HK1127490B - Solubilization and targeted delivery of drugs with self-assembling amphiphilic polymers - Google Patents

Description

Solubilization and targeted delivery of drugs using self-assembling amphiphilic polymers

Technical Field

The present invention relates to the field of amphiphilic polymers, in particular biocompatible micelle-forming comb polymers. The invention also relates to the field of drug solubilization and targeted drug delivery.

Background

In recent years, amphiphilic block copolymers comprising a hydrophobic segment and a hydrophilic segment have been intensively studied because of their ability to self-assemble into various nanostructures with changes in the surrounding solvent. See Cameron et al, can.j.chem./rev.can.chim.77: 1311-1326(1999). In aqueous solutions, the hydrophobic segments of the amphoteric polymers tend to self-assemble to avoid contact with water and minimize the free interfacial energy of the system. At the same time, the hydrophilic segments form hydrated "corona-like structures (corona)" in the aqueous environment, and thus the aggregates maintain a thermodynamically stable structure. The result is a stable latex-like colloidal suspension of polymer aggregate particles having a hydrophobic core and a hydrophilic corona.

Comb ampholytic copolymers differ from block copolymers in that the backbone is predominantly hydrophobic or hydrophilic, while the polymer chains of opposite polarity are suspended from the backbone rather than incorporated into the backbone. Comb copolymers have been prepared from hydrophobic backbones and hydrophilic branches (Mayes et al, U.S. Pat. No. 6,399,700), and from hydrophilic backbones and hydrophobic branches (Watterson et al, U.S. Pat. No. 6,521,736). The former is used to provide multivalent ligand presentation to cell surface receptors, while the latter is used to solubilize drugs and deliver them to cells.

Amphoteric polymer aggregates have been studied as carriers for solubilizing insoluble drugs, targeted drug delivery vehicles, and gene delivery systems. They have a more stable structure than conventional low molecular weight micelles due to chain entanglement and/or crystallization of the internal hydrophobic region. The polymeric nature of the carrier makes the aggregates relatively less susceptible to degradation that occurs when ordinary liposomes are diluted below their critical micelle concentration. They also have the advantage over traditional liposomal delivery compositions that no bilayer membrane exists, making them more easily fused to the cell membrane and delivering their payload directly to the cell.

Because of the excellent biocompatibility of polyethylene glycol (PEG) and the remarkable ability of PEG-coated "stealth" particles to evade the reticuloendothelial system, micelles, liposomes, and polymers comprising PEG have been widely considered as materials for drug delivery systems. There are many reports on the use of polyethylene glycol (PEG) as the hydrophilic component of PEG-lipids (forming liposomes and micelles); see, e.g., Krishnadas et al, pharm. Res.20: 297-. Self-assembling amphiphilic block copolymers that self-assemble into more robust "polymer assembly aggregates" (Photos et al, J. controlled Release, 90: 323-. See also Gref et al int.Symp.controlled Release Mater.20:131 (1993); kwon et al, Langmuir, 9:945 (1993); kabanov et al, J.Controled Release, 22:141 (1992); allen et al, J.controlled Release, 63:275 (2000); inoue et al, J.controlled Release, 51:221 (1998); yu and Eisenberg, Macromolecules, 29:6359 (1996); discher et al, Science, 284:113 (1999); kim et al, U.S. patent No. 6,322,805; seo et al, U.S. Pat. No. 6,616,941 and Seo et al, European patent No. EP 0583955. There have also been reports of the use of Polyethyleneimine (PET) for this function, which has focused on delivery to oligonucleotides (Nam et al, U.S. Pat. No. 6,569,528; Wagner et al, U.S. Pat. Pub. No. 20040248842). Similarly, Luo et al describe PEG-conjugated polyamidoamine ("PAMAM") dendrimers suitable for delivery of polynucleotides in Macromolecules35:3456 (2002).

In addition to the need to solubilize, disperse and deliver drugs, there is also a need for targeted drug delivery systems that specifically target tissues, tumors or organs. This is usually accomplished by the attachment of antibodies or other ligands with specific affinity for the cell wall at the target site. However, PEG lacks functional groups except at the polymer chain ends, and most of the end groups are inevitably occupied by linkages to other block copolymer components. Thus, attachment of targeting moieties such as antibodies or cell adhesion molecules to PEG block copolymers is generally limited to non-PEG segments, which unfortunately are not the portion of the copolymer that is typically exposed to the corona of the self-assembling aggregate.

The phase separation phenomenon, which causes self-assembly of block copolymers into polymer aggregates, can be easily reversed, and attempts have been made to improve the stability of the aggregates by crosslinking the hydrophobic core (see european patent No. EP 0552802). Covalent attachment of drugs to the hydrophobic component of block copolymers has also been attempted (Park and Yoo, U.S. Pat. No. 6,623,729; european patent No. EP 0397307).

There remains a need for a delivery system that is stable, biodegradable, can attach a targeting moiety to the exterior of an aggregate on demand, and efficiently deliver a drug to a desired cellular target.

Disclosure of Invention

The present invention provides biocompatible comb polymer molecules comprising a hydrophilic backbone having branch point moieties and hydrophobic branches attached to these branch point moieties. The present invention provides aqueous suspensions of polymer aggregates formed from such polymers, and provides methods of solubilizing insoluble or sparingly soluble organic compounds (e.g., drugs, dyes, vitamins, and the like) by incorporating such compounds into the hydrophobic core of the polymer aggregates. The process for solubilizing water-insoluble organic materials in aqueous solvents generally involves contacting the water-insoluble organic materials with the polymers of the present invention in an aqueous or mixed aqueous solvent.

In particular embodiments, the branch point moiety further comprises a reactive functional group that can serve as an attachment point for a targeting moiety. In particularly preferred embodiments, a targeting moiety, such as a ligand or antibody, is covalently attached to the branch point moieties of the polymers of the invention, and the drug is incorporated into the inner core of the aggregate to form a targeted drug complex.

The invention further provides methods of making comb polymers, aggregates, and the targeted drug complexes. The polymers of the present invention can self-assemble into polymer aggregates that can efficiently solubilize, disperse, and deliver drugs in vivo, are non-toxic, biocompatible, and stable, and can have multi-cellular targeting moieties on their outer surfaces.

Drawings

FIG. 1 shows 20ul samples of saturated solutions of three lipophilic dyes (A, Sudan IV; B, dichlorofluorescein; C, alcohol soluble eosin Y) in deionized water spotted onto silica gel TLC plates. The previous line: contains 50mg/ml of the π -polymer of example 1; the next line: no pi-polymer is present.

FIG. 2 shows 50ul samples of saturated solutions of four insoluble drugs (line 1, rhodopsin; line 2, camptothecin; line 3, amphotericin B; line 4, doxorubicin) in deionized water spotted onto a silica gel TLC plate. Column a, folate polymer a; column B, Polymer A; column C, no polymer. The three pencil marks around each dot illustrate the spread of solvent on the plate. The middle circles in columns a and B diffused uniformly in the silica gel, indicating the clarity of the solution; the middle circle of column C consists mainly of solid precipitates.

Detailed Description

The polymer of the present invention (referred to herein as "pi-polymer") has a comb-like structure having a skeleton formed of alternating branch point moieties B and hydrophilic water-soluble polymer segments a, and having a plurality of hydrophobic side chains C attached to each branch point moiety. They consist essentially of the structure shown in formula 1. The side chain C is a relatively short hydrophobic moiety and may be an aliphatic molecule, chain or oligomer. The ideal value of p is the integer 2, 3 or 4. In practice, side chains are often introduced by chemical reactions with non-ideal efficiency, resulting in the average value of p not being an ideal integer in the overall polymer produced. Non-integer averages may also be obtained by design, as described below. Thus, the average value of p in the polymer of the invention is greater than 1 and not more than 4(1< p.ltoreq.4). In preferred embodiments, p ranges from about 2 to 4, and most preferably 1.5< p.ltoreq.2.

The backbone polymer segment a is selected from hydrophilic and/or water-soluble polymer chains including, but not limited to, polyethylene glycol, polypropylene glycol, polyethyleneimine, polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides, and the like. Preferably, polymer unit A is of the formula- (CH)₂CH₂O)_m-wherein m is between 1 and 10,000, preferably between 3 and 3,000, and may be between 4 and 700. The terminal functional groups of the polymer chains are not characterized, which is not relevant to the present invention.

In the manufacture of different grades of polyethylene glycol, it is known in the industry to attach a divalent linking moiety (e.g., bisphenol a diglycidyl ether) to two polyethylene glycol chains to effectively double the polymer molecular weight while maintaining a relatively narrow molecular weight range. Thus, the resulting "polyethylene glycol" molecule is interrupted at the midpoint of the polymer chain by a non-glycol linker moiety (see, e.g., polyethylene glycol-bisphenol a diglycidyl ether adduct, CAS accession No. 37225-26-6). Higher oligomers, i.e. those having three PEG chains separated by two bisphenol A diglycidyl ether moieties, are also known, see for example International patent application WO 00/24008. Thus, the terms "polyethylene glycol" and "polypropylene glycol" as used herein encompass polyethylene glycol and polypropylene glycol polymer chains incorporating non-glycol linking units including, but not limited to, bisphenol a diglycidyl ether, bisphenol B diglycidyl ether, bisphenol S diglycidyl ether, hydroquinone diglycidyl ether, and the like. For the purposes of this specification, any such linking moiety is not counted as a "monomer unit".

Most preferably, the average length of the polymer segments a is between 20 and 50 monomer units. One or both ends of the polyethylene glycol chain may be substituted with functional groups suitable as linkers for other moieties, including, but not limited to, amino, thiol, acrylate, acrylamide, maleate, maleimide, and the like. The value of n ranges between 1 and 1000, preferably between 3 and 100. The total molecular weight of the pi-polymers ranges from 1,000 to 100,000 daltons or more; preferably above 2,000 daltons and more preferably above 7,000 daltons.

The hydrophobic moieties C may be the same or different and may be, for example, straight chain hydrocarbons (optionally substituted with one or more hydrophilic substituents), polycyclic hydrocarbons (optionally substituted with one or more hydrophilic substituents), hydrophobic amino acids, peptides and polymers. Suitable hydrophilic substituents include, but are not limited to, hydroxyl, ether, cyano, and amide functional groups. In particular, contemplated substituents are C with omega-hydroxy, omega-cyano, omega-amido or omega-alkoxy substituents₈To C₂₀An alkyl group. As used herein, the term "substituent" includes the substitution of a moiety with a heteroatom (e.g., O, N or S)C hydrocarbon chain or carbon atom in a ring system. Thus, ether and amide linkages as well as heterocycles can be incorporated into C.

The hydrophobic moiety C is preferably relatively short (C)₈-C₂₀) But also shorter oligomers. Suitable oligomers include oligomeric hydroxy acids, for example, polyglycolic acid, poly (DL-lactic acid), poly (L-lactic acid), and copolymers of polyglycolic acid and polylactic hydroxy acids, as well as polyamino acids, polyanhydrides, polyorthoesters, and polyphosphoesters, polylactones, for example, poly (epsilon-caprolactone), poly (delta-valerolactone), poly (gamma-butyrolactone), and poly (beta-hydroxybutyrate). The C moiety may also be selected from hydrophobic molecules such as cholesterol, cholic acid, lithocholic acid, hydrophobic peptides and the like. The molecular weight of each C moiety is greater than 40, preferably between 50 and 1,000, and most preferably between 100 and 500. In general, any moiety C that is not significantly soluble in water when in molecular form C-H is considered suitable for use in the present invention.

The comb polymer of the invention is distinguished in that the side chains C are not regularly and homogeneously distributed along the polymer chain, but rather occur in clusters C]_pIn (1). These clusters are almost regularly spaced from each other along the polymer chain, depending on the degree of monodispersity of the polymer units a. Thus, the distance between two side chains C attached to a common branch moiety B is different from the distance between two side chains attached to different branch moieties.

In a second embodiment of the invention, the branch point moiety B further comprises one or more reactive functional groups X and the polymer consists essentially of the structure shown in formula 2.

In formula 2, the individual reactive groups X may be the same or different and may be selectively blocked or protected as desired during assembly of the polymer 2. The average value of r can range from 0 (no X groups) to about 4. In particular, the reactive groups may be selectedFunctional groups useful for forming covalent bonds between molecules are known from the art. The group X serves as an attachment site for a drug molecule, tissue or cell targeting moiety, viral targeting moiety, or matrix attachment moiety (e.g., for the purpose of coating the surface of a stent or other medical device). In particular embodiments, there may be a single attachment site X. In another embodiment, there may be three or four different types of reactive groups. The substrate linking moiety may be attached to the substrate by covalent bonds, specific non-covalent interactions (e.g., antibody-antigen), or non-specific interactions (e.g., by ionic pairing or "hydrophobic" interactions). Suitable reactive groups X include, but are not limited to, -OH, -NH₂、-SH、-CHO、-NHNH₂、-COOH、-CONHNH₂Haloacyl, acetoacetyl, -CN, -OCN, -SCN, -NCO, -NCS and the like; reactive double bonds such as vinyl, acrylic, allyl, maleic, styryl, and the like; and groups having a reactive triple bond such as an ethynylcarboxyl group and an ethynylcarboxamido group (suitable for Michael addition reactions, Diels-Alder reactions, and free radical addition reactions).

Examples of cell targeting moieties include, but are not limited to, receptor specific ligands, antibodies, and other targeting moieties, such as peptides bearing the arginine-glycine-aspartic acid (RGD) amino acid sequence or the tyrosine-isoleucine-serine-arginine-glycine (YISRG) motif; growth factors, including epidermal growth factor, vascular endothelial growth factor, and fibroblast growth factor; viral surface ligands such as sialic acid and N-acetylneuraminic acid derivatives; cellular receptor ligands such as folic acid, methotrexate, pteroic acid, estradiol, estriol, testosterone, and other hormones; mannose-6-phosphate, sugars, vitamins, tryptophan, and the like. The antibody is preferably a monoclonal antibody against a cell-specific surface antigen; suitable targeting moieties include not only intact antibodies, but also antibody fragments that contain active antigen binding sequences, such as Fab '2 fragments, Fab' fragments, or short chain peptide analogs of the active antigen binding sequences of such antibodies.

Examples of viral targeting moieties include small molecule ligands that bind to viruses, such as aminoalkyl adamantane, Fuzeon^TMPRO-542, BMS-488043, sialic acid, 2-deoxy-2, 3-didehydro-N-acetylneuraminic acid, 4-guanidino-Neu 5Ac2en (zanamivir ), oseltamivir (oseltamivir), RWJ-270201 and the like; oligopeptides, oligosaccharides and glycopeptides that bind to viral surfaces, and antibodies and antibody fragments against virus-specific surface antigens. In a preferred embodiment, the invention provides pi-polymers loaded with viral neuraminidase or hemagglutinin ligands. Such polymers have been determined to have antiviral properties themselves; see, e.g., T.Masuda et al, Chemical&Pharmaceutical Bulletin51:1386-98 (2003); itoh et al, Virology212:340-7(1995) and Reece et al, U.S. Pat. No. 6,680,054 (2004). The hydrophobic core of the antiviral polymers and polymer aggregates of the present invention may be selectively loaded with one or more conventional antiviral drugs that are advantageously released in the vicinity of the viral particle.

Other pharmaceutically suitable linking groups may be small chemicals, peptides, antibodies or antibody fragments, enzymes or active pharmaceutical ingredients that may affect biological processes such as hormones or hormone agonists or antagonists; substances that interfere with viral binding; substances and the like that interfere with the cell cycle or cellular processes upon entering the cell. Cells of unicellular and multicellular organisms, including bacteria, fungi, higher animals and plants, can be targeted. Biotin can be attached to pi-polymers and used as attachment sites for avidin-and streptavidin-coupled proteins, peptides, and other targeting or pharmacologically active agents (e.g., antibodies, growth hormones, imaging agents, and the like).

"matrix" refers to organic or inorganic substances, surfaces and deposits, such as glass, silica or metal surfaces, extracellular matrix, protein deposits (e.g., various amyloid plaques), cell surfaces, viral surfaces, and generally homogeneous or heterogeneous surfaces, including prions, that are or have not been characterized in detail.

Examples of glass or silica matrix attachment moieties include various halosilanes, alkoxysilanes, acylsilanes, and chemicals having such functional groups, including polymers. Other linking groups may be designed according to the particular physico-chemical properties of the substrate. Suitable attachment moieties, such as those used to coat stents, are well known to those skilled in the art.

In a third aspect of the invention, the branch point moieties B are linked to other branch point moieties on the polymer chain to form a crosslinked hydrogel structure. Such crosslinking may be achieved by reacting the polymer with a multifunctional moiety containing homofunctional or heterofunctional groups, at least one of which reacts with X located on the first branch point moiety or a reactive group on C, and at least one of which reacts with X of the second branch point moiety or a reactive functional group present on C. Crosslinking may also be carried out by linking to the terminal functional groups of the polymer chain A. Such crosslinked polymers may optionally contain reactive functional groups suitable for attachment of drug molecules or targeting moieties.

The branch point moieties B are typically derived from multifunctional molecules having a plurality of reactive groups, two of which are suitable for linking to the hydrophilic polymer unit a and two of which are suitable for linking to the hydrophobic moiety C. Part B may optionally have an additional reactive group X as described above.

Particularly preferred branch point moieties are Dithiothreitol (DTT), Dithioerythritol (DTE) or a conjugate of 2, 3-diaminobutane-1, 4-dithiol with two molecules of maleic acid. The combination of this branch point moiety with polyethylene glycol as moiety a creates a polymer backbone of formula 3 and formula 3 a.

Wherein Y and Y' are the same or different and are preferably selected from OH, NH₂、ONH₂NHOH and NHNH₂. In a preferred embodiment, the reactive group X is a hydroxyl or amino group of a dithiol as the attachment site for the targeting or drug moiety, and the functional groups Y and Y' as the attachment site for the C moiety. Alternatively, the groups Y and Y' serve as attachment sites, and a hydroxyl or amino group is used to attach the C moiety.

Formulas 3 and 3a are intended to indicate that each sulfur atom can be attached to the alpha or beta position, respectively, of the PEG ester carbonyl. The present invention encompasses single isomer compositions as well as mixtures of regioisomers at one or both C-S bonds. Furthermore, due to the four asymmetric carbon atoms in formula 1, all chiral, meso, and diastereoisomers and mixtures thereof are encompassed by the present invention.

Diels-Alder reaction adducts of acetylenedicarboxylic acid and furan may also be suitable branch point moieties. For example, it is known that polyesters 4 derived from PEG and acetylenedicarboxylic acid undergo Diels-Alder reactions with furans (M. Delerba et al, Macromol Rapid Commun.18(8):723-728 (1997)).

Scheme 1

Thus, it can undergo diels-alder reactions with 3, 4-disubstituted furans to produce the species shown in fig. 5, and polymer 5 can be modified by hydroxylation or epoxidation to provide reactive groups (e.g., X and X' in scheme 1).

Likewise, reaction of PEG with ethylenediamine tetraacetic dianhydride can provide a polyester of formula 6:

other suitable branch point moieties may be derived from tartaric acid, acetylenedicarboxylic acid, nitrilotriacetic acid, 3, 4, 3 ', 4 ' -diphenylsulfone tetracarboxylic dianhydride, 3, 4, 3 ', 4 ' -diphenylether tetracarboxylic dianhydride, pyromellitic dianhydride, alkanedithiols (e.g., 1, 2-ethanedithiol and 1, 4-butanedithiol), bis (2-mercaptoethyl) ether, 2-mercaptoethyl sulfide, dimercaptopropanol, dimercaptopurine, dimercaptothiadiazole, dimercaptosuccinic acid, benzenedimethylmercaptan, benzenedithiol, dihalogenated benzenedimethylmercaptan, dihalogenated 4, 4 ' -thiobisbenzenethiol, and the like.

In the case where Y and Y' are OH, the hydrophobic group C may be attached to the polymer by amidation or esterification of the carboxylic acid group. Preferably, the hydrophobic group C is relatively small (C)₈-C₂₀) And is predominantly a hydrocarbon moiety, and may be straight or branched chain or contain one or more rings. Examples include, but are not limited to, covalently attached dodecylamine, pentadecylamine, cholesterol, and cholic acid moieties. Although the polymers of the present invention may be characterized as having up to two different hydrophobic side chains for convenience, it will be appreciated that mixtures of two or more hydrophobic compounds may be used to introduce a variety of hydrophobic side chains into a particular polymer.

In one particular embodiment, the polymer of formula 2 (wherein X ═ OH and r ═ 2) is prepared by reacting polyethylene glycol with maleic anhydride to form polyester 7, which is subsequently reacted with dithiothreitol to form 8. Subsequently, the acid 7 was amidated with octadecylamine to form the desired comb polymer 9 (scheme 2). The DTT-derived amide comb polymer of formula 9 is referred to herein as "π -Polymer A"; the specific polymer 9 in scheme 2 may be referred to as "C₁₈-pi-polymer a ".

Scheme 2

The corresponding amino-functionalized pi-polymer 9b is produced after deprotection using 2, 3-bis (tert-butoxycarbonylamino) butane-1, 4-dithiol (prepared as described by DuPriest et al, U.S. Pat. No. 4,755,528) in place of dithiothreitol (scheme 3).

Scheme 3

The use of butane dithiol 10c can likewise result in a polymer of general structure 9c, wherein a spacer group L is used for subsequent attachment of the targeting moiety (scheme 4). Spacer group L may be any spacer group known in the art for linking a ligand or tag to a substrate molecule, including but not limited to C₂To C₂₀Alkylene and oligo (ethylene glycol) spacers.

Scheme 4

In another embodiment, PEG polymers with terminal amino groups can be used to prepare examples having amide linkages between unit a and unit B as shown in structures 10-14 below. Can be synthesized by PEG diamine H₂N-(CH₂CH₂O)_mCH₂CH₂-NH₂Each such polyamide is derivatized with a suitable cyclic anhydride:

under mild conditions, the amic acid described above is the desired product. It is expected that upon heating, an imide may be formed, thereby rendering the polymer less reactive groups, but still suitable for attachment of hydrophobic C moieties. Alternatively, the side chain C may be added to the end of the polymer a segment, and a branch point moiety may be introduced during polymerization (scheme 5).

Scheme 5

In addition to the simple diamines of 1, 3-propanediamine shown in scheme 5, diamines having (optionally masked) reactive functional groups X may also be used, making polymer 15 suitable for attachment of targeting moieties (scheme 6). In the formula below, p can range from 0 to 4, and each X group is independently the same or different from any other X group that may be present. The reactive X groups need not be pendant groups but may be, for example, NH groups in the chain of atoms making up the diamine, such as monomer H₂N-(CH₂)₃-NH-(CH₂)₃-NH₂。

Scheme 6

Certain pi-polymers prepared according to the above protocol carry reactive groups X suitable for further derivatization, to attach targeting moieties, such as small molecules, peptides, nucleotides, sugars, antibodies, etc., or to crosslink polymer chains by bifunctional or multifunctional crosslinking agents. In particular embodiments, reactive groups on a polymer chain are partially derivatized to produce pi-polymers having a plurality of different reactive groups that allow attachment of a plurality of targeting and drug moieties to a polymer chain. Thus, addition reaction of the π -polymer of example 1 with a substoichiometric amount of acryloyl chloride (or maleic anhydride) can provide a polymer with acryloyl (or maleoyl) groups and residual hydroxyl groups. Subsequently, a sub-stoichiometric amount of a mercaptocarboxylic acid (e.g., HS- (CH)₂)₃Michael addition reaction of-COOH) to provide a compound having a hydroxyl group and a C groupPolymers of alkenoyl and carboxyl groups. The addition reaction of cysteine introduces amino and carboxyl groups in addition to the remaining reactive groups left by the sub-stoichiometric reagent.

Another method for obtaining a multifunctional pi-polymer involves careful removal of part of the hydrophobic chain C. For example, the π -polymer of example 1 with unreacted carboxylic acid groups can be prepared by limiting the amount of side chain-forming alkylamines in the acylation step by simple means. Yet another method is an amidation reaction with a mixture of amines, a portion of which contains a reactive group X. In addition, under appropriate conditions (maleic anhydride excess in step A and DTT excess in step B), a polymer preparation having the desired free thiol group population can be produced.

By design, the pi-polymer of example 1 contains hydroxyl groups derived from DTT moieties in the backbone, which function as reactive groups X. Esterification of these groups with acryloyl chloride or methacryloyl chloride in an aqueous medium in the presence of a carbonate/bicarbonate buffer can result in substitution of the acryloyl group on the-OH group. The acrylate polymer is susceptible to free radical polymerization (with or without the presence of additional free radical monomers, such as acrylic compounds or cross-linking agents such as bisacrylic compounds) to provide hydrogels suitable for controlled drug release (as a polymer depot or depot) and topical administration (e.g., a dermal patch or ointment).

Scheme 7

The acryloyl group can also undergo a Michael addition reaction, particularly with a thiol group, e.g., of a cysteine residue in a protein, enzyme, peptide, antibody, Fab '2 fragment or Fab' fragment, or of another targeting moiety (scheme 7). Maleic anhydride may also be used to esterify a dried pi-polymer bearing reactive hydroxyl groups to link it with a maleate group (michael reaction acceptor) while generating free carboxyl groups. In the resulting polymer, the maleic double bond can be used for Michael addition reactions, particularly with sulfhydryl groups, such as cysteine residues in proteins, enzymes, peptides, antibodies, Fab '2 fragments or Fab' fragments, or other targeting moieties (scheme 8), and carboxyl groups can be used for coupling to amino groups of drugs or ligands, or lysine residues in proteins and peptides.

The different moieties may be further attached to the newly introduced (or previously available) carboxyl group by amidation reactions. In this way, at least two different targeting moieties can be attached even under saturating reaction conditions (i.e., a stoichiometric excess of the moiety to be attached).

Polymers having pendant carboxylate groups may be amidated using amines under typical coupling conditions, or may be converted to isocyanate groups by Curtius rearrangement and subsequently coupled with amines or alcohols to form ureas or carbamates, respectively. Such reactions may be used to introduce a hydrophobic group C or to attach a targeting moiety.

The free amine may be introduced into the polymer by at least partially reacting one of the reactive groups with a diamine. The diamine must be selected so that one of the amino groups is protected or does not react under the reaction conditions. The latter is mostly done by using ethylenediamine at a pH of about 7.5, since the pKa values of the two amino groups differ greatly. Preferably, this amidation reaction is carried out as a separate step after the introduction of the hydrophobic side chain group. Subsequently, peptides or other molecules having carboxyl groups can be attached by amidation of this free amine.

Scheme 8

Thus, even under saturated conditions, up to 3 different peptides or other targeting moieties can be attached to the pi polymer: one through the thiol group, one through the amine or hydroxyl group, and one through the carboxylic acid group.

Hydroxyl and mercapto groups can also be converted to primary amines by reaction with aziridines or halogenated alkylamines such as bromoethylamine or chloroethylamine. Amidation of cysteamine may introduce a disulfide, which may react directly with cysteine of the peptide or antibody to attach the peptide or antibody; or reduced first using, for example, aminoethanethiol or DDT, followed by further reaction with the peptide or antibody.

By performing a partial reaction, one can introduce additional reactive functional groups to the polymers of the present invention, including but not limited to (1) thiol reactive groups, such as acrylic acid or maleic acid derivatives, (2) carboxylic acid reactive groups, such as amino or hydroxyl groups, (3) amine reactive groups, such as carboxyl groups, and (4) disulfide reactive groups, such as mercapto groups. The number of such additional functional groups per polymer molecule ranges from 1/r to several times r, depending on the reagents used and the amounts used.

Alternatively, two or more specific ligands may be linked to improve specificity of binding to, for example, a virus or cell surface. Two or more specific ligands may also be used to facilitate interactions between different cellular targets, e.g., one ligand may target a viral particle while another ligand may facilitate binding to phagocytes, thus bringing the viral particle into proximity or contact with the phagocytes and promoting phagocytosis.

Such derivatization may attach three or more different targeting and/or therapeutic moieties to the polymer by attaching different functional groups (e.g., amines, carboxylic acids, and thiols). Thus, one can attach tissue-specific targeting agents, imaging agents, and therapeutic agents to a single polymer and then obtain targeted therapeutic agents through self-assembly of the polymer, the distribution and targeting efficiency of which can be monitored.

Linking the ligand to the repeating units of the polymer of the invention provides for multivalent display of the ligand on the polymer chains and on the surface of the nanoparticles. Multivalent display typically results in a significant increase in affinity for the target. For example, multivalent antibodies are much more effective at clearing their target than normal bivalent antibodies. Carbohydrate-binding proteins and carbohydrates are known to be multivalent in nature, and their monovalent state does not play a role. Similarly, multivalent peptides and carbohydrate targeting moieties are significantly more efficient than monomers alone. Increased MW due to attachment to the polymer results in decreased renal clearance of peptides and other ligands. In addition, the benefits of the PEG backbone for peptides are similar to pegylation, including evading immune surveillance.

Furthermore, multivalent targeting moieties can modify and neutralize multivalent targets (e.g., viral particles) significantly more efficiently than monovalent targeting moieties. The ability to display multiple (different) peptides in multivalent form will lead to enhanced specificity. For example, a true HIV-specific (HIV virus-binding) polymer can be constructed by linking a peptide corresponding to the CD4 binding region to another peptide corresponding to the CCR-5 or CXCR-4 binding region of the virus, and possibly a third polypeptide corresponding to another receptor (CXCR-4 or CCR-5, respectively). The polymer can completely mask the binding region of the virus and prevent the virus from attaching to the cell and losing infectivity. Furthermore, the surfactant nature of the polymer can cause instability of the virus structure itself through binding. Instead of peptides, small molecules or a mixture of peptides and small molecules (preferably with complementary activity) that interfere with the same binding format (CD4, CCR-5, CXCR-4) may be used. The resulting polymers are ineffective against any free virus and therefore infection transmission may be desirably prevented by their use as condom lubricant components or the like. In addition, such polymers may be injected into patients to reduce HIV load.

Generally, when a polyfunctional agent such as DTT is used, partial crosslinking of the polymer chains can be produced by esterification reaction of carboxylic acid with DTT or similar side reactions. Secondary hydroxyl groups in the central region of the PEG chain (e.g., those associated with bisphenol a diglycidyl ether residues), if present in the PEG starting material, also lead to crosslinking reactions. The resulting crosslinked hydrogel structures are also useful materials. For example, by appropriately increasing this degree of crosslinking or by explicit crosslinking using alternative crosslinking agents (e.g., dioxiranes), flexible hydrogel materials can be made that can be used as drug depots. By appropriate modification of the material (e.g., shortening PEG length, adding open carboxyl groups (open carboxyl groups), and incorporating suitable acrylic groups), it is possible to make a linear or cross-linked hydrogel material that can serve as a depot, supported by fixation on a device such as a stent or by absorption into a device such as a pad for an adhesive patch or subcutaneous insertion patch. Typically, such crosslinked materials are suitable for controlled release rather than enhanced or targeted release.

The comb polymers of the present invention are useful for solubilizing sparingly water-soluble materials in aqueous solvent systems. A method of solubilising a substance in an aqueous solvent comprises contacting the sparingly soluble substance with a comb polymer of the invention in the presence of water to form a water-soluble complex of the substance and the polymer. Alternatively, the polymer is combined with the substance to be solubilized in a two-phase aqueous-organic emulsion and the organic solvent is removed by evaporation. An exemplary process is described in U.S. Pat. No. 6,838,089, incorporated herein by reference. It is believed that in most cases, the polymers self-assemble into nanoparticles with the sparingly soluble species dissolved in hydrophobic C chains that join at the particle core, while the a segments form a hydrophilic corona that can sufficiently lower the interfacial free energy to maintain stability of the aqueous suspension of particles.

In some cases, the sparingly soluble substance may not be completely dissolved in the core, but rather may be present in the particle core as a solid nanoparticle surrounded and suspended by C chains. For the purposes of the present invention, this is a degree of difference, since the practice of the invention is not dependent on a particular degree of mixing between chain C and the sparingly soluble substance. In some cases, the substance is dissolved at the molecular level between the C chains, while in other cases any degree of phase separation from the C-chain environment may occur. In some cases, it is expected that the system will transition from one state to another as a function of temperature.

The solvating power of the hydrophobic core of the polymeric particles can be improved by modifying the hydrophobic moiety C. Suitable modifications include, but are not limited to, the introduction of one or more hydrophilic substituents (e.g., hydroxyl, ether, amide, and cyano functionalities) to increase the polarity and/or polarizability of the hydrophobic core.

Sparingly soluble materials that can be solubilized by these polymers include fat soluble vitamins and nutraceuticals including, but not limited to, vitamins A, D, E and K, carotene, cholecalciferol, and coenzyme Q; insoluble drugs such as docetaxel, amphotericin B, nystatin, paclitaxel, doxorubicin, epirubicin, rubitecan, teniposide, etoposide, daunorubicin, methotrexate, mitomycin C, cyclosporin, irinotecan metabolite (SN-38), statins, and steroids; dyes, photodynamic and imaging agents, and nucleic acids, nucleic acid analogs and nucleic acid complexes. Nucleic acid analogs include species such as phosphorothioate and peptide nucleic acids; the nucleic acid complex is an ionic complex of an oligonucleic acid and a substantially charge-neutralizing amount of a cationic or polycationic species.

For the purposes of this disclosure, drugs that are insoluble at neutral pH are considered "sparingly soluble" because neutral pharmaceutical compositions are required in many cases. For example, ciprofloxacin is soluble in water at a pH below 4.5, but this pH is very irritating when used to prepare a medicament for ocular administration. The polymers of the present invention solubilize ciprofloxacin in physiological saline at pH7. Also, for the purposes of the present invention, "sparingly soluble" should be understood as referring to any substance which: their solubility in aqueous media is such that an increase in solubility may result in improved or more useful compositions. Thus, a drug of moderate solubility (e.g., solubility up to 2 g/liter) is "sparingly soluble" when administered intravenously at a unit dose of 5 g.

The ability of the polymers of the present invention to solubilize a pharmaceutically active species allows the present invention to also provide pharmaceutical compositions comprising one or more pi-polymers of the present invention and a therapeutically effective amount of one or more pharmaceutically active agents. The polymers of the present invention may render otherwise ineffective an amount of the pharmaceutically active agent. Thus, for the purposes of this disclosure, a "therapeutically effective amount" is that amount of drug that makes the composition effective overall.

All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

Examples

1. General procedure

The invention also provides a process for preparing the comb polymers of the invention. The synthesis of these polymers will be readily carried out by those skilled in the art of organic synthesis by the procedures described below. The main starting material is polyethylene glycol, which is preferably dried before use. Drying can be easily performed by stirring the melted PEG at high temperature in vacuum until the foam stops generating. This may take 8-12 hours, depending on the quality of the PEG. After drying, the PEG can be stored under argon for long periods. Commercially available commercial or research grade PEGs may be used to make the polymers of the present invention, such as the commercial polydispersed "PEG 1500" having a 1430-1570 molecular weight distribution. Such materials may incorporate bisphenol a diglycidyl ether, which introduces secondary hydroxyl groups in the middle of the PEG chain. To ensure that the polymers of the invention have the most reproducible and consistent properties, the PEG is preferably free of bisphenol a and of low dispersity. Most preferred is>PEG Polymers of 95% monodispersity, such AS those commercially available from Nektar Therapeutics (formerly Shearwater Polymers), Huntsville AL and Polypure AS, Oslo, Norway. An example of a particularly preferred PEG is "PEG-28" from Polypure, which is>95% HO (CH)₂CH₂O)₂₈H, molecular weight 1252.

All reactions are carried out under an inert atmosphere, such as nitrogen or argon, with magnetic or preferably mechanical stirring.

In step a, dry PEG was melted and maleic anhydride (2 moles per mole of PEG) was added with stirring. The amount of maleic anhydride should match as closely as possible the number of PEG terminal hydroxyl groups. Insufficient maleic anhydride will result in hydroxyl-terminated polymer chains, whereas excess maleic anhydride will consume thiol groups in the next step, which results in premature chain termination and terminal carboxyl groups. The reaction temperature is not critical and the process can conveniently be carried out at a temperature between 45 ℃ and 100 ℃. The preferred reaction temperature is between 65 ℃ and 90 ℃. If high temperatures are used, the maleic anhydride tends to sublime and steps should be taken to ensure that the maleic anhydride remains in solution. Minimizing headspace and submerging the reaction vessel in an oil bath are effective methods.

Depending on the temperature selected, the reaction may be completed in 2 hours or less, or the reaction may be carried out overnight. The reaction can be monitored by TLC on silica gel plates and continued until after maleic anhydride disappeared. Visual contrast, UV and iodine staining can all be used to check TLC plates.

In step B, the crude PEG di-maleate produced in step A is combined with Dithiothreitol (DTT) and N, N, N ', N' -Tetramethylethylenediamine (TEMED) (with water added if flow is required) and the mixture is stirred at 70 ℃. The reaction was complete within 30 minutes as indicated by the rapid increase in viscosity. If more or less than the optimum amount of DTT is used, the molecular weight of the product will be reduced. The molecular weight of the product can also be reduced, if desired, by replacing TEMED with a less efficient tertiary amine base such as TEA.

In step C, sufficient water is added to the reaction mixture to reduce viscosity, and 0.1mol N-hydroxysuccinimide (NHS) and 1.05mol hexadecylamine per mol carboxylic acid groups in the polymer are added (this amount NHS appears to optimally minimize the extent of side reactions). Then, an excess of N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) (1.4 mol EDC per mol of carboxylic acid groups) was added in portions, with additional water added as necessary to maintain stirring. The pH of the reaction mixture is maintained above 7 and preferably between 9 and 11 to optimize the reactivity of the alkylamine. Containing dodecylamine, the reaction can be carried out at about 40-45 ℃, whereas containing octadecylamine, the temperature is about 55-57 ℃. The reaction was monitored by TCL until a constant level of residual alkylamine was observed, which was typically after overnight reaction.

The reaction mixture was acidified to a pH of about 3.0 to about 4.5 and stirred at room temperature for about 24 hours to destroy unreacted EDC, then titrated to pH7.0 using 1N NaOH. The final reaction mixture was centrifuged at about 800Xg for 1 to 3 hours to remove solid contaminants and by-products.

After centrifugation, the mixture was purified on a GPC column (Toyopearl)^TM、Sephadex^TM、Sephacryl^TM、Biogel^TMEtc.) the supernatant can be chromatographed. However, pi polymers are amphiphilic substances and will exhibit affinity for most GPC column packings, which complicates contaminant removal. Alternatively, the column may be a macroporous hydrophobic interaction column (e.g., TOYOPEARL)^TMPhenyl650C, tosoh Biosciences, Montgomeryville, PA, u.s.a.), eluting with a methanol/water gradient. Preferably, the reaction mixture is dialyzed against several changes of acidified and neutral water to remove low molecular weight starting materials and reaction byproducts.

The reaction mixture may also be extracted with butanone, isopropanol, butanol, or other polar organic solvents to remove organic impurities, but substantial amounts of the amphiphilic polymer are lost in the extraction solvent. Preferably, the reaction mixture is subjected to ultrafiltration using a suitable membrane to separate the product into molecular weight fractions, such as 5kDa to 10 kDa; 10kDa to 30 kDa; 30kDa to 50kDa, etc., depending on the cut-off (cutoff) of the used filtration membrane. Aqueous solutions of the polymer may be dead-end filtered to produce a sterile or virus-free solution, depending on the choice of filtration membrane or media.

2. Synthesis of pi-polymers

Example 1: PEG-bis (alkylamidosuccinyl) disulfide medium molecular weight polymer (C16-. pi. -Polymer A)

Polyethylene glycol (PEG-1500, Sigma Chemical Co.) was dried in vacuo at 80 ℃ until bubbles cease to form (8-12 hours, depending on the quality of the PEG). The dried PEG can be stored for long periods in a dry environment under argon.

The dried PEG was melted on an oil bath under argon and maleic anhydride (2 moles per mole of PEG, corrected for impurities) was added gradually with stirring. The mixture was stirred under argon at 90 ℃. Because maleic anhydride tends to sublime, the headspace is minimized and the entire reaction vessel is maintained at reaction temperature. Any condensed maleic anhydride on the walls of the vessel is scraped back into the reaction mixture. The progress of the reaction was monitored by TLC on silica gel plates using ethanol and hexane as solvents, respectively, for UV observation and iodine staining. The reaction was continued for one hour after the disappearance of the maleic anhydride.

The crude PEG dimaleate was diluted with two volumes of water. Then, with stirring, dithiothreitol (DTT, 1.01 equivalents per equivalent of PEG) and an aqueous solution of N, N' -tetramethylethylenediamine (TEMED, 1.02 equivalents) (2 volumes water per volume of TEMED) were added to the reaction mixture. The reaction was stirred at 70 ℃ under argon for 2.5hr, left at room temperature overnight, and then stirred again at 70 ℃ for 2 hours. TLC monitored the reaction and judged complete after complete disappearance of DTT.

Water was added to the above reaction mixture to reduce viscosity until the mixture could be stirred (approximately 25% solids), the mixture was stirred under argon at 65 ℃, and N-hydroxysuccinimide (0.1 moles per mole of carboxylic acid groups in the PEG-dimaleate-DTT polymer) was added followed by hexadecylamine (1.05 moles per mole of carboxylic acid groups in the polymer) and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide (EDC, 0.56 moles per mole of carboxylic acid groups in the polymer). The mixture was stirred under argon for 1 hour and a second portion of EDC (0.56 moles per mole of carboxylic acid groups in the polymer) was added. After another hour, a third portion of EDC (0.28 moles per mole of carboxylic acid groups in the polymer, for a total of 1.4 moles per mole of carboxylic acid) was further added to compensate for the loss of EDC hydrolysis. When the added solids made the suspension difficult to stir, additional water was added as needed to maintain fluidity and the pH was maintained between 8 and 10 by the addition of 1N NaOH when needed. The mixture was stirred at 65 ℃ under argon overnight, monitored by TLC (silica, ethanol) until the alkylamine appeared to reach a stable concentration, and then stirred for an additional 4 hours. The reaction mixture was then acidified with 1N HCl to pH about 4.5, stirred for 24 hours to destroy unreacted EDC, and adjusted to pH7.0 by dropwise addition of 1N NaOH. Containing dodecylamine, the reaction is carried out at about 40-45 c, whereas with octadecylamine, the temperature is preferably 55-57 c.

The mixture was transferred to a centrifuge tube and spun in a bench top centrifuge at approximately 800Xg for 2 hours to separate the residual solids. After centrifugation, the reaction mixture was extracted with isopropanol to remove organic impurities. Ultrafiltration is preferred as an alternative to isopropanol extraction.

By this method, the following amino compounds were attached to the polymer:

example 1 a: undecane amine

Example 1 b: octadecaneamine

Example 1 c: 4-nonyl benzylamine

Example 1 d: 3- [ (4-phenoxy) phenyl ] propanamine

Example 2: PEG-bis (alkylamidosuccinyl) disulfide high molecular weight polymers

The procedure outlined in example 1 was followed except that 0.55mol DTT and 0.55mol TEMED per mole maleic anhydride were used. Because the viscosity increases rapidly, vigorous stirring is required. Indicating that most of the reaction was completed in 5-10 minutes, and then slowly completed through the next 4 hours as the temperature was increased from 55 c to 80 c.

Example 3: PEG-bis (alkylamidosuccinyl) disulfide polymers

The procedure outlined in example 1 was followed except that 1.5mol dodecylamine was used per mole of carboxylic acid groups in the polymer. N-hydroxysuccinimide (NHS, 1.0mol per mol carboxylic acid groups) and 1, 1' -carbonyldiimidazole (CDI, 3.0mol per mol carboxylic acid groups) were added, followed by stirring reaction at 80 ℃ for 4 hours and post-treatment as described above.

By this method, the following amino compounds were attached to the polymer:

example 3 a: undecane amine

Example 3 b: tetradecylamine

Example 3 c: octadecaneamine

Example 3 d: dehydroabietylamine

Example 3 e: cholesterol 2-aminoethylether

Example 3 f: 10-phenoxy decylamine

Example 3 g: sebacic acid hydrazide

Example 3 h: oleic acid hydrazide

Example 3 i: dehydroabietic acid hydrazide

Example 3 j: cholic acid hydrazide

Example 3 k: palmitic acid hydrazide

Example 4: PEG- (alkylamido succinate) copolymer

A solution of PEG (6.66mmol) and triethylamine (2.32ml, 16.65mmol) in anhydrous diethyl ether (10ml) was cooled at 0 ℃ under argon and treated dropwise with methanesulfonyl chloride (1.03ml, 13.32 mmol). At 0 ℃, stirring 1 continued to disappear, then at room temperature for 2 hours. Evaporating the ether and adding the anhydrous propaneKetone (15ml) was added to the residue to precipitate triethylamine hydrochloride, which was filtered from the solution. The filtrate was treated with lithium bromide (2.31g, 26.64mmol) and heated to reflux for 20 h. The mixture was then diluted with hexane and passed over Celite^TMA (0.5cm) pad of silica (3cm) was filtered and eluted with hexane. The filtrate was dried, filtered and evaporated to leave α, ω -dibromo-PEG, an oil.

By Godjoian et al, tetrahedron Letters, 37: 433-6(1996) alpha, omega-dibromo-PEG was reacted with one equivalent of 2, 2-dibutyl-4, 5-bis (methoxycarbonyl) -1, 3, 2-dioxastannolane (dioxastannolane). The resulting dimethyltartaric acid-PEG polyether was saponified with KOH in methanol and then amidated with dodecylamine or hexadecylamine as described above in examples 1 and 3, or with the amines in examples 3a-3 k.

Example 5: copolymerization of PEG and EDTA dianhydride

Anhydrous PEG was reacted with 1, 2-ethylenediaminetetraacetic dianhydride by the method described in example 1, and then amidated with dodecylamine as in example 1 or hexadecylamine as in example 3, or with the amines in examples 3a-3 k.

In the same way, the following dianhydrides were copolymerized with PEG and subsequently amidated:

example 5 a: naphthalene tetracarboxylic acid dianhydride

Example 5 b: perylene tetracarboxylic dianhydrides

Example 5 c: benzophenone tetracarboxylic dianhydride

Example 5 d: 4, 4' - (Hexafluoroisopropylidene) diphthalic anhydrides

Example 5 e: butane tetracarboxylic acid dianhydride

Example 5 f: bicyclo (2, 2, 2) oct-7-ene-2, 3, 5, 6-tetracarboxylic acid dianhydride

Example 5 g: diethylenetetraminepentaacetic dianhydride

Example 5 h: 3, 4, 3 ', 4' -diphenylsulfone tetracarboxylic dianhydride

Example 5 i: 3, 4, 3 ', 4' -Diphenyl Ether Tetracarboxylic dianhydride

Example 5 j: pyromellitic dianhydride

Example 6A: PEG-diamine copolymers with pendant thioethers

Using the same procedure used for DTT in example 1, PEG dimaleate, as prepared in example 1, was reacted with dodecanethiol (two equivalents per equivalent of PEG dimaleate). When no polymerization occurs, no dilution is required and the reaction is carried out in molten PEG-dimaleate. TEMED catalyst was added followed by mercaptan. After the reaction, the starting material disappeared and was monitored by TLC. Temperatures at which the loss of alkylmercaptan by evaporation is significant (up to about 100 ℃) can be used at most. A slight excess of alkyl mercaptan may be used to fully saturate the maleic acid groups. By charging with nitrogen and argon and/or heating in vacuo, at the end of the reaction, the excess alkylthiol is driven off until no alkylthiol can be detected by odor or by TLC.

By this method, the following thiols were attached to PEG dimaleate:

example 6 Aa: mercapto succinic acid di-t-butyl ester

Example 6 Ab: tetradecanethiol

Example 6 Ac: hexadecane thiol

Example 6 Ad: 2-mercaptoethanesulfonic acid

Example 6 Ae: 3-mercaptopropanesulfonic acid

Example 6 Af: 6-Mercaptohexanoic acid t-butyl ester

Example 6 Ag: 4-Mercaptobenzoic acid-t-butyl ester

Example 6 Ah: thioglycolic acid-t-butyl ester

Example 6 Ai: 4- (t-Butoxycarbonylamino) butanethiol

Example 6 Aj: 3- (t-Butoxycarbonylamino) benzylthiol

Example 6 Ak: 4-decyl benzyl mercaptan

Thiols with reactive functional groups are suitable for attaching the C chain, and/or the reactive functional groups may serve as the point of attachment (X) to the targeting moiety.

Example 6B: copolymers of PEG-diamines and side chain thioethers

The thiol adduct obtained in example 6A was amidated with 1, 4-diaminobutane (one equivalent of diamine per two equivalents of COOH groups) using the same procedure as for dodecylamine of example 1, diluted with water as necessary to maintain the fluidity of the reaction mixture. Additional aliquots of EDC were added as needed to ensure complete polymerization. By this method, the thiol adducts of examples 6A and 6Aa to 6Ak were converted to PEG-diaminobutane polyamide.

By this method, the following diamines can be converted to PEG polyamides (BOC ═ t-butoxycarbonyl):

example 6 Ba: 2- (O-BOC) -1, 3-diamino-2-propanol

Example 6 Bb: n ', N' -Bis (BOC) hexaethylenetetramine

Example 6 Bc: n ', N' -Bis (BOC) spermine

Example 6 Bd: n' -BOC spermidine

Example 6 Be: n ', N' '' -tris (BOC) pentaethylenehexamine

Example 6 Bf: agmatine salt

Example 6 Bg: lysine t-butyl ester

Example 6 Bh: 1, 6-diaminohexane

Example 6 Bi: 1, 4-phenylenediamine

Example 6 Bj: 1, 3-phenylenediamines

Example 6 Bk: 1, 4-diaminobutane-2, 3-diol acetonide

Example 7: PEG-bis (alkyl succinate) disulfide

The 2, 3-di-O-hexadecyl ether of DTT (m-2, 3-dihexadecyloxutane-1, 4-dithiol) was prepared by modification of the method of S.Sasaki et al, chem.pharm.Bull.33(10):4247-4266 (1985). It was added to PEG-dimaleate by the method of example 1.

By this method, the following ether dithiols were attached to the PEG polymer:

example 7 a: m-2, 3-di (n-butoxy) butane-1, 4-dithiol

Example 7 b: m-2, 3-bis (4-nonylphenylmethoxy) butane-1, 4-dithiol

Example 7 c: m-2, 3-bis (biphenyl-4-methoxy) butane-1, 4-dithiol

Example 7 d: 4, 6-bis (decyloxy) benzene-1, 3-dimethanethiol

Example 7 e: 4, 5-di (decyloxy) benzene-1, 2-dimethanethiol

Example 7 f: 3, 4-di (decyloxy) thiophene-2, 5-dimethanethiol

Example 8A: substituted PEG succinates

The procedure of example 1 was followed except that 2-dodecen-1-ylsuccinic anhydride was used in place of maleic anhydride. The dodecenyl substituent provides the pendant C chain in the final polymer.

By this method, the following substituted succinic anhydrides were esterified with PEG:

example 8 Aa: isobutylene succinic anhydride

Example 8 Ab: 2-octen-1-ylsuccinic anhydride

Example 8 Ac: octadecylsuccinic anhydride

Example 8 Ad: 3-oxabicyclo-hexane-2, 4-dione

Example 8 Ae: cyclohexane dicarboxylic acid anhydrides

Example 8 Af: phthalic anhydride

Example 8 Ag: 4-decylphthalic anhydride

Example 8 Ah: hexahydromethylphthalic anhydride

Example 8 Ai: tetrahydrophthalic anhydride

Example 8 Aj: norbornene dicarboxylic acid anhydrides

Example 8 Ak: cantharidin

Example 8 Al: bicyclo-octene dicarboxylic acid anhydride

Example 8 Am: exo-3, 6-epoxy-1, 2, 3, 6-tetrahydrophthalic anhydride

Example 8 An: s-acetylmercaptosuccinic anhydride

Example 8B: PEG-bis (alkylamidosuccinyl) disulfide with pendant alkyl groups

Substituted PEG succinates obtained as described in examples 8A and 8Aa to 8An were reacted with DTT according to the procedure of example 1.

By this method, the following dithiols were reacted with any substituted PEG succinates obtained as described in examples 8A and 8Aa to 8 An:

example 8 Ba: ethane-1, 2-dithiol

Example 8 Bb: propane-1, 3-dithiol

Example 8 Bc: butane-1, 4-dithiol

Example 8 Bd: pentane-1, 5-dithiol

Example 8 Be: hexane-1, 6-dithiol

Example 8 Bf: 1, 4-benzenedithiol

Example 8 Bg: 1, 3-benzenedithiol

Example 8 Bh: 1, 4-benzenedimethylthiol

Example 8 Bi: 1, 3-benzenedimethylthiol

Example 8 Bj: 1, 2-benzenedimethylthiol

Example 8C: copolymers of PEG-diamines with pendant alkyl groups

The substituted PEG succinate obtained as described in example 8A was copolymerized with 1, 4-diaminobutane according to the method of example 6B.

By this method, the following diamines were copolymerized with any of the substituted PEG succinates obtained as described in examples 8A and 8Aa to 8 An:

example 8 Ca: 2O-BOC1, 3-diamino-2-propanol

Example 8 Cb: n ', N' -Bis (BOC) hexaethylenetetramine

Example 8 Cc: n ', N' -Bis (BOC) spermine

Example 8 Cd: n' -BOC spermidine

Example 8 Ce: n ', N' -tris (BOC) pentaethylenehexamine

Example 8 Cf: agmatine salt

Example 8 Cg: lysine t-butyl ester

Example 8 Ch: 1, 6-diaminohexane

Example 8 Ci: 1, 4-phenylenediamine

Example 8 Cj: 1, 3-phenylenediamines

Example 8 Ck: 1, 4-diaminobutane-2, 3-diol acetonide

Example 9: PEG transesterification using substituted acids

PEG xylene sulfonate: to 1mol of PEG (dissolved in DMF or its melt) was added 2.1mol of p-toluenesulfonyl chloride (5% molar excess) under argon and stirred. To this reaction mixture was added 2.2mol of Tetramethylethylenediamine (TEMED). Subsequently, the reaction was carried out at 45 ℃ for 2 hours. The product was isolated by TLC using ethyl acetate, toluene or ethanol as TLC solvent. The PEG xylene sulfonate was extracted from the reaction mixture using toluene. Other sulfonylating agents, such as methanesulfonyl chloride (see example 4), trifluoromethanesulfonic anhydride, or trifluoromethanesulfonyl chloride (tresyl chloride) may also be used in place of toluenesulfonyl chloride (see U.S. patent application 10/397332, publication No. 20040006051).

Polyesterification of PEG ditosylate: to 1mol of molten PEG ditosylate are added 1mol of S, S' -didecyl-m-2, 3-dimercaptosuccinic acid and 2mol of TEMED under argon and stirred. DMF was added as necessary to maintain fluidity. The reaction mixture was heated to 80 ℃ and stirred for 24 hours or until TLC indicated completion.

Example 10: medium molecular weight polymer PEG-bis (succinyl) -bis- (O-acylated) thioether (C16-. pi. -Polymer B)

PEG-dimaleate (10.24g, 6.1mmol) prepared as in example 1 was placed in a 125ml dry flask and heated to 70 ℃ under argon to melt the PEG-dimaleate. To this molten mass was added water (10mL) and a solution of DTT (0.961g, 6.168mmol) and TEMED (0.723g, 6.166 mmol) in water (3mL) with stirring. The solution was stirred at 70 ℃ for about 4 hours. Vacuum dehydration gave a polymer solid in about 90% yield.

The dried polymer (5g, 2.7mmol) was heated to 70-90 ℃ under argon to melt it and TEMED (0.635g, 5.5mmol) was added. Palmitoyl chloride (1.689g, 5.5mmol) was added with stirring, and the mixture was stirred under argon overnight. (the ratio of polymer to acid chloride can be varied to achieve a degree of substitution of 0-100% of stoichiometry.) Water is added to the reaction mixture to isolate "C16-. pi. -Polymer B".

The following acids were esterified by this method using the hydroxyl group of the bis (succinyl) PEG-DTT copolymer:

example 10 a: oleic acid

Example 10 b: cholesterol succinate

Example 10 c: biphenyl-4-carboxylic acid

Example 10 d: 4-octyl phenyl acetic acid

Example 10 e: hexadec-6-ynoic acid

As an alternative to the use of acid halides, it is also possible to activate the hydroxyl group derived from DTT in the pi-polymer and couple directly to the carboxylic acid using 1, 3-bis (2, 2-dimethyl-1, 3-dioxolan-4-ylmethyl) carbodiimide (BDDC); see, Handbook of Reagents for Organic Synthesis, Reagents for Glycoside, Nucleotide, and Peptide Synthesis, Ed. David Crich, Wiley, 2005p107-108 and references therein.

Example 11: dimaleate of C16-pi-Polymer A

Polymer A dimaleate was prepared by the reaction of maleic anhydride with the polymer A hydroxyl groups. Thiol-containing ligands can be added to the polymer using the introduced reactive double bonds. The ratio of polymer a to maleic anhydride can be varied to obtain varying degrees of substitution within 0-100% of full stoichiometric esterification.

C16-. pi. -Polymer A (2g) and maleic anhydride (0.85g) were ground in a dry mortar and transferred to a 50mL round-bottomed flask. The flask was heated to 90 ℃ under argon and stirred for 2-3 hours. Subsequently, the solid reaction mixture was transferred with water into a dialysis bag (molecular weight cut-off 3.5kDa) and dialyzed against water to remove excess maleic acid and low molecular weight by-products. Subsequently, the dialysis retentate was removed from the dialysis bag and dried to constant weight at 60 ℃ to obtain C16-. pi. -Polymer A dimaleate (1.79 g).

Example 12: cysteine adduct of C16-pi-Polymer A dimaleate

To water (5mL) was added powdered dimaleate of C16- π -polymer A (example 11) (253mg) and the mixture was stirred vigorously. Cysteine (24mg) and TEMED (30.5uL) were added to the reaction mixture, and the mixture was stirred at room temperature under argon. The progress of the reaction was monitored using TLC (silica gel plate, n-butanol-acetic acid-water, 3:1:1) and ninhydrin test. The reaction mixture showed ninhydrin positive spots that co-migrated with the polymer. Cysteine also gave a ninhydrin positive spot, whereas the starting polymer did not show colour for ninhydrin.

3. Solubilization of insoluble or sparingly soluble substances using pi-polymers

Example 1: solubilized dye

In a separate container (Flexexcel (TM) clear polypropylene weigh dish, model WB2.5, from Allexcel, Inc., West Haven, CT), to a 1.0mL aliquot of a 50mg/mL aqueous solution of PEG 1500-succinyl-DTT-bis-C16-amide copolymer (C16-Polymer A, example 1) that was centrifuged to remove insoluble material (without additional purification) was added excess dye eosin Y, dichlorofluorescein, and Sudan IV, and the components were stirred together to form a paste. The bottom of the container was then placed on the floor of a small ultrasonic jewelry cleaning tank using water-resistant double-sided adhesive tape. Just enough water was added to the clean tank to immerse the weigh dish at about 1/3 a height. Sonication was performed in steps of 15 minutes, 5 minutes each. The liquid was transferred to a centrifuge tube and centrifuged twice for 30 minutes in a bench top centrifuge to remove undissolved dye particles. The supernatant was transferred to a clean centrifuge tube and re-centrifuged to remove entrained solids. A suspension of an equal amount of dye in distilled water (same amount as the polymer solution) was treated in the same manner as a control. The resulting solution was spotted on (25ul) TLC plates to form rings originating from the droplets. Comparing the intensity of the spot with the intensity of a spot formed from a standard dye solution prepared from ethanol or ethanol/water to determine an approximate concentration; the spots are shown in figure 1. The solubility of the dye in water is determined by dissolving an appropriate amount of the dye in 1 liter or more of deionized water (unbuffered) at room temperature, and further adding (i.e., dropping) water as needed to obtain a saturated solution.

Relative to in H₂O (Sudan IV is insoluble at neutral pH) at a concentration of 0.000mg/ml and Sudan IV at a concentration of about 0.2mg/ml in 50mg/ml polymer. Relative to in H₂O at a concentration of 0.010mg/ml and dichlorofluorescein at a concentration of about 5mg/ml in 50mg/ml polymer. Relative to in H₂O at a concentration of 0.007mg/ml and eosin Y at a concentration of about 5mg/ml in 50mg/ml polymer. The payload rates (drug loading per unit amount of polymer, g/g) for sudan IV, dichlorofluorescein, and eosin Y were calculated to be approximately 1:250, 1:10, and 1:10, respectively.

For polar compounds with similar physicochemical properties as the active drug substance, the effective loading rate of 1:10 is higher than that of lipidPlastids, cyclodextrins, Cremophor^TMOr the payload rate that can be achieved with detergents or other solubilizing systems. Eosin Y is a highly efficient photoactive singlet oxygen generator, and such concentrated solutions of eosin Y prepared from the polymer of example 1 can be expected to have pharmacological activity as photoactive cytotoxic agents.

The change in fluorescence spectrum of dichlorofluorescein in the polymer solution (red yellow/orange) versus in water (yellow green) was visually observable and indicates that the dye is not in an aqueous environment, but is microencapsulated in the organic environment of the self-assembled polymer particle core. Changes in fluorescence spectra have indeed been used in methods to determine changes in the polarity of the microenvironment (e.g., "lipid probes"). The color of the sudan IV polymer solution was reddish brown relative to a brown powder when in ethanol solution and suspended in water. Eosin Y showed no significant visual change (pink in water and pink in polymer solution).

Example 2: solubilizing drug related substances

Rhodopsin, amphotericin B, camptothecin and doxorubicin were selected as representative sparingly soluble Active Pharmaceutical Ingredients (API). Amphotericin B is used in liposomal formulations as an injectable antifungal, while camptothecin and doxorubicin are anticancer agents. Rhodopsin is a DNA intercalating dye with pharmaceutical potential, while eosin Y is a photoactive singlet oxygen generator with potential use in photodynamic therapy. Each API was solubilized in water using C16- π -Polymer A, C18- π -Polymer B and/or C16- π -Polymer A-folate conjugates (see below). Solubilization was demonstrated by spotting solubilized API and unsolubilized controls on TLC plates as described above for the dyes.

The dried polymer was reconstituted with water, heated, stirred and subjected to the necessary sonication. When the solution viscosity is too high, dilution is performed. The C16-. pi. -polymer A used was 10% w/v, the folic acid C16-. pi. -polymer A used was 5% w/v, and the C18-. pi. -polymer B used was 2% w/v.

The drug (20mg) was added directly to 1mL of the polymer solution, resulting in mass ratios of C16- π -Polymer A, folated C16- π -Polymer A and C18- π -Polymer B to the API (except for doxorubicin, see below) of 5:1, 2.5:1 and 1:1, respectively. The mixture was sonicated at low power for 1 hour, followed by centrifugation twice at 2000Xg to remove undissolved solids. The amount of solid pellets was not significant. Spotting the solution onto a silica gel TLC plate showed that the drug was solubilized, with migration slower than the solvent front (figure 2).

Doxorubicin hydrochloride was combined with the above polymer at a mass ratio of C16-pi-polymer a to doxorubicin hydrochloride of 10:1, or at a mass ratio of folic acid C16-pi-polymer a to doxorubicin of 5:1, followed by addition of sufficient 3M sodium acetate to neutralize the doxorubicin hydrochloride. The mixture was shaken vigorously for 24 hours and then centrifuged twice at 2000Xg to remove undissolved solids. The amount of solid pellets was not significant.

The mass ratio of solubilized API to polymer is shown in table 1. No attempt was made to maximize polymer loading, and therefore these ratios represent a lower limit on the amount of API that can be solubilized by the polymer.

50ul samples of each solution were spotted onto Bakerflex^TMSilica gel TLC plate and allowed to diffuse. The aqueous solution forms the outer boundary of the circle and the migration of the polymer with the microencapsulated material forms the inner circle (fig. 2). In all cases, there was very little API at the peripheral edge of the fully aqueous zone, indicating successful solubilization and minimal leakage of the microencapsulated material.

Table 1: solubilization of API

Mass ratio of polymer to substrate

	C16- π -Polymer A10% w/v	Folation C16- π -Polymer A5% w/v	C18- π -Polymer B2% w/v
				Rhodopsin (purpurin)	5:1	2.5:1	Not tested
Camptothecin	5:1	2.5:1	Not tested
				Amphotericin B	5:1	2.5:1	Not tested
Adriamycin	10:1	5:1	Not tested
				Eosin Y	Not tested	Not tested	1:1

4. Biocompatibility of pi-polymers

Example 1: applicability to topical emollients, creams or ointments

The inventors rubbed the slack wax concentrate of the polymer of example 1 onto the skin in the wrist joint and observed its absorption. The absorption of the substance is similar to a pharmacological emulsion containing wax, with a slight softening of the area. No immediate or delayed allergic reactions, such as redness, rash or itching, based on this single topical administration were observed.

These polymers are mostly hygroscopic waxes at room temperature and have an expected mp of about 45 ℃ to 60 ℃ or higher, depending on the composition. Polymers prepared from lower molecular weight PEGs are even liquid at room temperature. Some polymers are solid at room temperature and melt at body temperature. Thus, the properties of these pi-polymers make them excellent substrates for the manufacture of lotions, creams, ointments, emollients and other modes of administration, either by themselves or in admixture with a wide variety of substances, including active pharmaceutical agents.

Example 2: suitability for parenteral administration

An aqueous solution of the polymer of example 1 was prepared in phosphate buffer and then filtered through a 0.22um filter into a sterile tube.

A maximum tolerated dose protocol was used in which CD-1 mice were injected intravenously with up to 5% w/v aqueous polymer solution at the tail in an amount of 10ml per kg body weight. Mice were observed continuously for 12 hours, then every 2 hours, and grouped according thereto until 48 to 72 hours. Blood samples were collected and analyzed. Some mice were sacrificed and first subjected to gross histological examination. The selected sections were then subjected to microscopic histological observations.

No significant difference in blood chemistry was found between the control and treated mice. No significant differences or injury was found in gross histological examination of multiple organs including heart, lung, kidney, spleen, liver, intestine, stomach, bladder, skin, muscle, bone, brain and lymph nodes compared to control animals. The same results were observed for studies on multiple samples from different animal groups. No significant differences were found in the cell tissue structure of the examined tissue. Some kidney tissues show reduced shedding with time of exposure to the polymer. This indicates that the shedding is temporary and can return to normal over time.

In summary, the polymers are safe for medical use as pharmaceutical agents in injectable and other parenteral formulations. It is reasonable to expect that the compounds will be safe for use in oral liquids, caplets and tablets, nasal sprays, oral/bronchial aerosols, sublingual administration, dermal creams/lotions/patches, eye drops, other topical routes of administration and other routes of administration.

5. Attachment of targeting moieties to pi-polymers

Example 1: linking galactosamine to C16-pi-polymer B by amide bond formation

Galactosamine (GA) targets the hepatocyte asialoglycoprotein receptor (ASGPR), while polymers with covalently linked galactosamine are transported to the liver; see l.seymour et al, "hepatodrug Targeting: phase I Evaluation of Polymer-Bound Doxorubicin (liver drug targeting: Phase I Evaluation of Polymer-Bound Doxorubicin), "J Clin. 1668-1676(2002) and references therein.

C16-. pi. -Polymer B (example 10 of the synthesis above) (461mg, 0.2mmol equivalents COOH per repeating unit) was dispersed in 14mL of water, and EDC HCl (0.485mmol) and N-hydroxysuccinimide (0.464mmol) were added to this dispersion. The mixture was stirred at room temperature for 15 minutes and a solution of galactosamine hydrochloride (0.386mmol) and TEMED (0.387mmol) in 1mL of water was added. The solution was stirred and developed using 1-butanol-acetic acid-water (3:1:1) to perform TLC on silica gel and the reaction was monitored. An additional amount of TEMED (0.079mmol), NHS (0.078mmol) and EDC HCl (0.193mmol) was added to drive the reaction to completion. When TLC showed steady state GA consumption, the reaction mixture was dialyzed (membrane cutoff 3500Da) against 3x1000ml deionized water to remove low molecular weight reactants and byproducts. The dialysis retentate was removed and dried at 60 ℃ to constant weight (348 mg).

TLC of the product showed no free GA (ninhydrin negative). A sample of the product was hydrolyzed at 100 ℃ using 6N HCl to hydrolyze the bound GA. TLC analysis showed the presence of GA (ninhydrin positive) and Rf was the same as reference GA.

Example 2: attachment of Folic acid to C18-Pi-Polymer A

BDDC (2.44g, 8.56mmol) was weighed out in a 125ml round-bottomed flask purged with a stream of argon (BDDC is very viscous and has a consistency similar to honey and is difficult to handle). The flask was charged with C18-. pi. -Polymer A (10g, 4.28mmol), the mixture was heated to 70 ℃ and the reactants were stirred together for about 30 minutes. Sufficient THF was added after addition of folic acid (3g) to make stirring possible. The reaction was stirred at 40-70 ℃ overnight, with moisture ingress prevented. The THF was then evaporated and water (80mL) was added and the mixture was stirred at 50 ℃ for an additional 2 hours. After cooling to room temperature, the mixture was transferred to a dialysis tubing area with a molecular weight cut-off of 3500Da and dialyzed against 0.1N HCl (2x2000ml), water (2000ml), 5% sodium carbonate (2x2000ml) and water (4x2000ml) to remove unreacted reagents and by-products. The bright yellow-orange dialysis retentate was removed. A portion was evaporated to constant weight to determine the solid concentration and used for the solubilization assay described above.

Example 3: attachment of N-acetylneuraminic acid (NANA) to C16-. pi. -Polymer B

Neuraminic acid derivatives hold promise for targeting moieties for influenza viruses because both hemagglutinin and neuraminidase coat proteins are known to bind sialic acid.

BDDC (2.44g, 8.56mmol) was combined with C18-. pi. -Polymer A (10g, 4.28mmol) and heated to 70 ℃ and co-stirred under argon for about 30 minutes. N-acetylneuraminic acid (3g) was added followed by THF as needed to maintain fluidity. The reaction was stirred at 40-70 ℃ overnight, with moisture ingress prevented. Water (80mL) was added and the mixture was stirred at 50 ℃ for an additional 2 hours. After cooling to room temperature, the mixture was dialyzed against a membrane with a molecular weight cut-off of 3.5kDa, and 0.1N HCl, 5% NaHCO3 and water (2 × 2000ml each).

Example 4: attachment of beta-O-methylneuraminic acid (MNA) to C16-pi-Polymer B

C16-. pi. -Polymer B (43. mu. mol based on COOH in 1mL of water) was mixed together with 40. mu. mol of neuraminic acid β -methyl glycoside (Toronto Research Chemicals) and added successively to 40. mu. mol of NHS in 0.1mL of water and 40. mu. mol of EDC hydrochloride in 0.1mL of water. The reaction mixture was shaken at room temperature for 48 hours and analyzed by TLC on silica gel using isopropanol-ethyl acetate-water (4:3: 2). Examination at 130 ℃ using 0.2% orcinol in 70% sulphuric acid did not result in a colour reaction of the starting polymer, but TLC of the reaction mixture gave a purple spot co-migrating with the polymer.

Example 5: attachment of zanamivir to C16-pi-Polymer B

Zanamivir (GG167) is a potent viral neuraminidase inhibitor, and the polymer loaded with this multivalent ligand molecule is an inhibitor of influenza virus replication.

C16-. pi. -Polymer B (920mg) was dispersed in 30mL of water, and EDC HCl (1.2mmol) and N-hydroxysuccinimide (1.1mmol) were added to the dispersion. The mixture was stirred at room temperature for 20 minutes and a solution of the trifluoroacetate salt of 5-acetamido-7- (6 '-aminohexyl) -carbamoyloxy-4-guanidino-2, 3, 4, 5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranone keto acid (5-acetamido-7- (6' -aminohexyl) -carbamyoxy-4-guanidono-2, 3, 4, 5-tetradeoxy-D-glycero-D-galacto-D-galactoto-non-2-enopyranosonic acid) (U.S. Pat. Nos. 6,242,582 and 6,680,054) (0.39g, 0.67mmol) and TEMED (0.67mmol) dissolved in 1ml of water was added. The solution was stirred at room temperature and the reaction was monitored by TLC. The reaction mixture was dialyzed using a 3500kDa cutoff membrane and 3x1000ml deionized water to remove low molecular weight reactants and byproducts. The dialysis retentate was removed and dried to constant weight at 60 ℃. Sugar addition levels were determined by colorimetric assay for guanidine groups (can.j. chem., 36:1541 (1958)). Neuraminidase detection can be carried out according to the method of Potier et al, anal. biochem., 29287 (1979).

Example 6: attachment of Fab fragments to C16- π -Polymer A dimaleate

Single chain variable antibody (scFv) against the surface glycoprotein high molecular weight melanoma-associated antigen (HMW-MAA) targets melanoma cells; see F.Martin et al, J.virology, 73:6923-6929 (1999).

The Disulfide bonds in this antibody fragment were reduced using immobilized TCEP Disulfide Reducing Gel (ImmobilitdCEP Disufide Reducing Gel) (Pierce Biotech, Rockford, Ill.) according to the manufacturer's protocol and reacted with C16-. pi. -Polymer A dimaleate by the method of example 12 in the synthetic procedures section.

Claims (31)

1. A comb polymer consisting essentially of the structure:

the polymer comprises a backbone formed of alternating branch point moieties B and hydrophilic water-soluble polymer segments a; and having hydrophobic side chains C attached to each branch point moiety, wherein each side chain C is independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers; wherein n ranges from 3 to 100; the average range of p is 1< p.ltoreq.4.

2. The polymer of claim 1, wherein p has an average range of 2 to 4.

3. The polymer of claim 1, wherein p has an average range of 1.5. ltoreq. p.ltoreq.2.

4. The polymer of claim 1, further comprising one or more reactive functional groups X associated with each branch point moiety and consisting essentially of the structure:

wherein r has an average range of 1 to 4.

5. A polymer as claimed in any one of claims 1 to 4 wherein the water soluble polymer segment A is selected from polyethylene glycol, polypropylene glycol, polyethylene imine, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides and copolymers thereof.

6. The polymer of claim 5, wherein the polymer segment A is selected from the group consisting of polyethylene glycol, polypropylene glycol, and copolymers thereof.

7. The polymer of claim 6, wherein the polymer segment A is polyethylene glycol.

8. The polymer of claim 7, wherein the average length of polymer segment A is between 4 and 700 monomer units.

9. The polymer of claim 4, having the structure,

wherein m is 4-700, and Y 'are independently selected from R, OR, COOR, SR, NHR, NRR', ONHR, NHOR, NRNH₂NHNHR, NRNHR ', and NHNRR ', wherein R and R ' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

10. The polymer of claim 4, having the structure,

wherein m is 4-700, and Y 'are independently selected from R, COR, COOR, CONHR, CONRR', CONHOR, CONRNH₂CONHNHR, CONRNHR ' and CONHNRR ', wherein R and R ' are independently selected from linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides and polymers.

11. The polymer of claim 1, having the structure,

wherein m is 4-700, and Y 'are independently selected from R, OR, COOR, SR, NHR, NRR', ONHR, NHOR, NRNH₂NHNHR, NRNHR ', and NHNRR ', wherein R and R ' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

12. The polymer of claim 3, having the structure,

wherein m is 4-700, and Y 'are independently selected from R, OR, COOR, SR, NHR, NRR' ONHR, NHOR, NRNH₂NHNHR, NRNHR ', and NHNRR ', wherein R and R ' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

13. The polymer of claim 3, having the structure,

wherein m is 4-700, and Y 'are independently selected from R, OR, COOR, SR, NHR, NRR', ONHR, NHOR, NRNH₂NHNHR, NRNHR ', and NHNRR ', wherein R and R ' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

14. The polymer of claim 3, having the structure

Wherein the moiety D is derived from a diamine having the general structure,

each X is independently a reactive functional group, p is 0 to 4, and m is 4 to 700; wherein R and R' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

15. The polymer of claim 4, having the structure,

wherein m is 4-700, and R' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

16. The polymer of claim 4, having the structure,

wherein m is 4 to 700, L is phenylene, C₂-C₆Alkylene or xylylene, and R' are independently selected from linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides anda polymer.

17. A composition resulting from a chemical reaction of polyethylene glycol with maleic anhydride, which results in substantially complete esterification of the terminal hydroxyl groups of the polyethylene glycol by maleic anhydride, and of the resulting material with dithiothreitol.

18. A composition resulting from a chemical reaction of polypropylene glycol with maleic anhydride, which results in substantially complete esterification of the terminal hydroxyl groups of the polypropylene glycol with maleic anhydride, and of the resulting material with dithiothreitol.

19. A polymer having the structure (I) wherein,

wherein m is 4 to 700 and n is 3 to 100, and wherein in each occurrence of a monomer unit having the structure shown, Y and Y' are independently selected from OH, COOH, SH, NH₂、NHR、ONH₂、NHOH、NHNH₂And NRNH₂Wherein R is selected from C₁To C₅Alkyl group, (CH)₂)_kOH、(CH₂)_kCOOH、(CH₂)_kSH、(CH₂)_kNH₂、(CH₂)_kONH₂、(CH₂)_kNHOH and (CH)₂)_kNHNH₂Wherein k is 2 to 5.

20. A polymer having the structure (I) wherein,

wherein m is 4 to 700 and n is 3 to 1000And wherein in each occurrence of monomer units having the structure shown, Y and Y' are independently selected from OH, COOH, SH, NH₂、NHR、ONH₂、NHOH、NHNH₂And NRNH₂Wherein R is selected from C₁To C₅Alkyl group, (CH)₂)_kOH、(CH₂)_kCOOH、(CH₂)_kSH、(CH₂)_kNH₂、(CH₂)_kONH₂、(CH₂)_kNHOH and (CH)₂)_kNHNH₂Wherein k is 2 to 5; w and W' are O or H respectively₂And, wherein in each occurrence of a monomer unit, Z and Z' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers.

21. A polymer having the structure (I) wherein,

wherein m is 4-700 and Y 'are independently selected from R, OR, COOR, SR, NHR, NRR', ONHR, NHOR, NRNH₂NHNHR, NRNHR ', and NHNRR ', wherein R and R ' are independently selected from the group consisting of linear hydrocarbons optionally substituted with one or more hydrophilic substituents, polycyclic hydrocarbons optionally substituted with one or more hydrophilic substituents, hydrophobic amino acids, peptides, and polymers; and wherein in each occurrence of the monomer unit, W and W' are independently selected from H, -COCH ═ CH₂、-COC(CH₃)＝CH₂、COCH＝CHCO₂H and-COC (CH)₃)＝CHCO₂H。

22. A pharmaceutical composition comprising a polymer as claimed in any one of claims 1 to 9, said pharmaceutical composition further comprising an effective amount of a pharmacologically active agent.

23. A method of increasing the solubility of a substance in an aqueous solvent, the method comprising contacting the substance with the polymer of claim 1 or 2 to form a water-soluble complex of the substance and the polymer.

24. A method of increasing the solubility of a substance in a non-aqueous solvent, the method comprising contacting the substance with the polymer of claim 1 or 2 to form a complex of the substance and the polymer, the complex being soluble in a non-aqueous solvent.

25. The method of claim 23 or 24, wherein the substance is selected from the group consisting of a nutritional agent, a drug, a dye, a nucleic acid complex, and an imaging agent.

26. The method of claim 24, wherein the substance is a drug.

27. A method of generating binding affinity for a biological target in a polymer according to claim 4, the method comprising the step of attaching a targeting moiety to one or more reactive functional groups X on the polymer.

28. The method of claim 27, wherein the biological target is a surface of a cell or virus.

29. The method of claim 28, wherein the targeting moiety is selected from the group consisting of receptor-specific ligands, antibodies, antibody fragments, peptides comprising an RGD amino acid sequence, peptides comprising a YISRG motif, growth factors, sialic acid derivatives, N-acetylneuraminic acid derivatives; folic acid, methotrexate, pteroic acid, estradiol, estriol, testosterone, mannose-6-phosphate, sugar, vitamin, tryptophan, aminoalkyl adamantane, and Fuzeon^TMPRO-542, BMS-488043, sialic acid, 2-deoxy-2, 3-didehydro-N-acetylneureMethionine, 4-guanidino-Neu 5Ac2en (zanamivir), oseltamivir and RWJ-270201.

30. The method of claim 27, wherein the targeting moiety is a monoclonal antibody or antibody fragment.

31. The method of claim 25, wherein the substance is a vitamin.