TW201438741A - Self-assembling amphiphilic polymers as antiviral agents - Google Patents

Abstract

There are provided amphiphilic biodegradable copolymers comprings a hydrophilic backbone with pendant aliphatic groups as the hydrophobic component. The polymers form nanoscale molecular aggregates in aqueous environments, which have hydrophobic interiors that are capable of solubilizing insoluble organic compounds and disrupting viral coat proteins. The polymers optionally feature reactive functional groups that provide attachment points for antibodies, ligands, and other targeting moieties which mediate adherence of the aggregate to a viral target.

Description

Self-aggregating amphiphilic polymer as an antiviral agent

This invention relates to the field of amphiphilic polymers, and in particular to biocompatible, capsule-forming comb polymers. The invention also relates to the field of targeted administration and antiviral agents.

In recent years, amphiphilic block copolymers comprising hydrophobic blocks and hydrophilic blocks have been well studied because of their ability to self-assemble into a variety of nanostructures as the surrounding solvent changes. See Cameron et al, Can. J. Chem. / Rev. Can. Chim. 77: 1311-1326 (1999). In aqueous solutions, the hydrophobic compartment of the amphiphilic polymer has a tendency to self-assemble in order to avoid contact with water and to minimize the free interface energy of the system. At the same time, the hydrophilic block forms a hydrated "corona" in an aqueous environment, so the aggregate remains a thermodynamically stable structure. The result is stable, and the latex-like alternating suspension of the polymer aggregates into particles having a hydrophobic core and a hydrophilic shell.

The comb-type amphiphilic copolymer differs from the block copolymer in that its main chain is largely hydrophobic or hydrophilic, and a polymer chain having a side chain opposite to the main chain is not incorporated therein. Comb copolymers have been prepared using hydrophobic backbones and hydrophilic branched chains (Mayes et al., U.S. Patent No. 6,399,700), also prepared from hydrophilic backbones and hydrophobic branched chains (Watterson et al., U.S. Patent No. 6,521,736). The former is used to provide multivalent ligand expression to cell surface receptors, while the latter is used to dissolve the drug and deliver it to the cells.

Amphoteric polymer aggregates have been investigated as carriers for the dissolution of insoluble drugs, targeted drug delivery vehicles, and gene delivery systems. They have a more stable structure than conventional low molecular weight capsules due to chain entanglement and/or crystallinity of the internal hydrophobic region. The polymeric nature of the carrier is such that the aggregate is relatively immune to decomposition by ordinary liposomes when dissolved at their critical capsule concentrations. The absence of bilayer membranes makes them easier to fuse with cell membranes and deliver their payload directly to the cells. The amphiphilic properties of the aggregates also confer detergent-like activity, and the appropriate targeted aggregates are shown to fuse with the virion protein and cause it to rupture.

Due to the good biocompatibility of polyethylene glycol (PEG), and the PEG-coated "stealth" particles to avoid the apparent ability of the reticuloendothelial system, capsules, liposomes and polymers incorporated into PEG have been It is widely considered as a material for a drug delivery system. There are many reports of the use of polyethylene glycol (PEG) as a hydrophilic component of PEG-lipids (forming liposomes and capsules); see, for example, Krishnadas et al, Pharm. Res. 20:297-302 (2003). Self-assembling amphiphilic block copolymers - self-assembled into more robust "polymersomes" - have been studied as carriers for drug dissolution and delivery (Photos et al., J. Controlled Release, 90: 323-334). (2003)). See also Gref et al, Int. Symp. Controlled Release Mater. 20: 131 (1993); Kwon et al, Langmuir, 9: 945 (1993); Kabanov et al, J. Controlled 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. Patent No. 6,616,941 and Seo et al., European Patent No. EP 0 583 955. In this capacity, the application of polyethylenimine (PEI), which focuses on the transport of oligonucleotides, is reported (Nam et al., U.S. Patent No. 6,569,528; Wagner et al., U.S. Patent Application Publication No. 20040248842). In a similar situation, Luo et al, in Macromolecules 35:3456 (2002), describes PEG-bound polyamine amines suitable for polynucleotide transport. ("PAMAM") tree-shaped molecule.

In addition to the need to dissolve, disperse, and deliver drugs, there is a need for targeted drug delivery systems that, in particular, target tissue, tumors or organs. This is usually done by attaching antibodies or other ligands with specific affinity to the cell wall to the target site. However, PEG lacks a functional group except at the end of the polymer chain, and a large portion of the terminal group is inevitably occupied by a bond bonded to another block copolymer component. For this reason, linking a targeting moiety such as an antibody or cell adhesion molecule to a PEG block copolymer is generally limited to a non-PEG block, unfortunately it is not part of the copolymer normally exposed to the shell of the self-assembled aggregate. .

The phase separation phenomenon that causes the block copolymer to self-assemble into the polymer aggregate is easily reversible, and attempts have been made to increase the stability of the aggregate by crosslinking the hydrophobic core (see European Patent No. EP 0552802). Covalent attachment of a drug to a hydrophobic portion of a block copolymer has also been attempted (Park and Yoo, U.S. Patent No. 6,623,729; European Patent No. EP 0397307).

Dendrimers readily bind to targeting moieties and also have the ability to target specific cells in vivo (Singh et al. (1994) Clin. Chem. 40: 1845) and bind viral and bacterial pathogen blocks to organisms. Substrate. The ability of comb-like branched chains and branched nucleic acid grafts with polysialic acid to inhibit viral hemagglutination and block infection of mammalian cells in vitro has been evaluated (Reuter et al. (1999) Bioconjugate Chem. 10:271). The most effective viral inhibitors are comb-like branched chains and branched graft macromolecules which show up to 50,000-fold increased activity against these viruses.

Recently, pharmaceutical company Starpharma announced the successful development of dendrimer - based (dendrimer-based) biocide (VivaGel ^TM), which is used to prevent HIV infection by binding to receptors on the virus surface (Halford (Chem & Eng 2005) . .News 83(24): 30). Chen et al. (2000) (Biomacromolecules. 1: 473) have reported that quaternary ammonium functionalized polyimine (polymethyl aziridine) dendrimers are very potent biocides.

There remains a need for a drug delivery system that is stable, biocompatible, that readily attaches the targeting moiety to the exterior of the aggregate, and that is effective in administering to a desired cellular target. Similar stable and biocompatible target antiviral agents are also needed.

The present invention provides biocompatible comb polymer molecules comprising a hydrophilic backbone having branch-point moieties, and a hydrophobic branched chain attached to the branch point moieties. The present invention provides an aqueous suspension of polymer aggregates formed from these polymers, and provides a method for dissolving insoluble or sparingly soluble organic compounds such as drugs, dyes, vitamins, etc. by introducing these compounds The hydrophobic core of the polymer aggregate is realized. The method for dissolving a water-insoluble organic species in an aqueous solvent essentially comprises contacting the water-insoluble organic species with a solvent of the present invention in water or a mixture of water.

The invention also provides a method for treating or preventing a viral infection in an animal comprising administering to the animal a comb polymer consisting essentially of the following structural formula.

This structural formula contains a main chain formed by alternating branch point portions B and a hydrophilic water-soluble polymer block A. The hydrophobic side chain C and the ligand Z are attached to the branch point portion. Preferably, the side chain C is a linear or branched chain hydrocarbon - which may optionally be substituted by one or more hydrophilic substituents, or a C ₆ -C ₃₀ ring or a ring of hydrocarbons - which may optionally be subjected to a Or substituted with multiple hydrophilic substituents. The side chain C can also be a hydrophobic amino acid, peptide or polymer. Suitable hydrophilic substituents for side chain C are hydroxyl, carboxyl and amine groups, as well as guanamine, amsulfoxine, anthracene and anthracene groups. Preferred hydrophilic substituents are polar aprotic groups such as tertiary amines, hydrazine and hydrazine.

Ligand Z is a ligand that has specific binding affinity for the surface of the virus. "Specific binding affinity" means that in the presence of many cell surfaces and macromolecules found in mammals, the ligand binds to the surface of the virus in vivo. The group s is a bond or a spacer moiety, and when s is a spacer, each s can carry 1 to 4 groups Z. The value of n ranges from 3 to about 100; the average of p ranges from 1 to 2, and the average of r ranges from 1 to 4.

The branch point portion B is a multivalent portion having a bond to two polymer blocks A, a bond to 1-2 side chains C (average), and one or more linkages to the spacer "s" and / or the bond of ligand Z. In a particular embodiment, the linkage to B and s and/or Z is produced by a plurality of reactive functional groups that can serve as attachment points. In a particularly preferred embodiment, a targeting moiety, such as a ligand or antibody, is covalently attached to the branch point portion of the polymer of the invention, and the drug is incorporated into the core of the aggregate to form a target drug complex.

The invention also provides a biocompatible comb polymer molecule as described above, which is inherently antiviral in the absence of a small molecule therapeutic. This antiviral activity is believed to be due to the detergent-like ability of the amphiphilic polymer to destroy the outer coating of the viral particles. In a preferred embodiment, the antiviral activity is enhanced by ligation of a targeting moiety having binding affinity for the surface of the target virus.

The invention further provides methods for preparing the comb polymers, aggregates, and target polymer aggregates and drug complexes described herein. The polymer of the present invention self-assembles into polymer aggregates which are effective for dissolving, dispersing and transporting drugs in vivo; having antiviral activity; being non-toxic, biocompatible and stable; and capable of being on the outer surface thereof There are multiple cell- and virus-targeting moieties.

Figure 1 shows the effect of administration of a composition of the invention on the mean survival time of influenza infected mice.

Figure 2 shows an increase in survival time of influenza-infected mice when treated with the compositions of the invention.

Figure 3 shows the loss of body weight over the course of 7 days when mice infected with influenza were treated with the compositions of the invention.

The polymer of the present invention - referred to herein as "π-polymer", has a comb-like structure in which the main chain is formed by alternating branch point portions B and hydrophilic water-soluble polymer blocks A; The hydrophobic side chain C connected to each branch point portion is as shown in Formula 1. Side chain C is a relatively short, hydrophobic portion which may be an aliphatic molecule, chain or oligomer. The value of P is ideally an integer: 2, 3 or 4. In fact, the side chains are most often introduced by chemical reactions at less than perfect efficiency, which results in the overall average of p of the polymer formulation being not the expected integer. The average of the non-integer can also be obtained by design, as discussed below. Therefore, the average value of p in the polymer of the present invention is greater than 1, and may be up to 4 (1<p 4). In a preferred embodiment, p ranges from about 2 to 4, most preferably 1.5 < p 2.

The backbone polymer block A is selected from the group consisting of 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 a polyethylene glycol chain of the formula (CH ₂ CH ₂ O) _m wherein m is between 1 and 10,000, preferably between 3 and 3,000.

In the manufacture of various grades of polyethylene glycol, it is known in the industry to couple a divalent linker moiety (such as bisphenol A diglycidyl ether) to a single polyethylene glycol chain, which effectively doubles The molecular weight of the polymer while maintaining a relatively narrow molecular weight range. The resulting "polyethylene glycol" molecule thus passes through the non-ethylene glycol linker moiety (see, for example, polyethylene glycol-bisphenol A diglycidyl ether adduct, CAS grade number 37225-26-6) The midpoint of the polymer chain is broken. Larger oligos, ie those having three PEG chains separated by two bisphenol A diglycidyl ether moieties, are also known, see for example international patent applications. WO 00/24008. Thus, as used herein, the terms "polyethylene glycol" and "polypropylene glycol" include polyethylene glycol and polypropylene glycol polymer chains incorporating non-ethylene glycol linker 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 link portion is not counted as a "single unit."

Polymer block A most preferably has an average length between 20 and 50 monomer units. The polyethylene glycol chain may be substituted at one or both ends with a functional group suitable for use as a linker to other moieties including, but not limited to, an amine group, a thiol group, an acrylate, a acrylamide, a maleate, a Malay.醯imine and so on. The value of n ranges from 1 to 1000, and is preferably between 3 and 100. The total molecular weight of the π-polymer may range from 1000 to 100,000 Daltons or more; preferably above 2,000 Daltons, more preferably above 7,000 Daltons.

The hydrophobic moiety C may be the same or different, and may be, for example, a branched hydrocarbon (which may be substituted by one or more hydrophilic substituents), a fused cyclic hydrocarbon (which may be substituted by one or more hydrophilic substituents), Hydrophobic amino acids, peptides and polymers. Suitable hydrophilic substituents include, but are not limited to, hydroxyl, ether, cyano and guanamine functional groups. Specifically contemplated is a C ₈ to C ₂₀ alkyl group, a hydroxyl group having a [omega], [omega] a cyano, [omega] [omega] acyl group or alkoxy substituents. As used herein, the term "substituent" includes a hydrocarbon atom in a heterocyclic atom such as O, N or S substituted moiety C or a carbon atom in the ring system. Thus, ether and guanamine linkages as well as heterocycles can be incorporated into C.

Hydrophobic moiety C is preferably a relatively short (C ₈ -C ₂₀ ) aliphatic chain, but may also be a short oligomer. Suitable oligomers include oligohydroxy acid (glycolic acid), poly(DL-lactic acid), poly(L-lactic acid), and copolymers of poly(glycolic acid) and poly(lactic acid) hydroxy acid; and poly(amino group) Acid), poly(anhydride), poly(orthoester), and poly(phosphate); polylactones such as poly(ε-caprolactone), poly(δ-valerolactone), poly(γ-butene) Ester) and poly(β-hydroxybutyl ester). 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 part C is greater than 40, preferably between 50 and 1,000, most preferably between 100 and 500. The value of the logP of the molecular CH (octanol-water) is greater than about 1.4, preferably greater than about 2.0, more preferably greater than about 2.5. In general, any moiety C is considered suitable for use in the present invention if the molecule CH is substantially insoluble in water. "Substantially insoluble" means that the liquid CH, when mixed with water, forms a separate phase.

The side chain C of the comb polymer of the present invention is not regularly and uniformly distributed along the polymer chain, but appears in clusters [C] _p , which is a distinguishing feature of the present invention. These clusters are more or less spaced along the polymer chain, depending on the degree of monodispersity of polymer unit A. Therefore, the distance between the two side chains C connected to the common branch portion B is different from the distance between the two side chains connected to the different branch portions.

In a particularly suitable embodiment of the invention, the branch point portion B further comprises one or more reactive functional groups X, as shown in Formula 2.

In Formula 2, the individual reactive groups X may be the same or different in others, and may be optionally blocked or protected if necessary during assembly of the polymer 2. The average value of R ranges from 0 (without X groups) to about 4. Typically, the reactive group is selected from functional groups known in the art that can be used to form covalent bonds between molecular species. The group X serves as a point of attachment for the drug molecule, tissue or cell targeting moiety, viral targeting moiety or matrix linking moiety (eg, for coating a scaffold or other medical instrument surface). In certain embodiments, there may be a single connection point X. In other embodiments, there may be three or four different types of reactive groups. The matrix linking moiety can be attached to the substrate by a covalent bond, a specific non-covalent interaction (eg, an antibody-antigen or a non-specific interaction (eg, via ion pairing or "hydrophobic" interaction). Suitable reactive groups X include However, it is not limited to -OH, -NH ₂ , -SH, -CHO, -NHNH ₂ , -COOH, -CONHNH ₂ , haloxime, acetamidine, -CN, -OCN, -SCN, -NCO, - NCS, etc.; active double bonds such as ethylene, acrylic, allyl, maleic, cinnamic; and reactive triple bonds such as ethynylcarboxy and ethynylguanidino (suitable for use in Michael) Addition, Diels-Alder reaction and free radical addition reaction).

Exemplary-targeting moieties include, but are not limited to, receptor-specific ligands, antibodies, and other targeting moieties, such as having arginine-glycine-aspartate (RGD) amino acid sequence or tyrosine a peptide of the isoleucine-serine-arginine-glycine (YISRG) motif; growth factors, including epidermal growth factor, vascular endothelial growth factor and fibroblast growth factor; viral surface ligands, eg Sialic acid and N-acetamidine neuraminic acid derivatives; cell receptor ligands such as folate, methotrexate, pteroate, estradiol, estriol, testosterone and other hormones; mannose-6- Phosphate, sugar, vitamins, tryptophan, etc. The antibody preferably is a monoclonal antibody that directs a cell-specific surface antigen; suitable targeting moieties include not only intact antibodies, but also antibody fragments comprising active antigen-binding sequences such as Fab'2 fragments, Fab' fragments, or such antibodies Short chain peptide analogs of the active antigen binding sequence.

Virus - Examples of targeting moieties include small molecules that bind to viral ligands, such as aminoalkyl adamantyl ^{amine, Fuzeon TM, PRO-542,} BMS-488043, sialic acid, 2-deoxy-2,3-dideoxy Hydrogen-N-acetyl ceramide, 4-mercapto-Neu5Ac2en (zanamivir), oseltamivir, RWJ-270201, etc.; oligopeptides, oligosaccharides and glycopeptides - which bind to the surface of the virus And antibodies and antibody fragments that target viral-specific surface antigens. In a preferred embodiment, the invention provides a π-polymer comprising a ligand for a viral neuraminidase or a hemagglutinin. It has been well established that these polymers have antiviral properties on their own right side; see, for example, T. Masuda et al, Chemical & Pharmaceutical Bulletin 51:1386-98 (2003); M. Itoh et al, Virology 212: 340-7 (1995) ), and Reece et al., U.S. Patent No. 6,680,054 (2004). The hydrophobic core of the polymers and polymer aggregates of the present invention may optionally be loaded with one or more conventional antiviral agents that are advantageously released near the viral particles.

Other medically relevant linking groups can be small chemical agents, peptides, antibodies or antibody fragments, enzymes or active pharmaceutical ingredients that can affect biological processes such as hormone or hormone promoters or antagonists, substances that interfere with viral binding, A substance that interferes with cells or cellular processes after entering the cell. Cells of single and multicellular organs including viruses, fungi, higher animals, and plants can be targeted. Biotin can be linked to a π-polymer and used as a point of attachment for anti-biotin and streptavidin-conjugated proteins, peptides or other targeted active agents such as antibodies, growth hormones, imaging agents and the like.

"Matrix" means organic or inorganic matter, surfaces and deposits such as glass, cerium oxide or metal surfaces, extracellular matrices, protein deposits such as various amyloid plaques, cell surfaces, viral surfaces and generally homogeneous or heterogeneous surfaces. It may or may not have features, including prions.

Examples of the glass or cerium oxide matrix linking moiety include various halogenated decanes, alkoxy decanes, decyl decanes, and chemical agents exhibiting such functional groups containing a polymer. Other linking groups can be designed based on the specific physicochemical properties of the substrate. Suitable attachment portions, such as those used in stent coatings, are known to those of ordinary skill in the art.

In a third aspect of the invention, the branch point portion B is attached to other branch point portions at other locations of the polymer chain to form a crosslinked hydrogel structural formula. Such cross-linking can be achieved by reacting a polymer with a multifunctional moiety comprising a homo- or hetero-functional group, at least one of which reacts with an X or a reactive group at C of the first branch point moiety, and At least one of them reacts with X or a reactive functional group on C located at the second branch point portion. Crosslinking can also be carried out via attachment to the terminal functional groups of the polymer chain A. Such crosslinked polymers may optionally contain reactive functional groups suitable for attachment to a drug molecule or targeting moiety.

The branch point portion B is typically derived from a multifunctional molecule having a plurality of reactive groups, two of which are adapted to attach a hydrophilic polymer unit A, two of which are adapted to attach a hydrophobic portion C. Part B may optionally have the above-mentioned reactive group X.

A particularly preferred branching point moiety is dithiothreitol (DTT), dithioerythritol (DTE) or 2,3-diaminobutane-1,4-dithiol (dithiols) with two horses A combination of acid molecules. The combination of this branch point moiety with polyethylene glycol as Part A produces the polymer backbone of Formulas 3 and 3a.

Wherein Y and Y' may be the same or different, and are preferably selected from OH, NH ₂ , ONH ₂ , NHOH and NHNH ₂ . In a preferred embodiment, the hydroxyl or amine group of the dithiol is the reactive group X as a point of attachment or drug moiety, and the functional groups Y and Y' serve as the point of attachment for the C moiety. Alternatively, the groups Y and Y' can serve as a point of attachment, while a hydroxyl or amine group can be used to attach the moiety C.

Formulas 3 and 3a are intended to be transported separately to each of the sulfur atoms of alpha or beta to the PEG ester carboxyl group. The invention includes a single isomer component and a position at one or two C-S bonds a mixture of isomers. Moreover, due to the four asymmetric carbons in Formula 1, the invention includes all chiral, meta- and diastereomeric isomers as well as mixtures thereof.

Diels-Alder adducts of ethynyl dicarboxylic acid and furan can also be used as suitable branch point moieties. For example, polyester 4 derived from PEG and ethynyl dicarboxylic acid is known to undergo a Diels-Alder reaction with furan (M. Delerba et al, Macromol. Rapid Commun. 18(8): 723-728 (1997) ). Thus, it can undergo a Diels-Alder reaction with a 3,4-disubstituted furan to produce a species such as 5, and the polymer 5 can also be modified by hydroxylation or slow oxidation to provide a reactive group. (for example, X and X' in Formula 1).

plan 1

Similarly, reaction of PEG with ethylenediaminetetraacetic acid dianhydride will provide a polyester of formula 6:

Other suitable branching points may be derived from tartaric acid, ethynyl dicarboxylic acid, ammonia triacetic acid, 3,4,3',4'-diphenylphosphonium tetracarboxylic dianhydride, 3,4,3',4' -diphenyl ether tetracarboxylic dianhydride, pyromellitic dianhydride, alkanedithiol such as 1,2-ethanedithiol and 1,4-butanedithiol, bis(2-mercaptoethyl) Ether, 2-mercaptoethyl sulfide, dimercaptopropanol, dimercaptopurine, dimercaptothiadiazole, dimercaptosuccinic acid, xylyl mercaptan, benzene dithiol, dihalophthalaldehyde, Dihalogenated 4,4'-thiobisbenzenethiol and the like.

Wherein Y and Y' are OH, the hydrophobic group C can be attached to the polymer by amide or esterification of a carboxylic acid group. The hydrophobic group C is preferably relatively small (C ₈ - C ₂₀ ) and is predominantly a hydrocarbon moiety and may be linear or branched or contain one or more rings. Examples include, but are not limited to, moieties derived from the covalent attachment of the CH molecule n-octanol, n-nonanol, n-dodecylamine, n-pentadecylamine, cholesterol, and cholic acid. Although the polymers of the present invention are shown for convenience to contain up to two different water-transporting side chains, it should be understood that a mixture of two or more hydrophobic compounds can be used to introduce a plurality of hydrophobic side chains into a particular polymer.

As a specific example, the polymer of Formula 2, wherein X = OH and r = 2, is formed by reacting polyethylene glycol with maleic anhydride to form polyester 7, and then reacting with dithiothreitol to form 8 preparation. The acid 7 is then aminated with n-octadecylamine to form the desired comb polymer 9 (Scheme 2). The DTT-derived guanamine comb polymer represented by Formula 9 is referred to herein as "π-Polymer A"; in Scheme 2, the specific polymer 9 is referred to as "C ₁₈ - π-Polymer A".

Scenario 2

2,3-bis(t-butoxycarbonylamino)butane-1,4-dithiol (prepared by the method of U.S. Patent No. 4,755,528 to DuPriest et al.) for the substitution of dithiothreitol Protection results in the corresponding amino-functionalized π-polymer 9b (Scheme 3).

Option 3

The application of butanedithiol 10c likewise results in a polymer of general formula 9c in which the spacer group L is used in the appropriate position for subsequent attachment of the targeting moiety (Scheme 4). The spacer group L can be any spacer group known in the art for attaching a ligand or label to a substrate molecule, including but not limited to C ₂ to C ₂₀ alkyl and having one to ten -CH The oligo(ethylene glycol) spacer of the ₂ CH ₂ O- unit.

Option 4

In other embodiments, a PEG polymer having a terminal amine group can be used to prepare an example having a guanamine bond between the A and B units, as shown in Structural Formulas 10-14 below. Each of these polyamines can be obtained by reacting PEG diamine H2N-(CH ₂ CH ₂ O) _m CH ₂ CH ₂ -NH ₂ with an appropriate cyclic anhydride:

Under moderate conditions, the above proline is the desired product. After heating, it may be desirable to form a quinone imine which results in a polymer with fewer reactive groups, but which is still suitable for linking the hydrophobic C moiety. In addition, side chain C can be added to the end of the polymer A block, and the branch point portion can occur at the time of polymerization (Scheme 5).

Option 5

In addition to a simple diamine such as 1,3-diaminopropane, as shown in Scheme 5, a diamine having a (optionally masked) reactive functional group X can be used, which results in the polymer 15 being suitable for use in attaching a target To the part (Scenario 6). In the formula below, p may range from 0 to 4, and each X is independently the same or different from other groups X which may be present. The reactive group X may not be chain-linked, but may be, for example, NH in the atomic chain constituting the diamine, as in the monomer H ₂ N-(CH ₂ ) ₃ -NH-(CH ₂ ) ₃ -NH ₂ .

Option 6

Certain π-polymers prepared as above have reactive groups X suitable for further derivatization to link targeting moieties such as small molecules, peptides, nucleotides, sugars, antibodies, etc., or cross-linking via dual or multifunctional The agent achieves cross-linking of the polymer chain. In a particular embodiment, partial derivatization of reactive groups on the polymer chain is carried out to produce a pi-polymer having a plurality of different reactive groups, which allows attachment of multiple targeting and drug moieties to a single polymer chain. Thus, the addition of stoichiometric amounts of acrylonitrile (or maleic anhydride) to the π-polymer of Example 1 will provide polymerization with both propylene (or maleate) groups and residual hydroxyl groups. Things. Subsequent Michael addition of a thiol-carboxylic acid such as HS-(CH ₂ ) ₃ -COOH at stoichiometric amounts will provide a polymer having a hydroxyl group, an acryl group, and a carboxyl group. In addition to any residual reactive groups left by the reagents at stoichiometric amounts, the addition of cysteine to the amine and carboxyl groups is introduced.

Another method of multifunctional π-polymer involves deliberate omitting the portion of hydrophobic chain C. For example, the π-polymer of Example 1 can be prepared by simply using an amount of an alkylamine which is limited to form a side chain in the amidation step using an unreacted carboxylic acid group. Yet another method is to amidoxilate with a mixture of amines containing a reactive group X. Likewise, under appropriate conditions (excess maleic anhydride in step A, excess DTT in step B), a polymer preparation having the desired amount of thiol groups can be produced.

According to the design, the π-polymer of Example 1 includes a DTT partial hydroxyl group derived from the main chain as a reactive group X. The esterification of these groups with acrylonitrile or methacrylofluorene in the presence of carbonates/bicarbonates in an aqueous medium results in the substitution of propylene on the -OH groups. An acrylated polymer can readily undergo free radical polymerization (with or without a added free radical monomer such as an acrylic compound or a crosslinking agent such as a diacrylic acid) to provide suitable control for A drug (as a polymeric storage agent or reservoir) and a hydrogel for topical administration (eg, a skin patch or ointment). Acryl sulfhydryl groups can also undergo Michael addition, particularly with sulfuric hydrocarbons, such as heptyl sulphates remaining in proteins, enzymes, peptides, antibodies, Fab'2 fragments or Fab' fragments, or other targeting moieties. (Scheme 7).

Option 7

a π-polymer having a reactive hydroxyl group which, after drying, can be esterified with maleic anhydride To attach to the maleate group, Michael accepts the body while producing a free carboxyl group. Among the polymers formed, the male double bond is feasible for Michael addition, especially with sulfur hydrocarbons, such as residues in proteins, enzymes, peptides, antibodies, Fab'2 fragments or Fab' fragments, or other targets. To the thiol of the cysteine in the moiety (Scheme 8), and the carboxyl group is a viable lysine residue for coupling to the amine or drug or ligand of the drug or ligand.

The different moieties can be further linked to the newly introduced (or previously available) carboxyl groups via guanidation. Thus, at least two different targeting moieties can be linked even under saturated reaction conditions (ie, the portion to be joined is present in a stoichiometric excess).

Option 8

Polymers having pendant carboxylated groups can be amidated with an amine under typical coupling conditions, and they can also be converted to isocyanate groups via Curtius rearrangement. The lumps are then coupled with an amine or alcohol to form urea and urethane, respectively. Such a reaction can be used to introduce a hydrophobic group C, or to attach a targeting moiety.

The free amine can be introduced into the polymer by reacting at least one of the reactive groups with the diamine moiety. The diamine must be chosen such that one of the amine groups is either protected or not reacted under the reaction conditions. The latter can usually be accomplished by using 1,2-ethylenediamine at a pH of about 7.5 because the pKa's of the two amine groups vary greatly. Preferably, the guanamine is carried out as a separate step after introduction of the hydrophobic side chain groups. The peptide having a carboxyl group or another molecule can then be attached to the free amine by guanidation.

Thus, even under saturated conditions, how many three different peptides or other targeting moieties can be attached to the π-polymer: one via a sulfur hydrocarbon, one via an amine or a hydroxyl group, and one via a carboxylic acid group.

Hydroxyl and thiol groups can also be converted to primary amines by reaction with aziridine or a haloalkylamine such as bromoethylamine or chloroethylamine. The guanylation with cysteamine will introduce a disulfide which can be directly reacted by the peptide or the cysteine of the antibody to link the peptide or antibody; or it can be first reduced, for example with aminoethane thiol or DTT, Further reaction is carried out with a peptide or antibody.

Additional reactive functional groups may be introduced into the polymer of the present invention by performing a partial reaction including, but not limited to, (1) a sulfur-reactive group such as an acrylic acid or a maleic acid derivative, and (2) a carboxylic acid reaction. The group is an amine group or a hydroxyl group, (3) an amine-reactive group such as a carboxyl group, and (4) a disulfide-reactive group such as a thiol group. The number of these added functional groups per polymer molecule ranges from 1/r up to several times r, depending on the reagents used and the amount used.

Additionally, two or more specific ligands can be ligated to increase binding specificity to a say, virus or cell surface. Two or more specific ligands can also be used to cause interactions between different cellular targets, for example, one ligand can target viral particles and another ligand can promote binding to phagocytic cells, thereby The virus particles are brought close to or in contact with the phagocytic cells and promote phagocytosis.

This derivatization is linked by different functional groups (eg amines, carboxylates and sulphur hydrocarbons) Or more different targeting and/or therapeutic moieties are attached to the polymer. Thus, tissue-specific targeting agents, imaging agents, and therapeutic agents can be attached to a single polymer chain, and subsequent polymer self-assembly will result in targeted therapy, the distribution and targeting efficacy of which can be monitored.

The attachment of a ligand to the repeating unit of the polymer of the invention provides a multivalent display of the ligand on the polymer chain and on the surface of the nanoparticle. Multivalent displays often result in a greater increase in affinity for the target. For example, multivalent antibodies are more effective than normal bivalent antibodies in clearing their targets. Sugar-binding proteins and carbohydrates are known to be multivalent in nature and are ineffective if they are monovalent. Similarly, multivalent peptides and carbohydrates are more effective at targeting individual monomers of a single coin. An increase in MW results in reduced renal clearance of peptides and other ligands due to attachment to the polymer. In addition, the benefits of peptides provided by the PEG backbone are similar to those of PEGylated peptides, including avoiding immune surveillance.

In addition, the multivalent targeting moiety will modify the multivalent target (say, viral particle) and neutralize it, which is much more effective than the monomer targeting moiety. In multivalent forms, the ability to display multiple (different) peptides will result in enhanced specificity. For example, a true HIV-specific (HIV virus-binding) polymer can be linked by a peptide corresponding to the CD4 binding region of the virus, and another peptide corresponding to the CCR-5 or CXCR-4 binding region of the virus, and possibly The third peptide corresponding to other receptors (CXCR-4 or CCR-5, respectively) was established. Such polymers completely mask the viral binding region and cause the virus to be unable to attach to the cell and thus be non-infectious. In addition, the surfactant properties of the polymer will cause the viral structure itself to lose stability upon binding. Instead of a peptide, a small molecule (CD4, CCR-5, CXCR-4) that interferes with the same binding mode or a mixture of peptides and small molecules - preferably having complementary activities, can be used. The formed polymer will render the free virus ineffective, and thus it is desirable to stop the spread of infection by using them as a component of a condom lubricant or the like. Additionally, such polymers can be injected into patients to reduce the burden of HIV.

In general, when a multifunctional reagent such as DTT is used, the polymer chain moiety can also be partially crosslinked via esterification or similar side reactions of the carboxylic acid with DTT. In the central region of the PEG chain Secondary hydroxyl groups, such as those attached to the bisphenol A diglycidyl ether residue, can also promote cross-linking if they are present in the PEG starting material. The resulting crosslinked hydrogel structure is also a useful material. For example, a material that is a pliable hydrogel can be made by appropriately increasing the extent of the cross-linking or by using an optional cross-linking agent such as, for example, dioxirane, which can be used as a material The container of the drug stores the depots. By suitably modifying the material (eg, a smaller PEG length, a larger open carboxyl group, and a suitable acrylic group), a linear or crosslinked hydrogel material can be made that can be used or immobilized in an instrument such as Repositories supported on the stent or attached to an instrument such as a pad for adhesive patch or subcutaneous insertion of a patch. In general, such crosslinked materials will be suitable for controlled release rather than enhanced target release.

The comb polymers of the present invention are useful for dissolving slightly water soluble materials in aqueous solvent systems. A method of dissolving a substance in an aqueous solvent comprises contacting a sparingly soluble substance with a comb polymer of the present invention in the presence of water to form a water-soluble complex of the substance and the polymer. Alternatively, the polymer and the substance to be dissolved may be combined in a two-phase water-organic emulsion, and the organic solvent is removed by evaporation. An exemplary method is described in U.S. Patent No. 6,838,089 which is incorporated herein by reference. It is believed that in most cases, the polymer self-assembles into nanoparticles that have dissolved, slightly soluble substances in the hydrophobic C chain that coalesce in the nucleus of the particles while the A block (block) forms a hydrophilic shell. It has a sufficiently low interface free energy to keep the aqueous suspension of particles stable.

In some cases, the sparingly soluble material may not be completely dissolved in the core, but may be solid nanoparticles that are surrounded or suspended in the C chain of the core of the particle. For the present invention, this is a difference in degree because the particles of the present invention are not dependent on any particular degree of mixing of the C chain with the sparingly soluble material. In some cases, the material may dissolve at the molecular level in the C chain, but in other cases it may exhibit any degree of phase separation from the C-chain environment. In some cases, it may be desirable for the system to move from one state to another as a function of temperature.

The solvating power of the hydrophobic core of the polymer particles can be modified by modifying the hydrophobic C moiety. Suitable modifications include, but are not limited to, the introduction of one or more hydrophilic substituents, such as hydroxyl, ether, guanamine, and cyano functional groups, in order to increase the polarity and/or polarizability of the hydrophobic core.

The slightly soluble materials that can be dissolved by these polymers include fat-soluble vitamins and nutrients including, but not limited to, vitamins A, D, E, and K, carotene, microorganism D3, and coenzyme Q; insoluble drugs such as docetaxel, Amphotericin B, mycin, paclitaxel, doxorubicin, epirubicin, rupidine, scorpion thiophene, etoposide, daunorubicin, methotrexate, mitomycin C, ring Sporomycin A, irinotecan metabolite (SN-38), statins and steroids; dyes, photodynamic agents and imaging agents; and nucleic acids, nucleic acid analogs and nucleic acid complexes. Nucleic acid analogs include species such as phosphorothioates and nucleic acid peptides; nucleic acid complexes are ionic complexes of oligonucleic acids with substantially neutralizing electrical quantities of cationic or polycation species.

For the present disclosure, a drug that is insoluble at neutral pH is considered "slightly soluble" because in many cases a neutral pharmaceutical composition is required. For example, ciprofloxacin is suitably soluble in water below pH 4.5, but when the drug is configured for ocular administration, the pH is highly irritating. The polymer of the invention will dissolve ciprofloxacin in normal saline at pH 7. Also, for the purposes of the present disclosure, "slightly soluble" should be understood to mean the solubility of any substance in an aqueous carrier such that an increase in solubility will result in an enhanced or more useful composition. Thus, a drug that is moderately soluble - for example to the extent of 2 g/l - is "slightly soluble" if the unit dose for intravenous administration is 5 g.

As a result of the ability of the polymers of the invention to solubilize pharmaceutically active species, the invention also provides pharmaceutical compositions comprising one or more π-polymers of the invention in combination with a therapeutically effective amount of one or more pharmaceutically active agents. The polymers of the present invention may be effective in otherwise effective amounts of pharmaceutically active agents. Thus, for the purposes of the present disclosure, a "therapeutically effective amount" is the amount of the agent that renders the total ingredient effective.

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

Example General process

The invention also provides methods 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 primary starting material is polyethylene glycol, which is preferably dried prior to use. The molten PEG can be easily stirred by stirring the molten PEG at a high temperature in a vacuum until the foam stops generating. This can take 8-12 hours depending on the quality of the PEG. After drying, the PEG can be stored in argon indefinitely. Commercially available industrial or research grade PEG can be used to make the polymers of the present invention, such as the commercial polydisperse "PEG 1500" having a molecular weight distribution of 1430-1570. This material can incorporate bisphenol A diglycidyl ether which introduces a secondary hydroxyl group in the middle of the PEG chain. To ensure that the polymers of the present invention have the most reproducible and consistent properties, PEG is preferably free of bisphenol A and is low in dispersion. Most preferred are >95% monodisperse PEG polymers, such as are commercially available from Nektar Therapeutics (formerly Shearwater Polymers), Huntsville AL and Polypure AS, Oslo, Norway. One example of particularly preferred PEG from Polypure of "PEG-28", which is> 95% of _{HO (CH 2 CH 2 O)} 28 H, molecular weight of 1252.

All reactions are carried out in an inert atmosphere such as nitrogen or argon with magnetic or preferably mechanical agitation.

In step A, the dried 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 to the number of PEG terminal hydroxyl groups. Insufficient maleic anhydride will result in a hydroxy-terminated polymer chain, whereas an excess of maleic anhydride will consume the thiol group in the next step, which results in premature chain termination and terminal carboxyl groups. The reaction temperature is not critical and the process can be suitably carried out at temperatures between 45 ° C and 100 ° C. The preferred reaction temperature is between 65 ° C and 90 ° C. If used high Temperature, maleic anhydride tends to sublimate, the steps should be noted that maleic anhydride remains dissolved. Minimizing the headspace and immersing the reaction vessel in an oil bath is an effective method.

Depending on the temperature selected, the reaction can be completed in 2 hours or less, or the reaction can be carried out overnight. The reaction can be monitored by TLC on a silicone board and continued until the maleic anhydride disappears. Visual contrast, UV and iodine staining can all be used to inspect 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). Combine (if flow is required, add water) and stir the mixture at 70 °C. The reaction was completed in 30 minutes as indicated by the rapid increase in viscosity. If more or less than the optimum amount of DTT used, the molecular weight of the product will be reduced. The molecular weight of the product can also be reduced, if desired by replacing the TEMED with a less effective tertiary amine such as TEA.

In step C, a sufficient amount of water is added to the reaction mixture to reduce the viscosity, and 0.1 mol of N-hydroxybutaneimine (NHS) and 1.05 mol of hexadecylamine per mole of carboxylic acid groups in the polymer are added. The amount of NHS performed best minimizes the extent of side reactions). Then, an excess of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) (1.4 mol EDC per mole of carboxylic acid group) was added in portions, and additional water was added. If you need to keep 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 to 45 ° C, whereas octadecylamine is contained, and the temperature is between about 55 ° C and 57 ° C. After the reaction, TCL was carried out until a constant level of residual alkylamine was observed, typically after an overnight run.

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, which was then titrated to pH 7.0 using 1 N NaOH. The final reaction mixture was centrifuged at approximately 800 xg for 1 to 3 hours to remove solid contaminants and by-products.

After centrifugation, the supernatant can be analyzed on a GPC column chromatography ^{(Toyopearl TM, Sephadex TM, Sephacryl} TM, Biogel TM , etc.). However, π polymers are amphiphilic materials and will exhibit affinity for most GPC column packings, which complicates the removal of contaminants. Alternatively, the polymer can be chromatographed on a macroporous hydrophobic interaction column (e.g., TOYOPEARL ^(TM) Phenyl 650C5, Toshoh Biosciences, Montgomeryville, PA, USA) and eluted with a gradient of methanol in water. Preferably, the reaction mixture is dialyzed against changes in several acidified and neutral waters to remove low molecular weight starting materials and reaction byproducts.

The reaction mixture can also be eluted with butanone, isopropanol, butanol or other polar organic solvent to remove organic impurities, but a large amount of amphiphilic polymer is lost in the elution solvent. Preferably, the reaction mixture is subjected to ultrafiltration using a suitable membrane to fractionate the product to a molecular weight grade, such as 5 kDa to 10 kDa; 10 kDa to 30 kDa; 30 kDa to 50 kDa, etc., depending on the cutoff of the filtration membrane used. The aqueous solution of the polymer can be filtered through dead end to produce a sterile or virus free solution, depending on the choice of filtration membrane or medium.

2. Synthesis of π-polymer

Example 1: PEG-bis(alkylguanamine amber) disulfide medium molecular weight polymer (C16-π-Polymer A)

Polyethylene glycol (PEG-1500, Sigma Chemical Co.) was dried in vacuo at 80 ° C until bubble formation ceased (8-12 hours depending on the quality of the PEG). The dried PEG can be stored in argon indefinitely.

The dried PEG was melted on an oil bath in argon, and maleic anhydride was gradually added with stirring (correction of impurities per mole of PEG 2 moles). The mixture was stirred at 90 ° C under argon. Because maleic anhydride tends to sublime, the headspace is minimized and the entire reaction vessel is maintained at the reaction temperature. Any concentrated maleic anhydride on the walls of the vessel is scraped back into the reaction mixture. The reaction was monitored by TLC on a silica gel plate, using ethanol and hexane as solvents, respectively, for UV visualization and iodine staining. Continue to react after the disappearance of maleic anhydride hour.

The crude PEG hydrogen maleate was diluted with two volumes of water. Then, with stirring, dithiothreitol (DTT, 1.01 equivalent per equivalent of PEG) and N,N,N',N'-tetramethylethylenediamine (TEMED, 1.02 equivalents) in water (per volume TEMED, 2 volumes of water) was added to the reaction mixture. The reaction was stirred at 70 ° C for 2.5 hrs under argon, left at room temperature overnight, and then stirred again at 70 ° C for 2 hours. The reaction was monitored by TLC and the reaction was completed after DTT completely disappeared.

Water was added to the above reaction mixture to reduce the viscosity until the mixture was stirred (about 25% solids), the mixture was stirred at 65 ° C under argon, and N-hydroxybutanediamine was added (in PEG-Malay) In the acid hydrogen ester-DTT polymer, 0.1 mole per mole of carboxylic acid group, then hexadecylamine (1.05 mole per mole of carboxylic acid group in the polymer) and N-(3-dimethylamine) Propyl)-N'-ethylcarbodiimide (EDC, 0.56 moles per mole of carboxylic acid groups in the polymer). The mixture was stirred for 1 hour under argon and a second portion of EDC (0.56 moles per mole of carboxylic acid groups in the polymer) was added. At another time, a third portion of EDC (0.28 moles per mole of carboxylic acid groups in the polymer, a total of 1.4 moles of EDC per mole of carboxylic acid) was added to supplement the loss of EDC hydrolysis. When the added solids make the suspension difficult to stir, if it is desired to maintain fluidity, additional water is added and the pH is maintained between 8 and 10 by the addition of 1 N NaOH when needed. The mixture was stirred overnight at 65 ° C under argon, and was monitored by TLC (c.c., ethanol) until the alkylamines showed a stable concentration and then stirred for an additional 4 hours. Then, the reaction mixture was acidified to pH 4.5 with 1N HCl, stirred for 24 hours to destroy unreacted EDC, and adjusted to pH 7.0 by dropwise addition of 1 N NaOH. Containing dodecylamine, the reaction can be carried out at about 40-45 ° C, conversely containing octadecylamine at a temperature of from about 55 ° C to 57 ° C.

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

By this method, the following amine compound is attached to the polymer:

Example 1a: undecylamine

Example 1b: Octadecylamine

Example 1c: 4-decylbenzylamine

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

Example 2: PEG-bis(alkyl guanamine amber oxime) disulfide high molecular weight polymer

According to the procedure outlined in Example 1, except that 0.55 mol of DTT and 0.55 mol of TEMED per mole of maleic anhydride were used. Because the viscosity grows quickly, strong agitation is required. It was shown that most of the reaction was completed in 5-10 minutes, and then, as the temperature was raised to 55 ° C to 80 ° C, the reaction was slowly completed over the next 4 hours.

Example 3: PEG-bis(alkyl guanamine amber oxime) disulfide polymer

According to the procedure set forth in Example 1, except that 1.5 moles of dodecylamine per mole of carboxylic acid group was used in the polymer. Add N-hydroxybutanediimide (NHS, 1.0 mol per mole of carboxylic acid group) and 1,1'-carbonyldiimidazole (CDI, 3.0 mol per mole of carboxylic acid group), then stir at 80 ° C The reaction was carried out for 4 hours, and the reaction was gradually established by incorporating the above.

By this method, the following amine compound is attached to the polymer:

Example 3a: undecylamine

Example 3b: Tetradecane

Example 3c: Octadecylamine

Example 3d: Dehydroabietylamine

Example 3e: cholesterol 2-aminoethyl ether

Example 3f: 10-phenoxyguanamine

Example 3g: bismuth sebacate

Example 3h: bismuth oleate

Example 3i: Dehydroacetic acid hydrazine

Example 3j: Bismuth cholate

Example 3k: Barium palmitate

Example 4: PEG-co-(alkyl guanylaminosuccinic acid) polymer

A solution of PEG (6.66 mmol) and triethylamine (2.32 ml, 16.65 mmol) in dry diethyl ether (10 mL) was evaporated in EtOAc (EtOAc) ) It is processed dropwise. Stirring was continued for 1 hour at 0 ° C and then at room temperature for 2 hours. The ether was evaporated, and dry acetone (15 ml) was added to residue to precipitate triethylamine hydrochloride which was filtered from solution. The filtrate was reacted with lithium bromide (2.31 g, 26.64 mmol) and heated to reflux for 20 h. The mixture was then diluted with hexane and covered with a silicon dioxide through Celite ^TM (0.5cm) of (3cm) filtered short column, and eluted with hexane. The filtrate was dried and evaporated to leave α,ω-dibromo-PEG as an oil.

α,ω-Dibromo-PEG and one equivalent of 2,2-dibutyl-4,5-di(methoxycarbonyl)- by the method of Godjoian et al., Tetrahedron Letters, 37: 433-6 (1996) 1,3,2-dioxastannolane reaction. The formed dimethyl tartaric acid-PEG polyether is saponified with KOH in methanol and then amidely aminated with dodecylamine or cetylamine as described above in Examples 1 and 3, or by examples The amine in 3a-3k is amidely aminated.

Example 5: Copolymerization of PEG with EDTA dianhydride

The PEG is reacted with 1,2-ethylenediaminetetraacetic acid dianhydride by the method described in Example 1, and then, as in Example 1, with dodecylamine or as in Example 3 with hexadecylamine or They were amidely aminated with the amines of Examples 3a-3k.

In the same manner, the following dianhydride was copolymerized with PEG and subsequently amided:

Example 5a: naphthalenetetracarboxylic dianhydride

Example 5b: Terpene tetracarboxylic dianhydride

Example 5c: benzophenone tetracarboxylic dianhydride

Example 5d: 4,4'-(hexafluoroisopropylidene)diphthalic anhydride

Example 5e: Butane tetracarboxylic dianhydride

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

Example 5g: Diethylenetetraamine pentaacetic acid dianhydride

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

Example 5i: 3,4,3',4'-diphenyl ether tetracarboxylic dianhydride

Example 5j: pyromellitic dianhydride

Example 6A: PEG-diamine copolymer with side chain thioether

The PEG maleic acid ester, prepared as in Example 1, was reacted with dodecanethiol (per equivalent of PEG maleic acid hydrogenate, two equivalents) using the same procedure as used for DTT in Example 1. When no polymerization occurs, no dilution is required and the reaction is carried out in molten PEG-hydrogen maleate. A TEMED catalyst was added and then the mercaptan was added. After the reaction, the starting material disappeared and TLC was used. The temperature up to this point - at which the loss of alkyl thiol by evaporation becomes sufficient, can be used (up to about 100 ° C). A slight excess of alkyl mercaptan can be used to fully saturate the maleic acid group. Excess alkyl mercaptan is driven off at the end of the reaction by impinging with nitrogen and argon and/or heating in vacuo until no alkyl mercaptan is detected by odor or by TLC.

By this method, the following thiols are attached to PEG hydrogen maleate:

Example 6Aa: Di-t-butyl decyl succinate

Example 6 Ab: tetradecyl mercaptan

Example 6Ac: Hexadecanethiol

Example 6 Ad: 2-mercaptoethanesulfonic acid

Example 6Ae: 3-mercaptopropanesulfonic acid

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

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

Example 6Ah: mercaptoacetic acid-t-butyl ester

Example 6Ai: 4-(Tertibutoxycarbonylamino)butanethiol

Example 6Aj: 3-(Tertibutoxycarbonylamino)benzylthiol

Example 6 Ak: 4-mercaptobenzyl thiol

A thiol having a reactive functional group is suitable for linking the C chain, and/or a reactive functional group can serve as a point of attachment (X) to the targeting moiety.

Example 6B: Copolymer of PEG-diamine and side chain thioether

The thiol addition obtained in Example 6A was added using 1,4-diaminobutane (per two COOH groups, one equivalent of diamine) using the same procedure as used for the dodecylamine of Example 1. The product is amidated and diluted with water as needed 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 polydecylamine.

By this method, the following diamine can be converted to PEG polyamine (BOC = third butoxycarbonyl):

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

Example 6Bb: N',N"-di(BOC)hexaethylenetetramine

Example 6Bc: N', N"-di(BOC) spermine

Example 6Bd: N'-BOC spermidine

Example 6Be: N', N", N"'-Tris(BOC) pentaethylene hexamine

Example 6Bf: agmatine

Example 6Bg: tert-butyl lysine

Example 6Bh: 1,6-Diaminohexane

Example 6 Bi: 1,4-styrene diamine

Example 6Bj: 1,3-styrene diamine

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

Example 7: PEG-bis(alkyl succinic acid) disulfide

Preparation of DTT (m--2,3-di(hexadecyloxy)butane-1,4 by modification of the method of S. Sasaki et al., Chem. Pharm. Bull. 33(10): 4247-4266 (1985) -Dithiol) 2,3-di-O-hexadecyl ester. By the method of Example 1, it was added to PEG-hydrogen maleate.

By this method, the following ether dithiol is attached to the PEG polymer:

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

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

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

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

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

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

Example 8A: Substituted PEG succinate

The procedure prepared in Example 1 was followed except that 2-dodec-1-ene succinic anhydride was used in place of maleic anhydride. The dodecene substituent provides a side chain C chain in the final polymer.

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

Example 8Aa: Isobutenyl succinic anhydride

Example 8 Ab: 2-octene-1-yl succinic anhydride

Example 8Ac: octadecyl succinic anhydride

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

Example 8Ae: cyclohexane dicarboxylic anhydride

Example 8Af: Phthalic anhydride

Example 8 Ag: 4-mercaptophthalic anhydride

Example 8Ah: hexahydromethyl phthalic anhydride

Example 8Ai: tetrahydrophthalic anhydride

Example 8Aj: norbornene dicarboxylic anhydride

Example 8Ak: Cantharidin

Example 8 Al: Bicyclooctene dicarboxylic anhydride

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

Example 8 An: S-Ethylmercaptosuccinic anhydride

Example 8B: PEG-bis(alkylguanamine amber oxime) disulfide having a pendant alkyl group

The substituted PEG amber oxime ester obtained as described in Examples 8A and 8Aa to 8An was reacted with DTT according to the method of Example 1.

By this method, the following dithiol (dithiol) is reacted with any of the substituted PEG amber oxime esters obtained as described in Examples 8A and 8Aa to 8An:

Example 8 Ba: Ethane-1,2-dithiol

Example 8Bb: Propane-1,3-dithiol

Example 8Bc: Butane-1,4-dithiol

Example 8Bd: Pentane-1,5-dithiol

Example 8Be: Hexane-1,6-dithiol

Example 8Bf: 1,4-benzenedithiol

Example 8Bg: 1,3-benzenedithiol

Example 8Bh: 1,4-benzenedithiol

Example 8 Bi: 1,3-benzenedithiol

Example 8Bj: 1,2-benzenedithiol

Example 8C: Copolymer of PEG-diamine having a side chain alkyl group

The substituted PEG succinyl ester obtained as described in Example 8A was copolymerized with 1,4-diaminobutane according to the procedure of Example 6B.

By this method, the following diamines are copolymerized with any of the substituted PEG amber oxime esters obtained as described in Examples 8A and 8Aa to 8An:

Example 8Ca: 2O-BOC 1,3-diamino-2-propanol

Example 8Cb: N', N"-di(BOC) hexaethylenetetramine

Example 8Cc: N', N"-di(BOC) spermine

Example 8Cd: N'-BOC spermidine

Example 8Ce: N', N", N'''-Tris(BOC) pentaethylene hexamine

Example 8Cf: agmatine

Example 8Cg: tert-butyl lysine

Example 8Ch: 1,6-Diaminohexane

Example 8 Ci: 1,4-styrene diamine

Example 8Cj: 1,3-styrene diamine

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

Example 9: Transesterification using a substituted acid PEG

PEG xylene sulfonate: To 1 mol of PEG (dissolved in DMF or melted), 2.1 mol of toluenesulfonium chloride (5% molar excess) was added while stirring under argon. To the reaction mixture was added 2.2 mol of tetramethylethylenediamine (TEMED). The reaction was then incubated at 45 °C for 2 hours. The product was resolved to ethyl acetate, toluene or ethanol using TLC as solvent. The PEG xylene sulfonate can be eluted from the reaction mixture with toluene. Tolsulfonium chloride, other sulfonating agents such as methanesulfonate chloride (see, Example 4), trifluoromethanesulfonic anhydride or trifluoroethanesulfonyl chloride can also be used (see U.S. Patent Application Serial No. 10/397,332, Publication No. 20040006051).

Polyesterification of PEG xylene sulfonate: 1 mol of S,S'-dimercapto-m- 2,3-didecyl amber was added to 1 mol of molten PEG-xylene sulfonate with stirring under argon. Acid and 2 mol of TEMED. When needed, DMF is added to maintain fluidity. The reaction mixture was heated to 80 ° C and stirred for 24 hours or until TLC monitoring was completed.

Example 10: PEG-bis(amber oxime)-di-(O-deuterated) thioether medium molecular weight polymer (C16-π-polymer B)

The PEG-hydrogen maleate prepared according to Example 1 (10.24 g, 6.1 mmols) was placed in a dry 125 ml bottle and heated to 70 ° C in argon to melt the PEG-hydrogen maleate. To the molten material, water (10 mL) and DTT (0.961 g, 6.168 mmols) and TEMED (0.723 g, 6.166 mmols) in water (3 mL) were added with stirring. The solution was stirred at 70 ° C for about 4 hr. Water was removed in vacuo to give a solid polymer of approximately 90% yield.

The dried polymer (5 g, 2.7 mmols) was heated to 70-90 ° C under argon to melt it, and TEMED (0.635 g, 5.5 mmols) was added. With stirring, palmitoyl chloride (1.689 g, 5.5 mmols) was added and the mixture was stirred overnight under argon (the ratio of polymer to ruthenium chloride could be varied to give a stoichiometric degree of substitution of 0-100%). Water was added to the reaction mixture to separate "C16-π-Polymer B".

By this method, the following acids can be deuterated with the hydroxy group of the bis(amber) PEG-DTT copolymer:

Example 10a: oleic acid

Example 10b: Cholesterol succinate

Example 10c: Diphenyl-4-carboxylic acid

Example 10d: 4-octylphenylacetic acid

Example 10e: Hexadeca-6-acynoic acid

As an alternative to the sulfhydryl halide application, the DTT-derived hydroxyl group of the π-polymer can also be used as 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl). Carbodiimide (BDDC) and is directly linked to the carboxylic acid; see Handbook of Reagents or Organic Synthesis, Reagents for Glycoside, Nucleotide, and Peptide synthesis, Ed. David Crich, Wiley, 2005 p 107-108 and references) .

Example 11: Carboxy-substituted ester of C16-π-polymer A

The carboxylic acid-substituted polymer is used to attach a ligand having a reactive amine group using a standard peptide bond formation method (for example, via a carbodiimide reagent) to attach the amine group to the carboxylic acid functional group of the polymer. These materials are readily obtained by esterifying the hydroxyl group of the π-polymer with a cyclic anhydride. For example, a C16-π-polymer A hydrogen maleate is prepared by reacting a cyclic anhydride with a C16-π-polymer A hydroxyl group as follows: In a dry mortar, C16-π-Polymer A (2 g) and maleic anhydride (0.85 g) were ground and transferred to a 50 mL round bottom flask. The bottle was heated in argon at 90 ° C for 2-3 hr with stirring. The solid reaction mixture was then milled and slurried with water and the mixture was transferred to a dialysis bag (3.5 kDa cut off). The mixture was dialyzed against water to remove excess maleic acid and low molecular weight by-products, and the retentate was removed from the bag and dried to a very constant weight at 60 ° C to give C16-π-polymer A hydrogen maleate. Ester (1.79 g). The ratio of polymer A to maleic anhydride can be varied to give a 0-100% substitution of a fully stoichiometric amount of esterification.

Example 11a: C16-π-Polymer A diglycolate

By the method of the above Example 11, C16-π-polymer A (2 g) and dihydroxyacetic anhydride (1.0 g) were reacted to give C16-π-polymer A diethylene glycol ester. When maleic anhydride is present, the ratio of polymer A to anhydride can be varied to give a 0-100% substitution of a fully stoichiometric amount of esterification.

Example 11b: C16-π-Polymer A (aconitate)

C16-π-Polymer A (2 g) and aconitic anhydride (1.35 g) were reacted by the method of the above Example 11 to give C16-π-polymer A bis(aconate).

In a similar manner, the following anhydrides were combined with C16-π-Polymer A. When a low solubility anhydride is used, the pH as a purification aid can be adjusted to between 4.5 and 6.5 prior to dialysis. A second dialysis of 0.1 N HCl provides the acidic form of the polymer, if desired.

Example 11c: Succinic anhydride

Example 11d: glutaric anhydride

Example 11e: Phthalic anhydride

The reactive double bond introduced by esterification with maleic anhydride or cis-aconitic anhydride can also be used to add a sulfur-containing hydrocarbon complex to the polymer, as described in Example 12, below.

Example 12: Cysteamine adduct of C16-π-polymer A hydrogen maleate:

Powdered C16-π-polymer A hydrogen maleate (Example 11) (253 mg) was added to water (5 mL), and the mixture was vigorously stirred. Cysteine acid (24 mg) and TEMED (30.5 ul) were added to the reaction mixture, and the mixture was stirred under an argon atmosphere at room temperature. The progress of the reaction was monitored by TLC (silicone plate, n-butanol-acetic acid-water, 3:1:1) using ninhydrin. The reaction mixture shows a ninhydrin-positive spot co-transferred with the polymer. Cysteine also gives a ninhydrin-positive point, whereas the starting polymer does not give any color with ninhydrin.

Using a thiol having a polyhydroxy substituent, the above method is used to introduce an additional carboxyl group serving as a point of attachment. For example, decyl succinic acid is added to the following C16-π-polymer A diester:

Example 12a: C16-π-Polymer A hydrogen maleate;

Example 12b: C16-π-Polymer A diacrylate (dicrylate)

Example 12c: C16-π-Polymer A (di) aconate

Example 12c

In a similar manner, 3-mercaptoglutaric acid was added to the following C16-π-polymer A diester:

Example 12d: C16-π-Polymer A hydrogen maleate

Example 12e: C16-π-Polymer A Diacrylate

Example 12f: C16-π-Polymer A (di) aconate

3. Apply π polymer to dissolve insoluble or slightly soluble substances

Example 1: Dye dissolution

In a separate vessel (FlexExcel ^TM transparent polypropylene weighing boat (weigh-boats), WB2.5 size, AllExcel, Inc., West Haven, CT products) in, PEG1500- to co 1.0ml of 50mg / ml of - An aliquot of an aqueous solution of amber oxime-DTT-di-C16-guanamine polymer (C16-Polymer A, Example 1), which was insoluble by centrifugation but not otherwise purified - was added in excess The dyes Eosin Y, dichlorofluorescein and Sudan IV, and these ingredients are stirred together to form a paste. Use waterproof double-sided tape and connect the bottom of the container to the bottom of the small gem ultrasonic cleaner bath. Just enough water is added to the bath to submerge the weighing boat to approximately 1/3 height. The sonication was carried out for 15 minutes in a 5 minute step. The liquid was transferred to a centrifuge tube and centrifuged twice in a bench top centrifuge for 30 minutes to precipitate an insoluble dye. The supernatant was transferred to a clean tube and centrifuged again to remove entrained solids. A suspension of the same amount of dye in distilled water in the same amount as the amount of the polymer solution was treated in the same manner as a control. The resulting solution was spotted (25 ul) on a TLC plate to form a circle from the drop. The density of the change is compared to the point made from the standard of the dye solution made in ethanol or ethanol/water to determine the approximate concentration; the change is shown in FIG. The dye is determined in water by dissolving the appropriate amount of dye in 1 L or more of deionized water (buffer-free) at room temperature and if further water is added (ie, titrated) to obtain a saturated solution. Solubility.

Sudan IV in the 50 mg/ml polymer is approximately 0.2 mg/ml, and in contrast, 0.000 mg/ml in H ₂ O (Sudan IV is insoluble at neutral pH). The dichlorofluorescein in the 50 mg/ml polymer was about 5 mg/ml, and in contrast, it was 0.010 mg/ml in H ₂ O. Eosin Y in the 50 mg/ml polymer was approximately 5 mg/ml, and in contrast, 0.007 mg/ml in H ₂ O. The effective load ratio (g/g of polymer per unit amount of polymer) was calculated to be about 1:250 for Sudan IV, 1:10 for dichlorofluorescent yellow, and 1:10 for Eosin Y.

For polar compounds - physiochemical properties which is assembled pharmaceutically active substance, 1: 10 is higher than the ratio of payload payload ratio liposome, cyclodextrins, Cremophor ^TM detergent or other solubilizing or available systems. Eosin Y is a highly efficient photoactivatable monooxygen generator. This concentrated Eosin Y solution, as prepared with the polymer of Example 1, can be expected to be a photoactivatable cytotoxic agent with pharmacology. active.

Fluorescence broad spectrum of dichlorofluorescein is visually perceptible in the polymer solution (dense yellow/orange) for changes in water (yellow-green) and gives the indication that the dye is not in an aqueous environment Instead, it is encapsulated in the organic environment of self-assembled polymer particle cores. In fact, changes in the fluorescence spectrum have not been used as a means of determining the polarity change of the microenvironment (e.g., "lipid probe"). The color of the Sudan IV solution in the polymer is reddish brown, relative, in B The alcohol solution is red when dissolved in water and is a brown powder. Eosin Y did not show a clear visual change (pink in water, reddish pink in polymer solution).

Example 2: Solubilization (dissolution) of medically relevant substances

Purpurin, amphotericin B, camptotheca and doxorubicin were selected as representative sparingly active pharmaceutical ingredients (APIs). Amphotericin B is used as an injectable antibacterial agent in liposome preparations, while camptotheca acuminata and doxorubicin are anticancer drugs. Purpurin is a DNA intercalating dye with effective pharmaceutical applications, and Eosin Y is a photosensitive monooxygen reagent that has an effective application in aerodynamic treatment. Each API was dissolved in water with C16-π-Polymer A, C18-π-Polymer B and/or C16-π-Polymer A-folate combinations (see below). Dissolution was demonstrated by spotting the dissolved API and the non-dissolved control on the TLC plate, as described above for the dye.

If needed, the dried polymer is reconstituted with water, heat, agitation and sonication. When the solution viscosity is too large, dilute it. C16-π-Polymer A was used at 10% w/v, folic acid C16-π-Polymer A was used at 5% w/v, and C18-π-Polymer B was used at 2% w/v.

The drug substance (20 mg) was added directly to 1 ml of the polymer solution, which formed a polymer: the API quality ratio was 5:1 for C16-π-polymer A and 2.5 for C16-π-polymer A for folic acid: 1, 1:1 for C18-π-Polymer B, except for doxorubicin (see below). The mixture was sonicated at low power for 1 hour and then centrifuged twice at 2000 xg to remove undissolved solids. The amount of precipitated solids is not significant.

Doxorubicin hydrochloride is combined with the polymer as described above: at a mass ratio of C16-π-polymer A to doxorubicin hydrochloride of 10:1, or a mass ratio of C16-π-polymer A to doxorubicin of folic acid 5: 1 then, add sufficient 3M sodium acetate to neutralize doxorubicin hydrochloride. The mixture was shaken vigorously for 24 hours and then centrifuged twice at 2000 xg to remove undissolved solids. The amount of precipitated solids is minor.

The quality ratio of dissolved API to polymer is shown in Table 1. Did not try The loading of the polymer is maximized, so these ratios represent the lower limit of the amount of API that the polymer can carry into the solution.

A 10 ul sample of each solution was spotted on a Bakerflex ^(TM) silicone TLC plate and allowed to spread. The aqueous solution forms an outer boundary of the annulus formed by the movement of the polymer having the encapsulating material and the inner ring. In all cases, there is still very little API around the water-only area, which indicates successful dissolution and minimal leakage of the dispensed material.

4. Biocompatibility of π-polymer

Example 1: Suitable for topical emollients, ointments or patches

The concentrated oil-containing wax of the polymer of Example 1 was rubbed on the skin inside the wrist by the inventors and observed for ingestion. The material was shown to be absorbed similar to a pharmaceutical waxy paste and slightly softened to this area. After this single topical application, no immediate delayed allergic reactions such as redness, rash or itching were observed.

Many of these polymers are hygroscopic waxes at room temperature, and the desired mp is from about 45 ° C to 60 ° C or higher, depending on the ingredients. Polymers made with low molecular weight PEG can even be liquid at room temperature. Some polymers can be solid at room temperature and melt at body temperature. The characteristics of these π-polymers therefore make them a good substrate for the manufacture of lotions, ointments, ointments, emollients and other forms of transport, either by themselves or in mixtures with a variety of substances, It includes active pharmaceutical preparations.

Example 2: Suitable for parenteral administration

An aqueous solution of the polymer of Example 1 was prepared in a phosphate-buffered saline solution and then filtered through a 0.22 um filter into a sterile tube.

The maximum tolerated dose regimen was used in which CD-1 mice underwent a 10 ml dose per kg body weight and a tail vein was injected with up to 5% w/v of aqueous polymer solution. Mice were observed continuously for 12 hours and thereafter every two hours until 48 to 72 hrs, depending on the group. Blood samples were extracted and analyzed. Kill some mice and first check the total histology. Microscopy histology is then performed on the selected sections.

No observable differences were found in the blood chemistry of control and treated mice. No observable differences were found in the total histology of various organs including heart, lung, kidney, spleen, liver, intestine, stomach, bladder, skin, muscle, brain and lymph nodes compared to control animals. . A variety of samples from different groups of animals were studied and similar results were observed. There were no observable differences in the cellular organization of the examined tissue. Some kidneys show some reduction in exposure to polymer time. This implies that the discharge is a temporary phase and that it will become normal as time progresses.

It is safe to include pharmaceutical agents for medical use as injectable formulations and other parenteral formulations. It is reasonable to expect that the polymer is safe in oral solutions, capsules and tablets, nasal sprays, oral/bronchial sprays, sublingual, skin ointments/lotions/patches, eye drops, other topical routes, and other routes of administration. .

5. Connect the targeting moiety to the π-polymer

Example 1: Formation of galactosamine to C-16-π-Polymer B via guanamine bond formation

Galactosamine (GA) targets hepatic sialoglycoprotein receptor (ASGPR), a polymer with covalently linked galactosamine delivered to the liver; see L. Seymour et al, "Hepatic Drug Targeting: Phase I Evaluation of Polymer-Bound Doxorubicin" J. Clin. Oncology, 20(6): 1668-1676 (2002) and references therein.

C16-π-Polymer B (Example 10 of the synthesis method section above) (461 mg, 0.2 mmols equivalent of COOH per repeat unit was dissolved in 14 mL of water, and EDC HCl (0.485 mmols) and N-hydroxybutylimine (0.464 mmols) were added to the dispersion. The mixture was stirred at ambient temperature for 15 minutes, and a solution of galactosamine HCl (0.386 mmols) and TEMED (0.387 mmols) in 1 ml of water was added. After stirring the solution and reacting, TLC was carried out on silica gel and developed in 1-butanol-acetic acid-water (3:1:1). Additional amounts of TEMED (0.079 mmols), NHS (0.078 mmols) and EDC HCl (0.193 mmols) were added to force the reaction to completion. For consumption of GA, when the TLC exhibited a steady state, the reaction mixture was dialyzed against 3 x 1000 ml of deionized water (3500 Da cut-off membrane) to remove low molecular weight reactants and by-products. The retentate was removed and dried to constant weight (348 mg) at 60 °C.

TLC of the product indicated no free GA (ninhydrin negative). A sample of the product was hydrolyzed with 6N HCl at 100 ° C to hydrolyze the bound GA. TLC analysis indicated the presence of GA (ninhydrin positive) at the same Rf as the reference GA.

Example 2: Attaching folic acid to C18-π-polymer A

BDDC (2.44 g, 8.56 mmols) was weighed and added to a 125 mL round bottom bottle flushed with argon (BDDC is very viscous, has a honey like consistency and is difficult to handle). C18-π-Polymer A (I0g, 4.28 mmols) was added to the bottle, the mixture was heated to 70 ° C, and the reaction was stirred together for about 30 minutes. Folic acid (3 g) was added followed by sufficient THF to stir as much as possible. The reaction was stirred at 40-70 ° C overnight to protect it from moisture. The THF was then evaporated and water (80 mL) was added and the mixture was stirred at 50 &lt After cooling to room temperature, the mixture was transferred to a portion of the dialysis tube, shut off with 3500 Dalton, and used for 0.1 N HCl (2 x 2000 ml), water (2000 ml), 5% sodium carbonate (2 x 2000 ml) and water (4 x 2000 ml). Dialysis to remove unreacted reagents and by-products. Remove the bright yellow-orange retentate. A portion was evaporated to a constant weight to determine the solid concentration, and it was used in the above dissolution (solubilization) experiment.

Example 3: Attaching N-acetamidine neurone acid (NANA) and analogs to -π-polymer

The neuraminic acid derivative is a targeted part of the influenza virus because of the hemagglutinin and neuraminidase-coated proteins, which are all known to bind to sialic acid. Several methods for attaching NANA and its derivatives to the π-polymers of the present invention were developed.

Example 3a: Attaching N-acetamidine neurone acid (NANA) to C18-π-polymer A via esterification

BDDC (2.44 g, 8.56 mmols) and C18-π-Polymer A (10 g, 4.28 mmols) were combined and heated to 70 ° C and stirred together under argon for approximately 30 minutes. N-acetyl ceramide (3 g) was added and then THF was added if necessary to maintain fluidity. The reaction was stirred at 40-70 ° C overnight to protect it from moisture. Water (80 mL) was added and the mixture was stirred at 50 ° C for an additional 2 h. After cooling to room temperature, the mixture was dialyzed against 0.1 N HCl, 5% NaHCO ₃ and water (2 x 2000 ml each time) and the membrane was shut off with 3.5 kDa. Spotting onto a silicone TLC plate and showing the addition of neuramic acid to the polymer with a 0.2% orcinol of 70% sulfuric acid at 130 °C.

Example 3b: Attaching N-acetamidine neurone acid (NANA) monomaleate to C16-π-polymer A

5-N-Ethylceramide (NANA) (0.86 mmols), maleic anhydride (0.93 mmols) and triethylamine (1.77 mmols) were dissolved in 1.5 mL of DMSO in a dry round bottom flask. The bottle was rinsed with argon and placed in an oil bath. The mixture was stirred at 65 ° C to 85 ° C and the progress was checked by TLC on a silica gel plate (i-PrOH-EtOAc-water, 4:3:2) until the reaction was completed (with orcinol/H ₂ SO ₄ or urea) /HCl reagent detection, no NANA). The reaction mixture was cooled to room temperature and water was added to hydrolyze excess maleic anhydride. The NANA monomaleate formed is used directly in the next reaction.

An aqueous solution of C16-π-polymer A diethylene glycol ester (see, "Synthesis of π-Polymer", Example 11a) (1.23 mmols repeat unit, 2.46 mmols-COOH) was adjusted to pH 4.5-6.5. Carbonodiimide (EDC HCl, 3.86 mmols) and N-hydroxybutylimine (2.6 mmols) were added and the mixture was stirred at room temperature for about 60 minutes. A solution of NANA monomaleate (2.49 mmoles) was added - prepared as above and pH adjusted to pH 6 with TEMED 7. Stirring was continued for up to 26 hours at room temperature. The product was purified by dialysis, first to 20 mmolar sodium acetate at pH 4.5, then to water. Remove the retentate and store it for use.

Example 3c: Attaching N-acetamidine neurone acid (NANA) to C16-π-polymer A via a spacer

Add cysteamine (2-aminoethyl mercaptan) hydrochloride (0.93 mmol in water) to an equimolar amount of NANA monomaleate (prepared as described above), then add an equimolar amount of TEMED to Helps add sulfur to the double bond. After the reaction, use TLC on vermiculite (i-PrOH-EtOAc-water, 4:3:2) until the reaction is complete (detected with orcinol/H ₂ SO ₄ or urea/HCl reagent, no O-ma The thiol-NANA is given to give the targeting moiety D.

5-N-Ethyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) was derivatized by the same method to give a targeting moiety E.

Add cysteine and glutathione to NANA and DANA by the same method Maleic acid monoester.

The thiosuccinate conjugate of C16-π-polymer A bis(aconitate) was aminated with a targeting moiety D by the method described in Example 3b above. The polymer contains up to 8 COOH groups per repeat unit (see "Synthesis of π-Polymers", Example 12c).

Example 3d: Attaching N-acetamidine neurone acid (NANA) to a C16-π-polymer A via a spacer

The targeting moiety E was attached to the C16-π-polymer A diethylene glycol ester by the method described above (see "Synthesis of π-Polymer", Example 11a) polymer.

Example 3e: Attaching thyminine β-methylglycoside (MNA) to a C16-π-polymer

(0.396 mmol) having an average of one carboxyl group per repeat unit was dissolved in water and allowed to react with NHS (0.4 mmol) and EDC-HCl (0.64 mmol). Neurone-β-methyl glycoside (MNA) (0.42 mmol) was added. The reaction mixture was stirred at ambient temperature (25-30 ° C) for 18-24 hours and then purified by dialysis.

Example 3f: A sample of a second C16-π-polymer A diethylene glycol ester having two carboxyl groups per repeat unit was combined with MNA in the same manner.

Example 3g: Attaching β-O-methylneuraminic acid (MNA) to C16-π-Polymer B

C16-π-Polymer B--43 micromoles COOH base, mixed in 1 ml of water-- and tranexamic acid-β-methylglycoside (Toronto Research Chemicals) -40 micromolar, and added to 0.1 ml of water 40 micromoles of NHS were then added to 40 micromoles of EDC hydrochloride in 0.1 ml of water. The reaction mixture was shaken at room temperature for 48 hours and then analyzed by TLC on EtOAc (EtOAc:EtOAc) No color reaction with the starting polymer was detected with 0.1% orcinol in 70% sulfuric acid at 130 ° C, but the TLC of the reaction mixture gave a purple spot co-moving with the polymer.

All of the polymer conjugates in the above examples (3a-3g) have a positive anti-neuraminic acid when used after dialysis, when developed with TLC on the tallow/sulfate or urea/HCL reagent. should.

Example 4: Attaching zanamivir to C16-π-Polymer B

Zanamivir (GG167) is a potent inhibitor of viral neuraminidase, and polymers with such molecules such as multivalent ligands are inhibitors of influenza virus replication.

C16-π-Polymer B (920 mg) was dispersed in 30 mL of water, and EDC HCl (1.2 mmol) and N-hydroxybutylimine (1.1 mmol) were added thereto. The mixture was stirred at ambient temperature for 20 minutes in 1 ml of water of 5-acetamido-7-(6'-aminohexyl)-aminocarbamyloxy-4-mercapto-2,3. 4,5-tetradeoxy-D-glyceryl-D-galactose-non-2-pyranosonic acid (U.S. Patent Nos. 6,242,582 and 6,680,054) (0.39 g, 0.67 mmol) and TEMED (0.67) A solution of mmols) of trifluoroacetate was stirred at room temperature and after the reaction, TLC was carried out. The reaction mixture (3500 kDa shut-off membrane) was dialyzed against 3 x 1000 ml of deionized water to remove low molecular weight reactants and by-products. The retentate was removed and dried to a constant weight at 60 °C. The level of sugar incorporation can be detected by colorimetric analysis of the thiol group (Can. J. Chem., 36: 1541 (1958)). Neuraminidase analysis can be performed according to the method of Potier et al., Anal Biochem., 29 287 (1979).

Example 5: Connecting Mimosa

C16-π-Polymer A diethylene glycol ester of 4.5% w/v solution (1 mmol repeat unit, approximately 2 mmol COOH group) (see "Synthesis of π-Polymer", Example 11a) and NHS (2.27 mmol) And reacting with EDC‧HCl (2.23 mmols), adding a solution of 1-Mimosa (2.14 mmol, prepared in 5 ml of water and adjusting pH with TEMED to increase solubility) to the resulting mixture, and stirring at ambient temperature - pH about 6.8-7, about 22-24 hours. The pH was adjusted to 3-4 with 6N HCl, the mixture was stirred for 15-30 minutes, and the pH was increased to 6.1 by the addition of TEMED. The mixture was then dialyzed against water (3.5 kD cut off) to remove impurities and low molecular weight products.

Example 6: Attaching peptides and proteins to π-polymer A hydrogen maleate and diacrylate:

For the general procedure of Fab fragments: the disulfide bond in the F(ab')2 fragment of the antibody is reduced using the immobilized TCEP Disulfide Reducing Gel (Pierce, product number 0077712) using the manufacturer's protocol; or alternatively DTT in solution or The TCEP was reduced and the depleted reagent was removed by ultrafiltration using a 30 kD filter. The reduced F(ab')2 fragment containing the free sulfhydryl group is then reacted with π-polymer A hydrogen maleate or diacrylate in the presence of TEMED.

General procedure for cysteine and cysteine-containing peptides : The acrylate or maleate of π-Polymer A is reacted with a cysteine residue using triethylamine as a catalyst. To a suspension of polymer diacrylate (0.3 mmols repeating unit, 0.6 mmols acrylate) in water, cysteine (0.66 mmol) and triethylamine (1.32 mmol) were added. The bottle was flushed with argon and stirred at ambient temperature overnight (about 18 h). The reaction mixture of TLC on vermiculite (i-PrOH-ethyl acetate-water, 4:3:2) shows the absence of cysteine in the polymer and the ninhydrin-positive point, suggesting the addition of cysteine Into the acrylate double bond.

Example 6a: Attachment of a pandemic antibody fragment to C16-π-polymer A diethylene glycol ester:

A C16-π-polymer A hydrogen maleate was prepared starting with a PEG having a molecular weight of 4500. The BayRab ^TM human rabies immune globulin (hlgG) with pepsin in a conventional manner in acidic pH buffer treated to produce F (ab ') 2 fragments, a filter using a 50kD purified by ultrafiltration. The F(ab')2 fragment was ligated to PEG 4500 C16-π-Polymer A diethylene glycol ester by the EDC method described in Example 5 above.

Example 6b: Attachment of an anti-rabies antibody fragment to C16-π-polymer A hydrogen maleate:

Reduction BayRab ^TM hIgG F (ab ') with DTT (or optional TCEP) 2 fragments (see Example 6a), use of reagent was removed by ultrafiltration filter 30kD depleted. The Fab'-SH fragment was ligated to PEG 4500 C16-π-polymer A hydrogen maleate by addition of the free alcohol hydrocarbon Michael to the maleic acid double bond at pH 7-8.3 (TEMED). The conjugate was purified by ultrafiltration using a 100 kD filter to remove low molecular weight contaminants.

Example 6c: The PEG 8500 C16-π- Polymer A dimaleate ester as connected to the reduced ^{BayRab TM hIgG F (ab ')} 2 fragments.

Example 6d: Attachment of peptide to C16-π-polymer A hydrogen maleate:

The peptide KDYRGWKHWVYYTC ("Rab 1") has been reported to bind to the rabies virus (T. L. Lentz, 1990, J. MoI. Recognition, 3(2): 82-88). The Cys at the end of the peptide was used to synthesize an anti-rabies peptide-π-polymer A conjugate. The C16-π-polymer A hydrogen maleate (per repeat unit, two maleic acid fractions) obtained from onion PEG 1500 (0.157 mmol) was dissolved in water (6 mL), and the pH of the solution was TEMED. Adjust to about 8. The peptide (0.157 mmol) dissolved in DMF (3.1 ml) was added, and the reaction mixture was stirred at ambient temperature under argon while maintaining the pH between 8 and 8.3. The progress of the reaction was examined by detecting the reaction mixture with Ellman's reagent. After about 45 hours, Ellman's test was almost negative. Water was added to lower the DMF concentration, and the reaction mixture was centrifuged to remove a small amount of precipitate. The clear supernatant was ultrafiltered through a 10 kD centrifugal filter element (Arnicon Ultra 10kD, Cat. No. UFC901024) and the retentate was washed repeatedly with water to remove low molecular weight contaminants.

The following three peptides (O=ornithine; NH ₂ refers to C-terminal decylamine) were prepared by automated solid phase analysis and bound to PEG1500 π-Polymer A hydrogenate in the same manner as the Rab1 peptide. :

Example 6d: KDYRGWKOWVYYTC ("Rab2")

Example 6e: KGWKHWVYC(NH ₂ )("Rab3")

Example 6f: KGWKOWVYC(NH ₂ )("Rab4")

6. Antiviral activity of π-polymer

Example 1: Fighting the effects of influenza

The ATCC VR-1520 (H2N1) human influenza virus was used for mouse protection analysis. In control animals, a single tail vein injection, 200 ul / 20 g BW resulted in 99.5% lethal infection (7 days untreated survival).

10 mice per group. A small dose (0.0375%) and a high dose (0.15%) solution of the above π-polymer B-MNA conjugate of Example 3 of 200 ul/20 g body weight was injected into the tail vein. mouse. Post-treated animals were administered 24 hours after infection, while pre-treated animals were administered 6 hours prior to infection. A positive control was injected with an equal amount of free ligand, while a negative control received saline buffer injection.

The survival time is used as an index efficacy end point. Track weight as a research parameter. The histology of internal organs is performed in both total and microscopic examinations. The result is shown in Figure 2.

The increased survival time for the high dose treatment group was 5.93 hours (+/- 0.48 h SD) compared to 2.94 hours (+/- 0.75 h SD) for the positive control (Figures 1 and 2). On the basis of the mass of the ligand, the high dose treatment corresponds at most 0.03% of the ligand, which assumes a maximum polymer-conjugate substitution ratio of 0.2 w/w. Thus, the polymer conjugate shows a significantly higher level of efficacy than the unbound ligand control.

In the high-dose polymer B-MNA conjugate treatment group, the total histology and microscopic examination of some mice showed normal patterns, except that mice treated partially in the bone marrow showed a slight decrease in the level of white blood cells, which was attributable to The impact of influenza infection. The reduction in body weight in the protection group (high dose -8.9%, low dose -6.2%) and treatment group (high dose -9.0%, low dose -9.4%) was less than the positive control (-9.8%), suggesting The body itself is not a combination of polymers (Figure 3). A 0.7% small weight gain occurred in untreated mice.

Example 2: Effect on madness

The group of ten white Swiss mice - approximately 20 g each, mixed sex - was used for in vivo protection assays. Mice were challenged by injection of LD50 of 3x rabies virus. The injection was 0.03 ml CVS (Attack Virus Standard) rabies strain, ^10-6 dilution (100 LD ₅₀ /ml). Survival, sputum and body quality are monitored day through. Drugs were administered intraperitoneally at 25, 48, and 72 hours, and drugs were administered intracerebally at 25 and 48 hours. The results are shown in Tables 1 and 2.

Claims (11)

A comb polymer for treating or preventing infection by a virus, the comb polymer consisting essentially of the following structural formula: It comprises a main chain formed by alternating branch point moiety B and a hydrophilic water soluble polymer block A; and a ligand Z having a hydrophobic side chain C and a moiety attached to the branch point, wherein: the water soluble The polymer block A is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), poly(ethyleneimine), poly(vinyl alcohol), poly(vinylpyrrolidone), and copolymers thereof; Point portion B is a combination of dithiothreitol, dithioerythritol or 2,3-diaminobutane-1,4-dithiol with two maleic acid molecules; each hydrophobic side chain C has a logP value greater than 1.4 and is independently selected from the group consisting of C ₆ -C ₃₀ straight or branched chain hydrocarbons which may optionally be substituted by one or more hydrophilic substituents; C ₆ -C ₃₀ cyclic Or a polycyclic hydrocarbon, which may optionally be substituted with one or more hydrophilic substituents; and a hydrophobic amino acid, a hydrophobic peptide or a hydrophobic polymer; each ligand Z is independently a ligand having a specific binding affinity for the surface of the virus. s is a bond or a spacer moiety; the value of n ranges from 3 to about 100; the average of p ranges from 1 to 4; and the average of r In the range of 1-8. A comb polymer according to claim 1, wherein at least one of the ligands is N-acetyl ceramide, glutamic acid β-methyl glucoside or 4-mercapto-2,4-dideoxy-2,3- Dehydro-N-acetyl ceramide. The comb polymer of claim 1, wherein at least one of the ligands Z is a peptide, antibody or antibody fragment having a specific binding affinity for the surface of the virus. The comb polymer of claim 1, wherein the polymer block A is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), and copolymers thereof. The comb polymer of claim 4, wherein the polymer block A is poly(ethylene glycol). The comb polymer of claim 4, wherein the polymer block A has an average length of between 4 and 700 monomer units. The comb polymer of claim 1, wherein the polymer has the following structural formula: Wherein m is 4-700, and Y and Y' are independently selected from the group consisting of R, OR, COOR, SR, NHR, NRR', ONHR, NHOR, NRNH ₂ , NHNHR, NRNHR', and NHNRR', wherein R And R' are independently selected from the group consisting of C ₆ -C ₃₀ branched or straight chain hydrocarbons which may optionally be substituted by one or more hydrophilic substituents; C ₆ -C ₃₀ cyclic or polycyclic hydrocarbons , which may optionally be substituted by one or more hydrophilic substituents; and a hydrophobic amino acid, hydrophobic peptide or hydrophobic polymer, provided that when Y and Y' are NHR and s is a bond, Z is not N-acetylene The nervonic acid is also not β-O-methylneuraminic acid. The comb polymer of claim 1, wherein the polymer has the following structural formula: Wherein m is 4-700, W is O or NH, and Y and Y' are independently selected from R, COR, COOR, CONHR, CONRR', CONHOR, CONRNH ₂ , CONHNHR, CONRNHR', and CONHNRR'; wherein R and R 'Independently selected from the group consisting of C ₆ -C ₃₀ branched or straight-chain hydrocarbons, which may optionally be substituted by one or more hydrophilic substituents; C ₆ -C ₃₀ cyclic or polycyclic hydrocarbons, Optionally substituted with one or more hydrophilic substituents; and a hydrophobic amino acid, hydrophobic peptide or hydrophobic polymer, provided that when Y and Y' are COR, W is NH and s is NH(CH ₂ ) ₆ Z is not Zanamivir. The comb polymer of claim 1, wherein the polymer has the following structural formula: Wherein m is 4-700, W is O or NH, and R and R' are independently selected from the group consisting of C ₆ -C ₃₀ branched or straight chain hydrocarbons which may optionally undergo one or more hydrophilic Substituent substitution; C ₆ -C ₃₀ cyclic or polycyclic hydrocarbons which may optionally be substituted with one or more hydrophilic substituents; and hydrophobic amino acids, hydrophobic peptides or hydrophobic polymers. The comb polymer of any one of claims 1 to 3, wherein the virus is influenza virus. A comb polymer according to any one of claims 1 to 10 for use in the preparation of a medicament for the treatment or prevention of a viral infection.