the required particle size in the case of dispersion, use of a thickener and by the use of surf actants. Prevention of the action of microorganisms can be achieved by various antibac terial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like, isotonic agents, for example, sugars, polyalcohols. such as, manmtol, sorbitol or sodium chloride, can be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, amminnm rnonosteatate or gelatin.

[00106] Steri le injectable solutions can be prepared by incorporating the active compound in the required amount of an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active

compound in a sterile vehicle that contains a basic dispersion medium and the required other ingredients, for example,, from those enumerated above, and as known in the art. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation can he prepared by, for example, lyophilizaiion, vacuum drying or freeze drying, thai yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of interest ca be stored and reconstituted with a suitable liquid for use.

[0010? ] Oral compositions generally include a inert diluent, flavorant, odorant or an edible carrier. The composition can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions also can be prepared using a fluid carrier to yield a syrup or liquid formulation, or for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

100108 ] Pharmaceutically compatible binding agents and/or adjuvant materials ca be included as part of the composi tion. Tablets, pills, capsules, troches and the like can contain a binder, such as, macrocrystalline cellulose, gum tragacanth or gelatin; an excipient, such as, starch or lactose, a disintegrating agent, such as, alginic acid, Primogel or corn starch; a lubricant, such as, magnesium stearate or Sterotes; a. glidant, such as, colloidal silicon dioxide; a sweetening agent, such as, sucrose or saccharin; or a flavoring agent, such as, peppermint, methyl salicylate or orange flavoring.

[0 109] For administration by inhalation., the compound is delivered in the form of, for example, a wet or dry aerosol spray from a pressurized container or dispenser that contains a. suitable propellent, e.g., a gas, such as, carbon dioxide or a nebulizer, or a mist.

[001 10 j Systemic administration also can be by transmucosai or transdermal means. For transmucosai or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants generally are known in the art and include, for example, for transmucosai administration, detergents, bile salts and fusidic acid derivatives. Transmucosai administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as generally known in the art. A suitable carrier includes diraethyisulfoxide.

[001 1 1 j The compound also can be prepared in the form of suppositories (e.g., with conventional suppository bases, such as, cocoa butter and other glycerides) or retention enemas for rectal delivery.

[DO 1 12] In one embodiment, the acti e compound is prepared with carriers that will protect the compound against rapid elimination from the body, such, as, a controlled release .formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycoiic acid, collagen, polyorthoe-sters and polylactic acid.

[00113] Methods for preparation of such formulations will be- apparent to those skilled in the art. The materials also can be obtained commercially, for example, from A .a Corporation and Nova Pharmaceuticals, Inc.

[001 14] The instant aggregates can be used in topical forms, suc as, creams, ointments, lotions, unguents, other cosmetics and the like. Pharmaceutically active agents (PAAs), such as, the taxanes of interest and other bioacltve or inert compounds, can be carried, and include emollients, bleaching agents, antiperspirants, pharmaceuticals, moisturizers, scents, colorants, pigments, dyes, antioxidants, oils, fatty acids, lipids, inorganic salts, organic molecules, opacifiers, vitamins. pharmaceuticals, keratol tk agents, UV blocking agents., tanning accelerators, depigmenting agents, deodorants, perfumes, insect repellants and die like.

[0 1 15] it can be ad vantageous to formulate oral or parenteral

compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitar dosages for a subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic eadpoint.

[001 1 j The dosages, for example, preferred route of administration and amounts are obtainable based o empirical data obtained from preclinical and clinical studies, practicing methods known in the art. The dosage and deliver}' form can be dictated by and ca be dependent on the characteristics of the PAA, the polymer, the partic ular therapeutic effect to be achieved, the characteristics and condition of the recipient and so on. For repeated administrations over several clays or longer, depending on the condition, the treatment can be sustained until a desired eadpoint is attained. An exemplary dosing regimen is disclosed i WO 94/041 8.

[001 17 j The progress of the therapy can be monitored by conventional techniques and assays, as well as patient input.

[001 18] The ph rmaceutical compositions can be included in a container, pack or dispenser together with instructions for administration.

[001 19} Another method of administration comprises the addition of a compound of interest into or with a food or drink, as a food supplement or additive, or as a dosage form taken on a prophylactic basis, similar to a vitamin. The aggregate of interest can be encapsulated into forms that will survive passage through the gastric environment. Such forms are commonly known, for example, enteric coated

formulations. Alternatively, the aggregate of interest can be modified to enhance half-life, such as, chemical modification, or combination with agents known to result in delayed, sustained or controlled release, as known in the art,

[00120) The instant disclosure now w ill be exemplified in the following non-limiting examples. EXAMPLES

Materials

[ 0012 ! J Symmetrically branched PPI deadriraers were purchased from Sigma- Aldrich. Symmetrically branched PEI dendrimers and dendri grafts were prepared according to procedures provided in U.S. Pat, Nos. 4,631,337, 5,773,527, 5,63.1,329 and 5, 19,442. All of the antibodies were purchased from Sigma-Aldrich, Biodesign or Fitzgerald. Different generation PAMAM dendrimers were purchased from Dendritech, Inc.

Modified Symmetrically Branched PPls with Amino Functional Groups (m-SB- PPi- H₂-U>)

[DDI 22 J The following reagents including sy.mmetTical.ly branched PPl (SB-PPM, 8, 16, 32, 64, W 316, 773, 1,687, 3,514 and 7,168), methyl acrylate (MA, FW ==== 86.09), EDA (FW == 60.10) and methanol were utilized.

[00123 j To a round bottom flask were added 1 .0 g PP1-64 dendrimer (MW 7168) and 20 ml methanol (solution A). To a separate round bottom flask were added 2.4 g methylacryiate (MA) and 10 mi methanol (solution B). Solution A was then slowly dropped into solution B while stirring at room temperature. The .resulting solution was allowed to react at 40° C for 2 hours. On completion of the reaction, the solvent and unreacted MA monomer were removed by rotary evaporation and the product, 2.5 g of MA-functionalized PPi, then was redissolved in 20 ml of methanol.

[00124 j To a round bottom flask were added 1 0 g EDA and 50 ml of methanol, followed by a slo addition of MA-functionaiized PPI at 0° C. The solution then was allowed to react at 4°€ for 48 hours. The solvent and the excess EDA were removed by rotary evaporation. The crude product then was precipitated from an ethyl ether solution and further purified by dialysis to give about 2.8 g of primary amine- ranciiotialized symmetrically branched PPI (m-SB-PPI~NH?- 1.0) with a molecular weight of about 21,760. The product was characterized by Ή and ^JiC nuclear magnetic resonance ( M.R) and size exclusion chromatography (SEC).

[00125] Other MA or primary amine-modified symmetrically branched PPI dendrimers and symmetrically branched PEi deadrigrafts with various molecular weights were prepared in a similar maimer. Modified Symmetrically Branched PPl^'s with Mixed Hydroxy! and Amino

Function al G roups (mix-m-SB-PPI-6 - H2/OH-2)

[0 1 6] Amino-functionaiized symmetrically branched PPI (m-SB-PPI- 64-NHj-l -0), MA, EDA, monoemanolamme (MEA, FW ^::: 61.08) and methanol were utilized.

[00127] To a round bottom ilask were added 1 .0 g amino-modified. PPI or m-SB-PPI-NHr! - produced from the previous procedure and 20 ml of methanol (solution A). To a separate round bottom flask were added 2.4 g of MA and 10 ml methanol (solution 8). Solution A then was dripped slowly into solution B while stirring at room temperature. The resulting solution was allowed to react at 40 C .for 2 hours. On completion of the reaction, the solvent arid unreacted monomer MA were removed by rotary evaporation and. the product, 2.5 g of MA-foncfionalized m-SB- PPi^«64-MA~1.5, then was redissolved in 20 ml of methanol.

[00128] To a round bottom Ilask were added 32 g EDA, 130 g MEA and 1 0 ml methanol (the mole ratio of EDA; MEA was 20:80), followed by slow addition of m-SB-PPJ-64-MA-l .5 at 0° C. The solution then was allowed to reac at ° C for 48 hours. The solvent and the excess EDA were removed by rotary evaporation. The crude product: then was precipitated from an ethyl ether solution and further purified by dialysis to give about 2.8 g of mixed hydroxy! and amino fuuctionaiized (mixed surface) SBP (mix-m-SB-PPi-64^«NH2/OH-2.0, with an average of 20 % NH.2 and 80 % OH surface groups and a molecular weight of about 21,862).

[00129 j Other modified random AB-PEI and regular AB-PLL molecules with varying ratios ofhydroxyl and amino groups, as well as different molecular weights, were prepared in a similar manner.

[00130] Random asymmetrically branched PEI's were purchased .from Akiricii and Polysciences. Regular ABP's were prepared according to procedures provided in U.S. Pat. No. 4,289,872. All of the antibodies were purchased from Sigma-Aldrich, Biodesign or Fitzgerald.

Modified Random Asymmetrically Branched PEIs with Amino Functional

Groups (m-ran-AB-PEI-NIIrl.O)

[00131 ] Random asymmetrically branched PEI (rati- AB-PEI,

MW 2,000, 25,000 and 75,000), MA, EDA and methanol were utilized. [001321 To a round bottom flask were added 1 .0 g PEI (MW 2,000) and 20 ml methanol (solution. A). To a separate round bottom flask were added 3.0 g MA and 1 ml methanol (solution B). Solution. A then was dripped slowly into solution B while stirring at room temperature. The resulting solution was allowed to react at 40* C for 2 hours. On completion of the reaction, the solvent and unreacied MA were removed by rotary evaporation and the product, MA-functionaiize PEL then was redissolve in 20 ml of methanol,

[00133 j To a round bottom flask were added 80 g EDA and 50 ml of methanol, followed by a slow addition of MA-funciionalteed PEI at (V C (1 g MA dissolved in 20 nil methanol). The solution then was allowed to react at 4° C for 48 hours. The solvent and excess EDA were removed by rotary evaporation. The crude product then was precipitated from an ethyl ether solution and further purified by dialysis to give about 3.0 g of primary amme-functionafced random

asymmetrically branched PEI {m-ran-AB-PEI-NHj-L ) with a molecular weight of about 7,300. The product was characterized by ^!H and ⁵"€ N.M and SEC.

[0 134} Other MA or primar amine modified random asymmetrically branched PEI and regular asymmetrically branched PLL polymers with various molecular weights were prepared in a similar manner.

Modification of Branched Polymers with Hydrocarbon Chains

[00135 } The modification of a randomly branched PEI with 10% hydrocarbon chains is used as an example. One gram of branched PEI (FW ^:::: 25000) was dissolved in 10 mL methanol. To the solution were added 0.23 g of

1 ,2-epoxyhexane (FW =100, 16) and the mixture was heated at 40" C for 2 hours. The solvent then, was rotary evaporated and the residue redissolved in water. After dialysis (3,500 cutoff), the modified PEI was generated.

j 00136] Other MBP 's, such as, PAMAM, PEI and PP1 dendrimers and deiidri grafts, and asymmetric PLL with various percentages and lengths (e.g., C , C_S; , Cig and C22) of hydrocarbon chains were prepared in a similar manner. Modified Random Asymmetrically Branched PEis with Mixed Hydroxy! and Amino Functional Groups ( m- r a n- B- P I: I - N H j/O H -2 )

[0 137] Aini O-fuBctionalized random symmetrically branched ΡΕΪ (ro-raii-AB-PEl-Ni¾- Ϊ .0), MA, EDA, monoethanolamine (MEA, FW === 61 .08) and methanol were utilized.

[00138] To a round bottom flask were added 1 .0 g ammo-modified PES or m-ran-AB-PEI-NHj- ί ,0 produced from the previous procedure and 20 ml of methanol (solution A). To a separate round bottom flask were added 3.0 g of MA and 10 ml methanol (solution B). Solution A then was slowly dripped into solution B while stirring at room temperature. The resulting solution was allowed to react, at 40^s C for 2 hours. On completion of ihe reaction, the solvent and unreacted A were removed by rotary evaporation and the product, MA-functionalized m-ran-AB~PEI- MA-1.5, then was redissolved in 20 nil of methanol.

[00139] To a round bottom flask were added 60 g EDA, 244 g MEA and 1 0 ml methanol (the mole raiio of EDA; MEA was 20:80), followed by slow addition of m-ran-AB-PEJ-MA- i .5 at 0° C (1 g MA dissolved in 20 ml of methanol). The solution then was allowed to react at 4° C for 48 hours. The solvent and excess EDA were removed by rotary evaporation. The crude product then was precipitated from an ethyl ether solution and further purified by ^'dialysis to give about 2.4 g of mixed hydroxy! and amino functionaliml random ABP (m-ran-AB~PEi-NH₂ OHr2.0, with an average of 20% N¾ and 80% OH surface groups and the molecular weight was about 1 ,000).

[00140] Other modified random AB-PBl and regular AB-FLL polymers with various ratios of hydroxy 1 and amino groups, as well as different molecular weights were prepared io a similar manner.

Alkj i- odified Random Asymmetrically Branched Po y(2-etnyloxa oIitte) (PEOX) with Primary Amine Chain End Group

[00141 ] The synthesis of CHHCH₂)n~PEOX-ABP.tOO (C_i2.ABP100 is an arbitrary name to denote the ratio of monomer to initiator in the initial reaciion) is provided as a general procedure for the preparation of core shell structures. A mixture of€¾(€¾) i pBr (2.52 g) in 500 ml of toluene was azeoiroped to remove water with a distillation head under N_> for about 15 mm. 2~Ethyloxazoline (100 g) was added dropw.ise through an addition, funnel and the mixture was allowed to reflux between 24 and 48 hours. On completion of the polymerization, 12.12 g of EDA were added to the reactive polymer solution (A) to introduce the amine function group. The molar ratio of POX chain end to EDA was I to 20,

[00142] Alternatively, N-Boc-piperazine or water (e.g., with IN ajCOj) can be added to terminate the reaction. Motpholine or PE1^" also can be added to the reactive polymer solution (A) to terminate the reaction. The crude product was redisso^'lved in methanol and then precipitated from a large excess of diethyl ether. The bottom, layer was redissolved in methanol and dried by rotary evaporation and vacuum to give an asymmetrically random branched PEOX polymer as a white solid ( 101 g).

100143] Other asymmetrically randomly branched polymers, such as, CVPEOX ABP20, 50, 100, 200, 300 and 500, C_s PEOX ABP20, 50, 200, 300 and 500, C¾-PEOX ABP20, 50, 100, 200, 300 and 500, and polysiyrene-PEOX etc., as well as, non-modified and modified. poly(2~subsiituted oxazoline), such as,

poly(2~meihy.oxazo1ine), were prepared in a similar manner. All the products were analyzed by SEC and R,

Alkyl- odified Random Asymmetrically Branched Poly(2-ethyloxaz0line) (PEOX) with Primary Amine Chain End Group

[0014 1 The synthesis of CH {CH₂),-PEOX-ABP60 (C_{! 8}ABP60 is an arbi rary name to denote the ratio of monomer to initiator in the initial reaction) is provided as a general procedure for the preparation of core shell structures. A mixture of <¾(<¾)ΙΤ-ΒΓ (5, 1 g) in 500 ml of toluene was azeotroped to remove water with a distillation head under N, for about 15 min. 2-EthyloxaxoIine (100 g) was added dropwsse through an addition funnel and the mix ure was allowed, to reflux between 2.4 and 48 hours. On completion of the polymerization, 1 .1 g of EDA were added to the reactive polymer solution (A) to introduce the amine function group. The molar ratio of polyoxazoline reactive chain end to EDA was 1 to 1.0.

[00145] Alternatively, N-Boc-piperazine or water (e.g., with IN

Na₂CO; can be added to terminate the reaction. Morphoiine or PEl also can be added to the reactive polymer solution (A) to terminate the reaction. The crude product was redissolved in methanol and. then precipitated from a large excess of diethyl ether. The bottom layer was redissolved in methanol and dried by rotary evaporation and vacuum to give an asymmetrically random braiiched PEOX pol mer as a white solid.

[00146] Other asymmetrically randomly branched polymers, such as, C K-PEOX ABP20.40, 50, 70, 80, 100, 120, 200, 300, 500 etc. as well as,

«οο-modified and modified po!y(2«$ubstitiued oxa oiine), such as,

poly(2~methyloxa2olme), were prepared in a similar manner. All the products were analyzed by SEC and NMR,

Mixed Surface Modified Symmetrical raii hed Polymer-l^'gG Conjugates

I ^'00147] The preparation of mixed, surface (OH/NHs. mix) modified, symmetrically branched ΡΡΪ-lgG conjugates (mix-m-SB-PPl-644 B₂/OH-24gG conjugates) is provided as a general procedure for the preparation of polymer antibody.

[00148] Other conjugates, such as, ra-SB-PPi-4-NH₂-l -IgG, ra-SB-PPI- 8-NHr i -igG, m-SB-PPl-16-NHrl-lgG, m-SB-PPf-32-NHri-!gG, m-SB-PPI- - NH 24gG, m-SB-PPi~8~NH2-2~iga m-SB-PPM^NH₂-2-l§G₅ m-SB-PPl-32-MH₂~ 2-IgG, m-SB~PPl-4- H 3 gG, m-SB-PPI-8- H₂~3~IgG, m4>B4>FI 6~NH 34gG, m-SB-PPI-32- H₂-3-IgG, mix-m-SB-PPi-4- H₂/OH-l (OH/N¾ mixHgG, mix-m-SB.PPl-8-Nli₂/01i-i (OH/NHfe mixHgG, raix-ra~SB~PPI«l 6-NH₂/OH-l (OH/NH₂ mixHgG, mix-m-SB~PFl-32- H₂/OH-l (O.H/ H, mixHgG,

niix-ffi-SB4^>Pi-4- ¾/QH-2 (QH Ffe mixHgG, msx-m-SB.PPl-8-NH₂/OH-2 (OH/NH2 mix)4gG, mix-ni-SB-PPI~16-NH₂ OH-2 (OH/NH₂ mixHgG,

mix-nr-SB-PPi-32-NH₂/0«~2 (OH/NIT mixHgG, mix-in-SB-FPi-4-NK₂/OH-3 COH H₃ mixHgG, mix-m^«SB^«PPl-8-NH₂/OH~3 (OH/NH₂ mix)4gG,

niix-m-SB-PPi-16-NH₂/OH-3 (OH/NH₂ mixHgG, mix-m-SB-PPi-32-Ne₂/Oe-3 (O.H NH₂ mixHgG, as well as primary amine and mix OH NH₂ modified combbursi PEi dendrigrafts (Generation 0-5) also were obtained in a similar maimer. The synthesis of other targeting moieties attached to a modified SBP of interest also was obtained in a similar manner. LC-SFDF-Mixed Surface ra-SB-PPI^- H^OH-2

[0 149 j To the mixed surface randomly branched mix-m~SB~PPl-64- N¾ OH-2 (4 x iO^'7 mot) in 400 μΐ of phosphate buffer (20 mM phosphate and 0.1 M NaCf pB 7.5) were added 4.0 x 10^"6 rnol of sulfo-LC-SPDP (Pierce, IL) in 400 μΐ of water. The mixture was vortexed and incubated at 30° C for 30 minutes. The i..C-SPDP-mix-m-SB-PPl-64-NH₂/OH-2 was purified by gel filtration

chromatography and equilibrated with buffer A (0. i M phosphate, 0.1 aCl and 5 mM E^'DTA, pH 6.8). The product was concentrated farther to yield 465 μ£ of solution with a concentration of approximately 0.77 nmoi.

Thiolated mix-iJi-SB-PPI-64-NH₂/OH-2

[00150 j The LC-SPDP mix-ro-SB-PPi-64-NB₂/OB-2 (50 nmoi in 65 μϊ of buffer A) was mixed with 100 μΙ_ of ditluothreitol (DTT) (50 mM in buffer A) and was incubated at room temperature for 15 minutes. Excess DTT and byproducts were removed by gel filtration with buffer A. The produc was concentrated in a 10 Centricon Concentrator to yield 390 μί,. of the thiolated niix~m-SB-PPI~64-N.H2/OH-2 that was used for conjugation with activated antibody.

Maieimide R (MAL-R)-Ac ivated Antibody

j 001511 To the antibody in PBS (31 μΤ, 5.1 nig or 3 nmoi ) were added 20.4 Τ of a MAL-R-NHS ( -hydroxysoccinimide) solution ( 10 mM in water). The mixture was vortexed and incubated at 30* C for 15 minutes. T he product was purified by gel filtration with buffer A. The maieimide-R -activated antibody was used for conjugation with the thiolated mix-m-SB-PPI-64-NB? OB-2. niix-i«-SB-PPI-64- H₂/OH-2-Antibody Conjugate

[001 2 ] To the thiolated mix-m-SB-PPI-64^«NB₃/OH-2 (310 μΐ or 35.7 nmoi) was added the MAL-R>activated antibody (4.8 ml, or 34 nmoi). The reaction mixture was concentrated to approximately 800 Τ and then allowed to incubate overnight at 4° C and/or at room temperature for about 1 nr. On completion, the reaction was quenched with 100 pL of ethy l maieimide (50 mmolar solution) and the conjugate then was fractionated on a carboxymethyl cellulose (CM) column (5 ml) with a sodi um chloride step gradient in 20 mM phosphate buffer at pH 6. The conjugate was el u ted with a sodium chloride gradient and characterised by eatioroc exchange chromatography, UV spectroscopy and polyacryiamide gel electrophoresis.

Con} li ation via Redncth e Coupling-Redaction of Antibody

[00133] To the antibody, 2.1 nig or 14 nmol iii 160 pL of buffer B

(containing 0.1 M sodium phosphate, 5 mM EDTA and 0.1 M NaCi, pH 6,0) were added 40 pL of DTT (50 mM in buffer B). The solution was allowed to stand at room temperature for 30 tain. The product was purified by gel filtration in a

Sephadex G-25 column equilibrated with buffer B, The reduced antibody was concentrated to 220 pL and was used for conjugation.

MAL-R-Mixed Surface Modified SBP

[00154] To the mixed surface modified SBP in 400 ill (400x1 i mols) at pH 7.4 were added 400 pL of AL-R-NHS ( 10 mM water). Thai was mixed and incubated at 30° C for i 5 min. On termination, the product was purified on a Sephadex G-25 column equilibrated with buffer B. The MAL- -mixed surface modified SBP was collected and stored to aliquois in the same buffer at -40° C.

Mixed Surface Modified SBP-A»tibody Conjugate

[00155 ] To the reduced antibody ( 14 amois in 220 uL) was added ihe MAL~R~mix~m-SB-PPI~64~NH₂/OIi-2 (1 54 pL, 16.6 umols) with siimug. The pH was adjusted to about 6.8 by the addition of 12.5 μΐ, of sodium carbonate (1.0 M solution), die reaction was continued for 1 hr at room temperature and terminated with the addition of 100 pL of cystearnine (0.4 mM solution). The conjugation mixture was purified on a CM cellulose column with, a sodium chloride gradient elation. igG-Asymrnetrieal Randomly Branched Polymer Conjugates

[001561 The preparation of randomly branched mixed surface {OH N.H₂ mix) m-ran-AB-PEl- HVOH-2-igG conjugates is provided as a general procedure for the preparation of polymer-antibody conjugates.

[DDI 57 J Other conjugates such as PEI-IgG, ra-fan-AB-PEl-Nl¾- 1-IgG, m-mn-AB-PEi- H₂-2 gG, m-rau-AB-PEi-NH₂-3 gG_!, m-ran-AB-PEI-NH2- -igG₅ as well as m~mn-AB-PE!.-N%/OH~l (OH/NH₂ rnixHgG, m-ran-AB-PEI-NH₂/OH-2 (O.H/NH₂ mixHgG, m-ran-AB-PFJ-NHa/OH-3 (OR/NH₃ mix)~lgG_:, regular polylysine polymer, alfcyl-modified randomly branched poly{2-ethylox.a?:ols:ne) with primary amine chain ends were all synthesized in a similar manner. The svtiihesis of various protein conjugates with asymmetrically randomly branched PEOX polymers also is conducted in a similar manner,

LC-SPDP-Mixed Surface m-raB-AB-PEi- H₂/OH-2

[001 8] To the mixed surface randomly branched m-ran~AB-P^'Ei~ H₂/OH~2 (4 x Κ mol) in 400 μΐ, of phosphate buffer (20 raM phosphate audi). I M NaCI, pH 7.5) were added 4.0 x 10* mol of sulfo-LC-SPDP (Pierce, IL) in 400 μ! of water. That was vortexed and incubated at 30° C for 30 minutes. The LC-SPDP-nv- ran-AB-PEl- H;/C)H-2 was purified by gel filtration chromatography and equilibrated with buffer A (0, i phosphate, 0.1 M NaCI and. 5 raM EDTA, pH 6.8), The product was concentrated, further to yield 465 μΐ of solution with a concentration of approximately 0.77 nmol/umol.

Thiolated m-ran-AB-PEl-NH₂/Oe-2

[00159] The LC-SPDP m-ran-AB-PEI-NH₂/OH-2 (50 nmol in 65 ml of buffer A) was mixed with 100 uL of dithioihreitol (D^'TT) (50 mM in buffer A) and was allowed to incubate at room temperature for 15 minutes- Excess DTT and byproducts were removed by gel filtration with buffer A. The product was concentrated in a. 10 K Centricon Concentrator to yield 390 μΐ, of the thiolated m~ran-AB-PEl- H₂/^'OH>2 that was used for conjugation with activated antibody.

[00160] Ma!eimide-R-activated antibody made as described above was ^•used for conjugation with the thiolated m-ran-AB-PEf-NHa/OH-2.

«i-ran-A^'B-PEI- H2 OH-2-Aati ody Conjugate

[00161 ] To the thiolated m-ran-AB~PEi~NH_a/OH-2 (310 Τ or 35.7 nmol) was added the MAL-R-activated. antibody (4.8 mL or 34 nmol). The reaction mixture was concentrated to approximately 800 μΕ and aliowed to incubate overnight at 4* C and/or at room temperature for about 1 hr. On. completion, the reaction was quenched with 100 pL of ethyl mafeimide (50 mmolar solution) and the conjugate then was fractionated on a CM cellulose column (5 ml) with a sodium chloride step gradient in 20 tnM phosphate buffer at pH 6. The conjugate was eluted with a sodium chloride gradient and characterized by cationic exchange

chromatography, UV spectroscopy and polyacrylamide gel electrophoresis.

Pa itaxel Formulation and anoparticle Preparation

[00162 j As & general procedure, paciitaxel was dissolved in methanol to a concentration of up to 40 mg/mL. A C j _sPEOX ASP60 polymer was separately dissolved to a concentration of up to 1 0 mg mL in methanol The two solutions were then mixed at various volumes to result in final polymer to paciitaxe! molar ratios in the mixtures ranging from 3: 1 to 0:1. The mixtures subsequently were lyophiJized for 20 to 96 hours depending on volume.

100163 ] The size of the aggregates as measured by light scattering ranged from about 70 .run to 90 nm in diameter before JyophiJization and .120-140 urn after lyophilization.

[00164] Alternatively, ^'both paciitaxel and the Cj_!¾PEOXABP60 polymer can be dissolved in a common sol ent, such as, acetone, methanol, or ethanol and then dropwise added to water while being stirred or sonicated, followed by sterile filtration with a 0.22 pm filter. The final product then can be generated by lyopMSization. and the size of the aggregates .measured by light scattering.

[00165 j Other taxane-induced aggregates or nanoparticles using various ydrophobicaity surface-niodified branched polymers, such, as,€4, C¾, C or tz hydrocarbon-modified randomly branched PEOX, FEI and PPI polymers;€ , C Cis d Cj₂ hydrocarbon-modified PAMAM, PEi and PPI dendrimers and dendri.gra.fts; and C4, Q, Cn, Cis and C₂₂ hydrocarbon-modified branched

P^'LL poIymers can be prepared in a similar manner, anoparttcle with a 7: 1 Ci8^'P.EOXABP66 PolyiaenPaciitaxei Ratio

[00166] Ci*PEO^'XABP60 (700 mg) was dissolved in 9.33 m^'L of methanol to yield a 75 mg/mL solution. A 15 mg/mL solution of paciitaxel was also prepared by dissolving 100 mg in 6.67 mL of methanol The two solutions were mixed for 20 minutes resulting in a solution containing 6.25 mg paciitaxel and 43,75 nig polymer per mL, providing a solution with a 7; I polyraer.drug ratio. The mixture was placed on a rotary evaporator and. the methanol, removed to dryness. The resultant solid was redissolved with stirring to 33.3 ml of water to a final paclitaxel concentration of 3 mg/mL The solution preparation, was passed through a 0.8 μτ« filter and then a 0.22 pm filter. The filtrate was lyophi!ked over a 24-72 hour period depending on the amount used. The vial was stoppered, and the ready-fo-use w ite powder was stored at room temperature. Thai preparation was designated as F1D-007.

Nauoparticle Measurement

[00167] The size of various polymers, polymer-only aggregates, a s wel l as drug-induced polymer aggregates was measured by a dynamic light scattering method using a Malvern Zetasizer Na.no~ZS Zeo.3600 particle size analyzer.

Activity Testing

[001681 Metabolism in viable cells produces, "reducing equivalents " such as, NADH or ADPIT Such reducing compounds pass electrons to an

intermediate electron transfer reagent that can. reduce the tetrazoliura product, MTS (Promega), into an aqueoas, soluble forrnazan product, which is colored. At death, ceils rapidly lose the ability to reduce tetxazolium products. The production of the colored formazan product, therefore, is proportional to the number of viable cei ls in culture.

[00169 j The CeliTiter 96 Aqueous products (Promega) are MTS assays for determining the number of viable cells in culture. The MTS tetra¾olium is Similar to MTT tetraxolhim, with the advantage that the f rma an product of MTS reduction is soluble in cell culture medium and does not require use of a solubilization solution. A single reagent added directly to the assay wells at a recommended ratio of 20 μΐ reagent to 100 μΙ of culture medium was used . Cells were incubated 1-4 hours at 37° C and then absorbance was measured at 490 nm.

Toxicity and Efficacy of !Nanoencapsu!ated PacSitaxeS/ABP60 (FlB-007)

[00170] As previously described, nanoencapsulated paclitaxel was prepared using CisABP60 polymer with a polymer to paclitaxel ratio of 7:1. That preparation, given the designation FID-007, was compared to TaxoJ and Abraxane in cytotoxicity studies with norma! human derma! fibroblast eel! lines and various cancer ceil, lines, and in in vivo studies of toxicity (maximum tolerated dose, MTD) and inhibition of tumor growth in three mouse xenograft models. la Vitro Activity of 110-667

[001 71 ] FID-007 was tested with Taxol and Abraxane on norma! hitman fibroblast cells and on various cancer ceil lines in in vitro cytotoxicity experiments. While FID-007 inhibits the proliferation of a range of human cancer cell lines in vitro including lines originating from breast, ovarian and lung cancer cells, FID-007 exhibited lower toxicity to norma! cells, similar to the levels observed with Taxol and Abraxane (Figure .15). Overall, FID-007 was .10 times less toxic to normal cells than to tumor cells, exhibiting a very high EC^'so greater than 100 μΜ, FID-007 was active in a. 72 h toxicity assay in human lung cancer cell line A549 with an !€¾> of

2.8 ng/roL (Figure ! 6). FID-007 cytotoxicity to normal cells was comparable to that of Taxol and Abraxane. FID-007 was cytotoxic to MDA-MB-231 (triple negati ve breast cancer cells) with an I.C50 of 4.9 ng/mL (Figure 17). FID-007 was cytotoxic to OV-90 (ovarian cancer cells) with an IC50 of 5.0 ng/mL (Figure 18). With all three cancer cell lines, FID-007 cytotoxicity was comparable to that of Taxol and

Abraxane. in Vivo Activity of FID-007

[001721 A series of experiments was performed to determine hi vivo tolerability, activity, and basic pharmacokinetics of FID-007 administered intravenously O.V.) in mice, as compared to Taxol and Abraxane. FID-007 was well tolerated up to 150 rag/kg daily dosing. To confirm antineoplastic activity, FID-007 was administered I.V. daily at we!l-to!eraied doses to mice in three different mouse xenograft models (including lung, ovarian and breas cancers), in genera!. FID-007 was better tolerated in mouse xenograft models than standard cytotoxic agents that have similar targets, such as Taxol and Abraxane, and selectively inhibited the growth of tumors.

[00173 ] Half-life of FID-007 in mice was determined using an optimized HPLC method to be approximately 9.3 hoars. Liver and spleen, followed by blood were the organs with the highest concentration of FID-0G7 at 1 hour. The PK profiles of FID-007., Taxol and Abraxane are shown in Figure .1 .

[001 74] The single dose MTD of FID-007 was compared to that of Taxol and Abraxane in a study wherein various doses of the drugs were adrainistered through the tail vein of healthy CD- I mice and SOD (immune deficient) mice over the course of several weeks. Control mice were administered, saline. The single dose MTD for Taxol, Abraxane, and FID-007 on CD-I mice was found to be 20 mg/kg, 240 mg kg, and 175 mg/kg., respectively. No major side effects were observed in ail the mice that survived. However, weight gain was observed in al the treatment groups of Abraxane and FID-007 as compared to the control groups (treated, with saline). Abraxane at .120 mg/kg and above caused a dose-dependent increase in weight. The same was observed with FID-007 at closes of 150 mg kg and higher.

1001 75] The multiple dose MTD of FID-007 was determined similarly by administering FID-007 ( 100 and 1 0 mg/kg) to healthy CD- 1 and SOD mice ( 10 weeks, females) via the tail vein at day 0, day 3 and day 6, Animals were monitored twic per day and weighed every 3 days. The multiple dose MTD for FID-007 in CD-I mice was determined to be 100 mg/kg and was 30 mg/kg in SOD mice with some side effects immediately after injection. The FID-007 multiple dose groups did not. have excessive weight gain as compared to the control group.

[ 001 76] The in vivo efficacy of ^'FID-007 in inhibiting tumor growth was compared to that of Taxol and Abraxane in tumor xe ograft mouse models of human lung, breast and ovarian cancer. Sixty female and male SOD mice (6-8 weeks, 20-26 g, Charles River, 40 female mice for breast and ovarian cancer, 20 male mice for lung cancer) were injected on each side of the torso (left and right) with 0,1 mL of suspension of long A549, breast MDA-MB-23 ! or ovarian OV 0 cells in serum-free medium. Cells were cultured previously in. a humidified incubator (37"C, 5% CO;., 95% air). Doses of 3 x 10* (A549), 10⁷ ( DA-MB-231), and 5 X 10* (OV-90) cells were used per mouse tumor. The tumors were allowed to grow for 7 to 9 days before treatment started, and all tumor volume measurements were obtained using a digital, c liper (VWR Inc.). The tumor volumes were calculated by the formula (^"W^" x L)/2, where W is the maximum tumor width and L is the maximum tumor length. Tumor and body weight measurements were obtained on the same day prior to the first treatment, then every three days. Day 0 was designated as the .first day of treatment. On day 0, the animals (hat developed tumors were di ided randomly into five groups [about 4 mice (8 tumors) per group], with each, treatment group representing a wide range of tumor sizes.

[00177] Abraxane (80 g/kg), HD-00? (20 rag/kg), Taxol {20 mg/kg) and Cj$ABP60 polymer starting material, designated as NanoCarrier 001 -B, (20 rog/kg) were prepared fresh for each injection. Saline was used as a vehicle control The drugs or saline were administered^* through tail vein injection ever}' three days. Drug doses were chosen to be equi toxic for all treatment groups based on the previously determined single and multiple dose MTD. Lung, breast and ovarian cancer groups each received a total of four injections. Injection volume for control, Abraxane and HD-00? was 0.1 mL er injection throughout the entire study . Due to the viscosity of the Taxol formulation, 0.2 mL per injection were administered for the 20 mg kg dose. Average body weight and tumor volume measurements were calculated by averaging all the animals within the same group. The mice were euthanized with, isoflurane 21 days from the last treatment for Sung cancer and ovarian cancer, and 10 days for breast cancer. Blood and isolated serum, as well as tumor tissues and li ver were collected and stored at -80^' C.

[001.78] For the lung cancer (Ά549) xenograft group, overall, no deaths occurred, in any of the treatment groups. Probably due to the toxicity of Taxol, heavy breathing and inactivity were observed i the first 30 minutes post, treatment in a couple of mice. Average body weight and tumor volume measurements were calculated by averaging all the animals within the same group. The overall average bod weight gains for saline control, Taxol, FID-007 and Nano vehicle control were 6.05%, 5.87%, 6,38% and .12.3 %, respectively. However, all mice in the Abraxane group developed bad .neurotoxicity and lost > 20% weight. Those mice were sacrificed at .13 days. Tumor volumes increased, by 1827 mm' for the saline control group and 131 1 mm³ for the ano-Carrier-W) 1 B vehicle control group, and by 305.8 mm'' for the Taxol group. However, FiD-007 groups had a reduction in tumor volumes by 39.7 mm³ (Figure 20). Figures 2.1 and 22 show representative images of tumors of the treatment, groups.

[00179] For the breast cancer {MDA- B-231 ) xenograft group, no deaths occurred in any of the treatment groups. Possibly due to the toxicity of Taxol, heavy breathing and. inactivity were observed in the first 30 minutes post treatment.

43 I« the Abraxane group, all mice showed side effects of weak hind legs and 20% body weight loss after three treatments, leading to a decision to stop the 4^th treatment for that group. Average body weight and tumor volume measurements were calculated by averaging al! die animals within the same group. The overall average body weight gains for saline, Taxol, FlD-007 and Nano-Catrier-001 B were 3.76%. 0.46%, 1.8%, and 4.2%, respectively. For the Abraxane group, average body weight drop was 7.66%. Tumor volumes increased by 328.6 mirr and 458.8 mm* in the saliae and Naoo-Camer-OO!B groups, respectively. In the FiD-007, Taxo! and Abraxane groups, tumor volumes decreased by 108.7 ira , 75.5 mn and 70.2 mm ,

respectively. Tumor volume observations are shown in Figure 23. Figures 24 and 25 are representative images of tumors of the treatment groups.

1 01 01 For the ovarian cancer (OV-90) xenograft: group, the Taxol treatment group showed some toxicity with heavy breathing and inactivity observed in the fust 30 minutes post treatment in two mice. The average body weight gain was 3,23%, 17, 1%, 13.5%, 15.4% and 2,24% in. the saline control, Taxol, Abraxane, FID-007 and Nauo-Carrier-OOl B control groups, respectively. Tumor volumes increased by 652.7 mm 271.9 mm^"' and 9.1 ranr' in saline control,

Nan.o-Carrier-00 ί B control and Taxol groups, respectively, while there was a decrease in tumor volume in ihe FID-007 groups by 93. i mm^*' and in the Abraxane group (80 mg/kg) by 72.4 mm ^' . Figure 26 summarizes tumor volume observations for each treatment group. Figures 27 and 28 are representative images of tumors before and after dissection.

[00181 j Fj^'D-007 demonstrated in vitro cytotoxicity to lung, breast and ovarian eel! hues similar to the established antineoplastic drugs Taxol and Abraxane while maintaining a low level of toxicity to normal cells. The in vivo efficacy of FID-007 in inhibiting tumor growth and. reducing tumor mass was as good as or significantly better than the two approved drugs in mouse xenograft models of human lung, breast; and ovarian cancers.

[001 2 j All references cited herein are herein incorporated, by reference in entirety,

[001 3] It will be appreciated that various changes and modifications can be made to the teachings herein without departing .from the spirit and scope of the disclosure.