HK1078097B - Cyclodextrin grafted biocompatible amphiphilic polymer and methods of preparation and use thereof - Google Patents

Description

Cyclodextrin grafted biocompatible amphiphilic polymer and preparation method and application thereof

Background

The present invention relates to novel polymeric bioactive agent carriers. More particularly, the present invention relates to cyclodextrin grafted biocompatible polymers useful as carriers for bioactive agents and methods for their preparation.

Many biologically active molecules such as antiviral agents, anticancer agents, peptides/proteins and DNA, with a variety of different therapeutic effects, have been offered as commercial products through advances in recombinant DNA and other technologies. However, there is always a need for certain ideal carriers for drugs and active agents to improve their solubility, release and therapeutic efficacy.

Cyclodextrins (CD) are cyclic oligosaccharides typically composed of 6-8 glucose units having a truncated cone shape with a broad open side composed of secondary hydroxyl groups (2-OH and 3-OH) and a narrower side composed of primary hydroxyl groups (6-OH). Cyclodextrins provide a unique micro-heterogeneous environment because the outside of the molecule is hydrophilic, while the voids appear hydrophobic due to their higher electron density. The inclusion property of cyclodextrin, i.e., the formation of a complex between a guest molecule and a cyclodextrin molecule, has been widely studied. The complex formed in the solid state or in solution consists of the guest molecule and the host cyclodextrin which holds the guest molecule in its cavity and is stabilized by van der waals forces and, to a lesser extent, dipole-dipole interactions. Inclusion complexes in aqueous solution are believed to be further stabilized by virtue of hydrophobic interactions, i.e., the tendency of the solvent water to push hydrophobic solutes of appropriate size and shape into the substantially hydrophobic cavities in order to bring the solvent to its "most likely structure" and to bring the overall system to the lowest energy.

The practical use of natural cyclodextrins (α -, β -and γ -CD) as drug carriers is limited by their low solubility in water. Safety is another major concern with cyclodextrins as drug carriers because cyclodextrins are toxic. Modification of parent cyclodextrins to improve safety while maintaining their ability to form complex complexes with various substrates has been the goal of many research groups. Some groups have focused on improving the interaction between drugs and cyclodextrins, while others have attempted to prepare materials that can be more precisely defined chemically.

The two most promising cyclodextrin derivatives suitable for parenteral administration are hydroxypropyl β -cyclodextrin (HP β CD or HPCD) and sulfobutyl ether- β -cyclodextrin (SBE β CD or SBE-CD). HP β CD is generally found to be safe when administered parenterally to animals or humans. [ Pitha et al, J.Med.Sci.84 (8), 927-32(1995) ]. Minor, reversible, histological changes were found in high dose animal studies (100-400 mg/kg), and more significant hematological changes suggesting red blood cell damage were observed in these high dose studies. In human studies, no adverse effects were observed. SBE beta CD, was found to be safe when administered parenterally to mice [ Rajewski et al, J.Med.Sci.84 (8), 927-32(1995) ]. However, like most modified cyclodextrins, the binding constant between a drug and HP β CD is generally less than its constant with the parent or unmodified cyclodextrin. The higher the degree of substitution of hydroxypropyl groups, the poorer the binding ability of the sample due to steric hindrance of the host molecule.

Hydrophobic modifications of cyclodextrins have also been prepared in an attempt to improve the formulation of certain CD-clathrable samples. It was found that partial methylation of the hydroxyl groups, usually at the 2-and 6-positions of beta-cyclodextrin (DM-beta CD or DMCD), leads to stronger drug binding capacity due to increased hydrophobic interactions. Although methylated cyclodextrins are highly soluble in water, they also have a greater toxicity. The toxicity of DM β CD can be greatly reduced by modifying the free 3-hydroxy groups with acetyl groups. This indicates that a water-soluble cyclodextrin derivative having excellent biocompatibility and inclusion ability can be prepared, provided that the substituent group is carefully selected. Controlling the degree of substitution also has an important role in balancing water solubility and complexing ability. When the substituent groups are more hydrophobic than the methyl groups, e.g., ethyl groups, acetyl groups, etc., the entire cyclodextrin derivative will become practically insoluble in water. These compounds have demonstrated potential use as sustained release carriers for water-soluble drugs. Among the alkylated cyclodextrins, hepta (2, 6-di-O-ethyl) -beta-cyclodextrin and hepta (2, 3, 6-tri-O-ethyl) -beta-cyclodextrin are the first sustained release carriers used in combination with water-soluble diltiazem, isosorbide dinitrate and cleupin acetate.

On the other hand, peracylation has a medium alkyl chain length (C)₄～C₅) Due to the cyclodextrinMultifunctional and biocompatible are thus particularly useful new hydrophobic carriers. They have a wide range of applicability to a variety of different routes of administration: for example, hepta (2, 3, 6-tri-O-butyryl) -beta-cyclodextrin (C)₄) The bioadhesive property of (A) can be used in oral or transmucosal formulations, and hepta (2, 3, 6-tri-O-valeryl) -beta-cyclodextrin (C)₅) The film-forming property of (a) is useful for transdermal preparations. In oral administration, molsidomine, a water soluble and short half-life drug, whose release is significantly slowed by the use of peracylated- β -cyclodextrin as its solubility decreases, particularly when those with a longer carbon chain than the butylated derivative are used. When the complex is administered orally to beagle dogs, hepta (2, 3, 6-tri-O-butyryl) -beta-cyclodextrin depresses the peak plasma concentration value of molsidomine and maintains adequate drug levels over a prolonged period of time, while other derivatives with shorter or longer chains than hepta (2, 3, 6-tri-O-butyryl) -beta 0-cyclodextrin demonstrate insufficient sustained release effects. This suggests that hepta (2, 3, 6-tri-O-butyryl) - β 1-cyclodextrin may be a useful carrier for orally administered water-soluble drugs, particularly drugs that are metabolized in the gastrointestinal tract. The excellent and sustained effect exhibited by hepta (2, 3, 6-tri-O-butyryl) - β 2-cyclodextrin may be the result of both increased hydrophobicity and mucoadhesion. Due to their hydrophobic nature, hepta (2, 3, 6-tri-O-butyryl) - β 3-cyclodextrin and other hydrophobic cyclodextrin derivatives can only be used in solid or oily formulations. On the other hand, like native β -cyclodextrin, its membrane toxicity, which causes tissue irritation and hemolysis in a concentration-dependent manner, is another limitation that hinders its use in pharmaceutical applications. For example, DM- β -CD induces 50% hemolysis in human erythrocytes at concentrations lower than the corresponding concentrations of so-called "biocompatible" CD derivatives such as 2-hydroxypropyl- β -CD, sulfobutyl ether of β -CD, and maltosyl- β -CD. The hemolytic activity of cyclodextrin is associated with the extraction of membrane components, mainly by inclusion with cholesterol. However, this disadvantage can be overcome by further structural modification of the alkylated CD, for example, hepta (2, 6-di-O-methyl-3-O-acetyl) -beta-CD (DMA-beta-CD), which has been found to be much weaker in hemolysis while maintaining inclusion capacity similar to that of MD-beta-CD [ Hirayama et al, J. Med. Sci. 88(10), 970-5(1999)]. Since cyclodextrins are poorly absorbed from the gastrointestinal tract after oral administration, oral administration of cyclodextrins rarely poses a safety hazard due to systemic absorption of the cyclodextrins themselves. However, cyclodextrins can cause secondary systemic effects by increasing gastrointestinal elimination of certain nutrients and bile acids. This effect is most pronounced in the fecal elimination of bile acids assisted by delta-cyclodextrin. However, this enhancement of the elimination effect is only observed at very high oral doses of cyclodextrin (up to 20% of the diet). The secondary effect of enhanced bile acid elimination increases the conversion of serum cholesterol to bile acids, with the consequence of a decrease in plasma cholesterol levels.

A wide variety of cyclodextrins have been prepared over the years to improve the physiochemical properties and inclusion capacity of the parent cyclodextrins, and certain cyclodextrin-containing drug products have been approved. Because large amounts of cyclodextrin are required to alter the solubility properties of the drug being carried, the toxicity of cyclodextrin must be very low in order to safely deliver the desired dose of drug. Thus, reducing the total dose or reducing the intrinsic toxicity of cyclodextrins can broaden the pharmaceutical applications of cyclodextrins.

In view of the foregoing, it can be seen that it would be an advancement in the art to provide an improved cyclodextrin-containing bioactive agent carrier and method of use thereof.

Summary of The Invention

The present invention provides a novel class of polymer-containing amphiphilic cyclodextrins wherein a plurality of hydrophobic cyclodextrin or derivatized cyclodextrin moieties are linked or grafted to a biocompatible hydrophilic polymer backbone by suitable biodegradable or non-biodegradable linkers. Optionally, one or more Targeting Moieties (TM) or mixtures thereof can also be covalently linked to the polymer backbone. The CD-grafted polymer of the present invention can be synthesized by coupling 2-30 CDs or their derivatives to a hydrophilic polymer, such as polyethylene glycol (PEG) or poly N- (2-hydroxypropyl) methacrylamide) (HPMA), through a suitable linker. If desired, one or more Targeting Moieties (TM) can optionally be covalently linked to the polymer backbone, as described above. The purpose of the targeting moiety is to target specific cells for drug delivery. The synthetic carrier, i.e., the hydrophobic CD-grafted hydrophilic polymer, improves the solubility and reduces the cytotoxicity of the drug/carrier complex.

Brief Description of Drawings

FIG. 1 is a graph showing the stability of paclitaxel/CD complex in 50% serum or 10xPBS dilution.

FIG. 2 shows a reaction scheme for the synthesis of PEG-SS-AcCD.

FIG. 3 shows a reaction scheme for the synthesis of PEG-SS-DECD.

FIG. 4 shows a reaction scheme for the synthesis of PEG-GFLG-DECD.

FIG. 5 shows the reaction scheme for the synthesis of PEG-C3-AcCD, PEG-C3-DECD and PEG-C3-BnCD.

FIG. 6 shows the reaction scheme for the synthesis of PEG-L8-AcCD, PEG-L8-DECD.

Detailed Description

Before the present compositions and methods for delivering drugs are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

"active agents" refer to those agents that can function as guest molecules of the present invention. Active agents include chemicals and other substances as long as they form inclusion complexes with cyclodextrins or derivatized cyclodextrin graft polymers, have inhibitory, antimetabolic or prophylactic effects against any disease (e.g., tumor, syphilis, gonorrhea, influenza and heart disease), or have inhibitory or toxic effects against any pathogenic agent. Active agents include many drugs, for example, anticancer agents, antineoplastic agents, antifungal agents, antibiotics, antiviral agents, cardiac drugs, neurological drugs, and drugs; alkaloids (e.g., camptothecins), antibiotics, bioactive peptides, steroids, steroid hormones, polypeptide hormones, interferons, interleukins, anesthetics, nucleic acids, including antisense oligonucleotides, pesticides, and prostaglandins. The active agent further comprises aflatoxins, ricin, cyclosporins, ipomoea, gancyclovir, furosemide, indomethacin, chlorphenamine maleate, methotrexate, acefenamide derivatives and analogs, including western medicines, desatrines, and Veratrum nigrum. It also includes various flavone derivatives and analogs, including dihydroxyflavone (chrysin), trihydroxyflavone, pentahydroxyflavone, hexahydroxyflavone, lamb salt, quercetin, fisetin; various antibiotics, including penicillin derivatives (e.g., ampicillin), cyclosporins (e.g., doxorubicin, daunorubicin), teramycins, tetracyclines, tetracycline hydrochloride, gentamicin, butoconazole, irinotecan, guanepetracycline, macrolides (e.g., amphotericin), felicidin, nystatin, various purine and pyrimidine derivatives and analogs, including 5 ' -fluoro-uracil, 5 ' -fluoro-2 ' -deoxyuridine, and allopurinol; various photoactive substances, especially those for photodynamic singlet and triplet oxygen generation, phthalocyanines, porphyrins, derivatives and analogs thereof; various steroid derivatives and analogs, including cholesterol, digitoxin; various coumarin derivatives and analogs, including dihydroxycoumarin (horsepower-psoralen), bishydroxycoumarin; anthelmintic soja, rhein, emodin, secalinic acid; various dihydroxyphenylalanine derivatives and analogs include dihydroxyphenylalanine, dopamine, epinephrine, and norepinephrine (norepinephrine hydrochloride).

By "parenteral" is meant, intramuscular, intraperitoneal, subcutaneous, and, where applicable, intravenous and intraarterial.

By "biocompatible" is meant that the substance is non-immunogenic, non-allergenic and will elicit minimal undesirable physiological reactions. They are biodegradable and they are "biologically neutral" in the sense that they lack specific binding properties or biorecognition properties.

"linker" or "ring" is defined as a specific chemical moiety or group used in chemical substances that is capable of covalently linking a cyclodextrin moiety to a polymer backbone and that may be biodegradable or non-biodegradable. Suitable linkers are defined in more detail below.

"drug" shall mean any organic or inorganic compound or substance, as long as it has biological activity and is suitable or used for therapeutic purposes. Proteins, hormones, antineoplastic agents, oligonucleotides, DNA, RNA, and gene therapy agents are all encompassed within the broad definition of drug.

"peptide," "polypeptide," and "protein" will be used interchangeably when referring to a peptide or protein drug, and will not be limited to any particular molecular weight, peptide sequence or length, biological activity, or field of therapeutic application, unless specifically indicated.

"targeting moieties" refer to those moieties that bind to a particular biological substance or site. The biological substance or site is considered to be a "target" for the targeting moiety bound to it. Examples of suitable targeting moieties include antigens, haptens, biotin derivatives, lectins, galactosamine, fucosamine moieties, receptors, substrates, coenzymes, cofactors, proteins, histones, hormones, vitamins, steroids, prostaglandins, synthetic or natural polypeptides, carbohydrates, lipids, antibiotics, drugs, digoxin, pesticides, anesthetics, neurotransmitters, and various nucleic acids.

"nucleic acid" is defined as any nucleic acid sequence taken from any source. Nucleic acids include all types of RNA, DNA and oligonucleotides, including probes and primers, antisense oligonucleotides and phosphorothioate oligonucleotides used in Polymerase Chain Reaction (PCR) or DNA sequencing. Also included are synthetic nucleic acid polymers, for example, methylphosphonate oligonucleotides, phosphotriester oligonucleotides, morpholino-DNA and Peptide Nucleic Acids (PNA), including PNA clips, DNA and/or RNA fragments and derivatives taken from any tissue, cell, nucleus, cytoplasm, mitochondria, ribosomes and other cellular sources.

"Cyclodextrin (CD)" is a cyclic oligosaccharide consisting of tapered, hollow molecules of glucose monomers coupled to each other, with a hydrophobic interior or void. The cyclodextrin of the present invention can be any suitable cyclodextrin, including alpha-, beta-, and gamma-cyclodextrins, and combinations, analogs, isomers, and derivatives thereof. Cyclodextrins may be natural or modified with hydrophobic groups, as will be described in more detail below.

In describing the present invention, the term cyclodextrin "complex" refers to a non-covalent inclusion complex. An inclusion complex is defined herein as a cyclodextrin or derivatized cyclodextrin, which functions as a "host molecule", which binds to one or more "guest molecules" and contains or confines them, in whole or in part, within the hydrophobic cavity of the cyclodextrin or derivative thereof- -the complex thus formed. The most preferred CDs are derivatives such as carboxymethyl CD, glucosyl CD, maltosyl CD, hydroxypropyl cyclodextrin (HPCD), 2-hydroxypropyl cyclodextrin, 2, 3-dihydroxypropyl cyclodextrin (DHPCD), sulfobutyl ether CD, acylated, ethylated and methylated cyclodextrins. Oxidized cyclodextrins capable of providing aldehydes, and oxidized forms of any derivative capable of providing aldehydes, are also preferred. Also included are the altered forms such as the crown ether-like compounds and higher homologs of cyclodextrins.

"controlled release" is defined as the release of the trapped guest molecule/drug from the CD polymer carrier only by cleavage of a bond used to synthesize the carrier.

The present invention relates to novel CD-grafted, biocompatible, amphiphilic polymers, methods for their preparation and use as carriers for bioactive agents. According to its most general definition, the present invention relates to a complex between a biologically active agent and at least one CD-grafted polymer conjugate comprising a biocompatible hydrophilic polymer backbone such as PEG and HPMA, poly-L-lysine (PLL) and Polyethyleneimine (PEI), onto which at least one, preferably a plurality of hydrophobically modified CDs have been grafted. Optionally, a Targeting Moiety (TM) may be covalently attached to the polymeric support.

Preferred cyclodextrin-containing polymers can be defined as a cyclodextrin-containing polymer wherein the cyclodextrin or derivatized cyclodextrin moiety is attached to a biocompatible hydrophilic polymer backbone via a single spacer arm attached to the 2, 3, or 6-position of the cyclodextrin in a manner represented by formula 1 below:

(1) p is a biocompatible hydrophilic polymer backbone having a molecular weight of 2,000 to 1,000,000 daltons, preferably 5,000 to 70,000 daltons, most preferably 20,000 to 40,000 daltons. Preferably, the biocompatible polymer backbone is a hydrophilic polymer selected from the group consisting of polyethylene glycol (PEG), N- (2-hydroxypropyl) methacrylamide polymer (HPMA), Polyethyleneimine (PEI) and polylysine (pLL), which may be suitably end-capped in a manner known in the art and may also be substituted with substituents that do not adversely affect the intended function of the polymer. Preferably, the biocompatible polymer backbone is a polyethylene glycol (PEG) polymer. When the cyclodextrin is attached at its 2, 3 or 6 position, then the corresponding R₁O-、R₂O-or R₃The O-group will be displaced and the 2, 3 or 6-carbon atom of the glucopyranose will be covalently linked to the linker X;

(2) r' is one selected from the group consisting of hydrogen, a tissue-Targeting Moiety (TM) or a cell membrane Fusion Moiety (FM), as described herein, provided that a mixture of hydrogen, a targeting moiety and a cell fusion moiety can be present on the same polymer backbone and/or within the polymer composition;

(3) x is a linker having the formula:

-Q-Z-Q’-

wherein Q is covalently bonded to the hydrophilic polymer chain, either directly or via alkyl side groups or other functional groups, and Q' is covalently bonded to the cyclodextrin. Q and Q' are independently selected from NR₄S, O, CO, CONH and COO. In other words, Q and Q' may comprise an amine, alkylamine, acylamine, thio, ether, carbonyl, amide or ester moiety. Z comprises a member selected from the group consisting of alkylene disulfides, [ - (CH)₂)_aS-S(CH₂)_a-]Alkylene [ - (CH)₂)_a]-]Alkylene oxide (- [ (CH)₂)_aO]_b(CH₂)_a-) or a short-chain peptide, wherein a is an integer of 1 to 10 and b is an integer of 1 to 20. Preferably, Q is an amide, Q' is an amine, alkylamine, or acylamine and the linker has the general formula: -CONH-Z-NR₄-. Most preferably, Q will be through an alkylene (-CH)₂)_aThe groups are attached to the derivatized polymer chain. When Z is an alkylene disulfide, alkylene oxide or peptide, the linker is biodegradable. When Z is alkylene, the linker is non-biodegradable;

(4)R₁、R₂、R₃and R₄Independently selected from H, alkyl (C)_n’H_2n’+1) Alkenyl (C)_n’+1H_2(n’+1)-1) Or acyl (C)_n’H_2n’+1CO), wherein n' is an integer from 1 to 16, preferably from 1 to 8, most preferably from 1 to 4. When R is₁、R₂、R₃And R₄When hydrogen, cyclodextrins are relatively hydrophilic in nature. When R is₁、R₂、R₃And R₄When one or more of them is an alkyl, alkenyl or acyl group, the derivatized cyclodextrin becomes more hydrophobic in nature. Therefore, when R is₁、R₂、R₃And R₄Each being alkyl, alkenyl or acylCyclodextrin is most hydrophobic. Acyl-derived cyclodextrins are more biodegradable than alkyl-or alkenyl-derived cyclodextrins;

(5) q is an integer of 5, 6 or 7, which renders the pendant cyclodextrin moiety an α -, β -or γ -cyclodextrin derivative, respectively. Preferably, q is 6 or 7, most preferably q is 6. In other words, the preferred cyclodextrin is β -cyclodextrin;

(6) w is an integer such that each polymer backbone contains 1.5 to 30, preferably 2 to 15 cyclodextrin moieties per 20KD (kiloDalton) polymer backbone. The integer "w" represents the average number of cyclodextrin moieties in the polymer composition, because the polymer composition is a mixture of polymer chains, wherein the chain length, molecular weight, and number of cyclodextrin moieties per polymer chain are variable. Thus, each polymer in such a polymer composition has a certain weight average molecular weight and an average number of cyclodextrin moieties per 20KD of the polymer backbone.

One embodiment of the present invention is a novel class of CD-grafted biocompatible polyethylene glycol (PEG) polymers, which can be represented by the following general formula 2:

wherein q, w, X, R₁、R₂、R₃And R₄As described in formula 1, m and n are integers satisfying the following conditions: when combined with w, they represent polyethylene oxide polymer chains having a molecular weight as described for the hydrophilic polymer in formula 1. In other words, as shown in formula 1, the molecular weight of the backbone of the biocompatible polyethylene oxide hydrophilic polymer is preferably in the range of 5,000 to 1,000,000, more preferably 5,000 to 70,000, and most preferably 20,000 to 40,000. As shown in formula 1, CD can be grafted onto the polymer via a single-arm linker X through the 2-, 3-or 6-position of the CD molecule, preferably through the 6-position of the CD molecule. When w has the same value as formula 1, it is understood that w is used to represent a cyclic paste per 20kD of polymer backboneThe number of fine units, not meaning that there are "w" polyethylene glycols (CH) linked in sequence₂CHXO) polymeric units of monomers. In other words, the polymer backbone contains "w" monomer units comprising pendant cyclodextrin groups spaced along the polymer backbone. The spacing may be random or uniform depending on the synthesis process.

Most preferably, the cyclodextrin-containing polymer is a polyethylene glycol polymer backbone containing pendant groups CD having the following general formula 3:

wherein Q, Q', Z, R, R₁、R₂、R₃、R₄And a and q are as described in formula 1, w is an integer such that each polymer backbone contains 1.5 to 30, preferably 2 to 15 cyclodextrin units per 20KD of polymer chain, and m and n are integers satisfying the following conditions on average: when combined with w, they represent polyethylene oxide polymer chains having a molecular weight as described for the hydrophilic polymer in formula 1. As explained in formula 2, the monomeric polyethylene glycol units containing pendant cyclodextrin groups are not contiguously linked, but may be randomly or uniformly spaced along the polymer backbone.

Specific beta-cyclodextrin copolymers falling within the scope of formula 3 are set forth in Table 1 below.

TABLE 1

Code of compound	Abbreviation of CD Polymer	w	Q	Z	Q’	R1	R2	R3	R4
										6	PEG-SS-CD	5	C(O)NH	SS	NR4	H	H	H	H
13	PEG-C3-CD	4.5	C(O)NH	C3	NR4	H	H	H	H
										18(a)18(b)	PEG-L8-CD	5.58.5	C(O)NH	L8	NR4	H	H	H	H
7	PEG-SS-DECD	1.5	C(O)NH	SS	NR4	C2H5	H	C2H5	C2H5
										11	PEG-GFLG-DECD	4.5	C(O)NH	GFLG	NR4	C2H5	H	C2H5	C2H5
14	PEG-C3-DECD	2.6	C(O)NH	C3	NR4	C2H5	H	C2H5	C2H5
										20	PEG-L8-DECD	3.9	C(O)NH	L8	NR4	C2H5	H	C2H5	C2H5
3	PEG-SS-AcCD	5	C(O)NH	SS	NR4	CH3CO	CH3CO	CH3CO	CH3CO
										15	PEG-C3-AcCD	4.5	C(O)NH	C3	NR4	CH3CO	CH3CO	CH3CO	CH3CO
19(a)19(b)	PEG-L8-AcCD	5.58.5	C(O)NH	L8	NR4	CH3CO	CH3CO	CH3CO	CH3CO
										16	PEG-C3-BnCD	4.5	C(O)NH	C3	NR4	C3H7CO	C3H7CO	C3H7CO	C3H7CO

In Table 1, SS is-CH₂CH₂SSCH₂CH₂-, C3 is-CH₂CH₂CH₂-, L8 is-CH₂CH₂OCH₂CH₂OCH₂CH₂GFLG is the tetrapeptide Gly-Phe-Glu-Gly.

As drug carriers, these novel CD-grafted polymers of the present invention have the following advantages over their monomeric precursors.

First, they are highly water soluble and less toxic. Polyethylene glycol (PEG) is a linear polyether diol that has many useful properties, e.g., good solubility, biocompatibility, because of minimal toxicity, immunogenicity, and antigenicity, and good excretory dynamics. These characteristics have led to PEG being most widely studied in pharmaceutical research and ultimately have led to FDA approval for oral administration. Thus, PEG can alter the physicochemical properties and toxicity of the conjugated cyclodextrin, making it more biocompatible.

In addition, these CD-grafted polymers also provide improved guest molecule binding stability. The hydrophobic modification of the CD provides a more hydrophobic interior and exterior of the cyclodextrin cavity, thereby improving the stability of the inclusion complex. In addition, having multiple CDs in one polymer backbone will increase the local CD concentration and produce a synergistic effect on drug binding. Thus, after binding to a suitable guest drug, the amphipathic copolymer may form a polymeric micelle through additional hydrophobic or ionic interactions. In addition, these CD-grafted polymer-containing drugs can be taken up by cells by endocytosis rather than by passive diffusion.

In addition, the CD-grafted polymers are useful for controlled release and targeted-delivery of bioactive agents. The polymers readily form specific types of polymeric micelles with the appropriate drug. Passive drug targeting can improve drug efficiency by targeting specific cells and organs, thereby reducing drug accumulation in healthy tissues and greatly reducing their toxicity, thus allowing higher doses of drug to be administered if desired. After intravenous administration, the polymeric micelles, it was found, have a long systemic circulation time, because of their small size and hydrophilic outer shell, which greatly reduces the possibility of uptake by the Mononuclear Phagocyte System (MPS), and also because of their higher molecular weight, which prevents renal excretion. Drugs that bind to polymeric micelles can accumulate in tumors to a greater extent than when free drug is used and show reduced distribution to non-target areas such as the heart [ Kwon et al, J Control Rel, 29, 17-23(1994) ]. The accumulation of polymeric micelles in unwanted or inflamed tissues may be due to increased vascular permeability and impaired lymphatic drainage (increased permeability and retention (EPR) effect). The EPR effect is considered to be a passive targeting method, but targeting of the drug may be further enhanced by binding to a targeting moiety such as an antibody or sugar, or by the introduction of a polymer that is sensitive to changes in temperature or pH. Targeting micelles or pH sensitive micelles may serve to deliver drugs to tumor, inflamed tissue or endosomal compartments because these sites are linked to lower pH values than normal tissue [ Litzinger et al, Biochim Biophys Acta 1113(20, 201-27 (1992); Tannock et al, cancer Res 49(16), 4373-84 (1989); Helmlinger et al, Nat Med 3(2), 177-82(1997) ].

PEG is commercially available in products of various molecular weights, low polydispersity (Mw/Mn < 1.1). They can be subjectively classified into low molecular weight PEG (Mw < 20,000) and high molecular weight PEG (Mw > 20,000) according to their molecular size. Recent applications of PEG have focused on the immobilization of cytotoxic anticancer drugs on PEG or the grafting of PEG onto proteins, micelles or liposomes, which can reduce systemic toxicity, prolong residence time in vivo, alter biological distribution and improve therapeutic efficacy [ Takakura et al, Crit Rev Oncol, Hematol 18(3), 207-31 (1995); duncan et al, anticancer drugs 3(3), 175-210 (1992). Recent studies found that renal clearance of PEG decreased with increasing molecular weight, with the sharpest change occurring at MW 30,000 after intravenous administration. The half-life of PEG circulating in the blood (t1/2) also showed a concomitant and dramatic increase. For example, as the molecular weight increases from 6,000 to 50,000, the t1/2 for PEG increases from about 18min to 16.5 h. Thus, conjugation of an anticancer drug to PEG of molecular weight 20,000 or more prevents rapid elimination of PEG-conjugated chemicals and provides a passive tumor-accumulating effect [ Greenwald et al, Crit Rev drug Carrier Syst 17(2), 101-61(2000) ].

In one embodiment of the invention, carboxyl grafted PEG (20,000 daltons or 25,000 daltons, containing 8-10 carboxyl groups per PEG molecule) is used as a starting material for coupling with cyclodextrins. To keep steric hindrance to a minimum, the CD moiety is attached to the PEG backbone via one of the 7 primary hydroxyl groups at the small open end (6-position) of its cavity. In addition, a flexible linear linker is used to keep the CD moiety at a distance from the polymer backbone and allow it to move freely. Due to the biocompatibility of the polymeric materials of the present invention and the flexibility of the polymers, their induced toxicity will be minimal, as well as minimal mechanical irritation to surrounding tissues.

Dosage forms consisting of a solution of the graft polymer containing the dissolved drug or the drug as a suspension or emulsion are administered to the body. The only limitation on how much drug can be loaded in a formulation is one of the functions, i.e., the drug loading can be increased until the required properties of the polymer are adversely affected to an unacceptable degree, or until the properties of the formulation are adversely affected to render administration of the formulation difficult to an unacceptable degree. Generally, it is expected that in most cases the drug will comprise about 0.01 to 50% by weight of the formulation, most often about 0.1% to 25%. These drug loading ranges are not to be construed as limiting the invention. It is within the scope of the invention even if the drug loading is outside these ranges, as long as functionality is maintained.

A significant advantage of the composition which is the object of the present invention is the ability of the graft polymer to increase the solubility and stability of the drug substance. The combination of hydrophobic CD and hydrophilic polymer imparts an amphiphilic nature to the polymer. In this connection, it functions to a large extent as a combination of cyclodextrin inclusion and a polymeric micelle system. This is particularly advantageous for the solubilization of hydrophobic or poorly water soluble drugs such as cyclosporin A, tacrolimus, thiaquinavir and paclitaxel.

Another advantage of the compositions of the present invention is the ability of the polymers to increase the chemical stability of many drug substances. A variety of drug degradation mechanisms have been observed to be inhibited when the drug is in the presence of the polymer. For example, paclitaxel and cyclosporin A are significantly stabilized in the aqueous polymer compositions of the present invention as compared to certain aqueous solutions of the same drug in the presence of an organic cosolvent. This stabilizing effect on paclitaxel and cyclosporin a is but one example of an effect that can be achieved equally well for many other drug substances.

The drug loaded on the CD-graft polymer of the present invention may be administered by various routes including parenteral, topical, transdermal, transmucosal, inhalation, or insertion into a body cavity, for example, by oral, vaginal, buccal, transurethral, rectal, nasal, oral, pulmonary, and aural administration.

The present invention is applicable to all types of biologically active agents and drugs, including nucleic acids, hormones, anti-cancer agents, and it provides an efficient route to delivery of polypeptides and proteins. The only limitation on the polypeptide or protein drugs that can be used is the problem of functional limitation. In some cases, the functional or physical stability of polypeptide and protein drugs may also be enhanced by the addition of various additives to aqueous solutions or suspensions of the polypeptide or protein drugs. Additives such as polyols (including sugars), amino acids, surfactants, polymers, other proteins and certain salts may be used. Advances in protein engineering may provide the potential to increase the intrinsic stability of a polypeptide or protein. While such designed or modified proteins may be considered new entities in terms of regulatory definition, this does not change their applicability to the present invention.

In addition to peptide or protein based drugs, other drugs from all therapeutically and medicinally useful classes can be used. These drugs are described in a number of well-known documents, such as the Merck Index, the physicians' own reference, and the pharmacology foundation of therapeutics.

Paclitaxel is a diterpenoid natural product that exhibits encouraging activity against ovarian, breast, head and non-small cell lung cancers. Recently, the paclitaxel form has been approved for the treatment of breast cancer and refractory human cancers. One of the major problems with paclitaxel is its very low water solubility. The current formulation of this drug contains 30mg of paclitaxel in 5mL50/50 cremophoreEL (polyethoxylated castor oil, a solubilizing surfactant) and ethanol mixture. When diluted in saline, the concentration of paclitaxel is 0.6-1.2 mg/ml (0.7-1.4ml) according to the administration recommendations. The dilute solution is expected to contain mixed "micellar" particles of paclitaxel/Cremophor and is reported to be physically unstable after prolonged storage, since dilution to a certain concentration would obviously result in a supersaturated solution. In addition, Cremophor, an uncharged surfactant, has been reported to cause histamine release and is associated with negative effects such as severe allergic reactions [ Sharma et al, journal of international cancer 71(1), 103-7(1997) ]. The question of whether cyclodextrin derivatives can solubilize paclitaxel has been investigated. Methylated cyclodextrins have been found to be much more effective than other hydrophilic cyclodextrin derivatives in improving the aqueous solubility of paclitaxel (HPCD and DMCD are capable of dissolving paclitaxel at about 0.7 and 33mg/ml, respectively, at 50% CD concentration). [ Sharma et al, J.Med.Sci.84 (10), 1223-30(1995) ]. However, the toxicity of DMCD and the high concentration required for complex therapeutic levels of paclitaxel have limited its clinical use. The CD-grafted amphiphilic polymers of the present invention provide significant advantages over prior art formulations due to ease of preparation and administration, low toxicity and rapid and controlled release of active agents and steerable delivery.

Antisense oligonucleotides and their analogs, e.g., peptide DNA (pna), morpholino-DNA, P-ethoxy DNA, methylphosphonate-DNA, etc., have proven to have important uses in biomedical research, but their use in pharmaceutical applications has been largely limited by stability and/or solubility, and cellular uptake behavior, etc. There is currently no effective means for safely and perfectly delivering antisense oligonucleotides to target sites in vivo, particularly for their neutral analogs, such as PNA, morpholino DNA, P-ethoxy DNA, and methyl phosphate-DNA, because they are not effective in binding to any antisense oligonucleotide carrier, which is currently mostly a polymeric-cationic polymer. However, the CD-grafted amphiphilic polymers of the invention can then be effective carriers for neutral analogues because each nucleoside unit has aromatic base residues that can be included as potential targets by cyclodextrins, thus enabling the CD-grafted polymers to bind oligonucleotides and their analogues through the intensification of the CD inclusion mechanism. Such binding may be made very strong by the binding interaction between a large number of CD moieties on the polymer and a large number of aromatic base rings on the antisense oligonucleotide. In addition, additional ionic interactions (for charged oligonucleotides) or hydrophobic interactions (for uncharged oligonucleotide analogs) can also enhance the binding between the antisense oligonucleotide and the CD-polymer carrier. As a result, the finally bound complex can form a loose or tight polymer micelle depending on its content, thereby safely delivering the antisense oligonucleotide and its neutral analogue into cells.

In summary, the CD-grafted polymers of the present invention improve the stability of the drug/binding complex through a number of CD-moieties synergistic interactions and external hydrophobic or ionic interactions. It is likely that inclusion is the primary mechanism of drug-binding ability of the polymers of the invention. However, ionic interactions and external hydrophobic interactions (outside the CD cavity) may also contribute significantly, depending on the molecular structure of the particular copolymer and guest. In addition, properly constructed PEG-CD copolymers of the present invention are excellent solubilizers of paclitaxel and safe therapeutic carriers. They may also be used as solubilizers and carriers for other hydrophobic drugs. The CD-grafted amphiphilic polymers of the invention are soluble in water and biocompatible and have very slow release kinetics, especially when they contain a high proportion by weight of hydrophobic moieties. In addition, the strong binding constant of the drug/polymer complex results in a slow release of the bound drug upon dilution, sometimes even requiring its replacement with other molecules. They are therefore useful as ingredients in oral formulations for the delivery of certain water-soluble drugs.

Furthermore, properly configured CD-grafted polymers of the invention can be used to deliver antisense oligonucleotides and their uncharged analogs, as well as hydrophobic peptides and proteins, since external hydrophobic interactions can provide sufficient stability for hydrophobic antisense oligonucleotides or hydrophobic peptides. Negatively charged oligonucleotides are also expected to be good guest molecules for certain specially constructed polymers, since the basic nitrogen in the linker of the polymer can neutralize the negative charge under appropriate conditions.

The following examples are given to illustrate methods of making and methods of using the compositions of the present invention.

Example 1

Materials and methods: PEG with pendant propionic acid groups (PEG-10PA and PEG-8PA Mw. about.20 KD, SunBio, Anyang, south Korean) was dried overnight at room temperature under vacuum. Beta-cyclodextrin (TCI (USA), Portland, OR) was dried overnight at 130 deg.C under vacuum before use. Other chemicals were from Aldrich chemical company (milwaki, WI) and were used as supplied in the original supply without further purification. HPLC (high pressure liquid chromatography) analysis was performed on a Waters system, the systemAn RI detector and Ultrahydrogel 120 and Ultrahydrogel 500SEC columns were fitted.¹H-NMR was recorded on a Varian 400MHz machine.

PEG-SS-CDSynthesis of (Compound 2)

Mono-6- (6-amino-3, 4-dithio-hexylamino) -6-deoxy-beta-cyclodextrin(Compound 1):

2, 2' -dithiobis-ethylamine dihydrochloride (10g, 4.44mol, Fw ═ 225.2) was dissolved in 30mL of distilled water, followed by addition of 1.0M KOH (8.88mol) and mono-6-tosyl- β -cyclodextrin (0.5g, Fw ═ 1289) powder. The resulting suspension was stirred overnight on a 70 ℃ oil bath and then concentrated to about 4 mL. The mixture was loaded onto a Sephadex G-25 column (2.5X 80cm) and eluted with 0.1M TEA. About 0.38g of Compound 1 is obtained.

PEG-SS-CD(Compound 2)

PEG grafted with carboxyl groups (2.24g, PEG-8PA, 20KD, polyethylene glycol containing about 8 pendant propionic acid groups, average molecular weight about 20,000) was dissolved in 25mL of anhydrous DMF and the mixture was cooled to 0 ℃ on ice under argon. To this was added 28 μ L of tributylamine (1.18mmol, Fw-185.36, d-0.778), followed by 175 μ L of isobutyl chloroformate (IBCF, Fw-136.6, d-1.053) in 1mL of DMF. The mixture was stirred at 0 ℃ for 1 h. Subsequently, the reaction mixture was slowly added to a solution of 1.75g of compound 2 in 100ml of ldmf at room temperature. After stirring overnight at room temperature, the reaction was stopped by adding 1mL of water. The mixture was concentrated and then diluted with 60mL of water. The product solution was purified by elution on a Sephadex G-50 column with 0.1M TEA followed by ether precipitation.¹H-NMR analysis indicated that about 5CD moieties were coupled to a PEG backbone of molecular weight about 20,000 daltons. The retention time of this product was about 0.45min longer than that of the starting PEG, as determined by HPLC chromatography [ GPC column, Rt (product) ═ 17.33min, versus Rt (PEG-8A) ═ 16.87min]。

¹H-NMR(400MHz，D2O)：δ，5.0(s，7H，H1’)，3.3-3.9(m，370H，41H-CD，329H-PEG).。

Example 2

PEG-SS-AcCDSynthesis of (Compound 3)

PEG-SS-CD (Compound 2, 1.0g, about 5CD/20 KD-PEG) in P₂O₅Drying was carried out in a desiccator, followed by co-evaporation with 50mL of anhydrous pyridine. The residue was dissolved in 30mL pyridine under argon protection and then added to 2.0mL acetic anhydride (Fw 102.1, d 1.08). The mixture was stirred at room temperature for 2 days and then dried on a rotary evaporator. The crude product was purified by repeated ether precipitation from methanol. Hplc (gpc) analysis showed the product to be 0.46min longer than the base polymer (Rt 19.70min for the product and 19.24min for the reactant polymer).¹H-NMR analysis indicated that every 20kD PEG contained about 5CD moieties and all hydroxyl groups were acetylated.

¹H-NMR(400MHz，D₂O)：δ，4.7-5.5(s，14H，H1’，H3’)，3.4-5.5(m，382H，35H-CD，347H-PEG)，2.05(m，20H，H-Ac).

Example 3

PEG-SS-DECDSynthesis of (Compound 7)

PEG-SS-NH2(Compound 4):

PEG grafted with carboxyl groups (PEG-8PA, 2.6g, about 2.0mmol COOH groups) was dissolved in 30mL of anhydrous DMF and cooled to 0 ℃ on ice. To this was added tributylamine (0.35mL, 1.5mmol, Fw-185.36, d-0.778), followed by isobutyl chloroformate (0.20mL, 1.5mmol, Fw-136.6, d-1.053). The mixture was stirred at 0 ℃ for 80min and then added carefully to a solution of 2, 2' -dithiobis-ethylamine (3.5g, Fw ═ 152.2, 23mmol) in 50mL anhydrous DMF. The mixture was stirred at room temperature for 20h, concentrated to about 20mL on a rotary evaporator at 40 ℃, then dialyzed against distilled water after dilution with 50mL of water (4 × 5L, 26 hours, Sigma D-0655, MWCO ═ 12,000). The dialyzed solution was concentrated by rotary evaporation at 40 ℃ to obtain 4.1g of a syrup. The slurry was dissolved in 10mL of methanol and then precipitated by the addition of 80mL of ethyl ether. The precipitate was collected by centrifugation and the precipitation process was repeated 2 times. The final product was a white powder weighing about 2.2 g. The product showed only one complete peak in its HPLC (GPC) chromatogram with a retention time (18.66min) that was about 1.5min longer than the starting PEG-8PA (17.11 min).

N- (. beta. -Cyclodextrin-6-yl) glycine methyl ester(Compound 5)：

Glycine methyl ester hydrochloride (1.5g, Fw-125.56, 12mmol, manufactured by Aldrich) was dissolved in 100mL of anhydrous DMF under argon protection. DIPEA (2.1ml, 12mmol, Fw-129.25, d-0.724) was added followed by 6-mono-tosylcyclodextrin powder (3.0g, Fw-1289,. -80% pure,. -1.8 mmol). The mixture was stirred at room temperature to a clear solution. The temperature was slowly raised to about 70 ℃ and subsequently stirred for a further 4 h. The mixture was then concentrated to a slurry on a rotary evaporator at 55 ℃. The crude product was dissolved in 40mL of hot water, cooled to room temperature and precipitated by addition of 80mL of acetone. The white precipitated powder was collected by filtration and dried under vacuum overnight. About 2.3g of the desired compound 5 are obtained. The product was used in the next step without further purification.

N- (hepta-2-O-ethyl-6)^B，6^C，6^D，6^E，6^F，6^G-hexa-O-ethyl-beta-cyclodextrin-6^A-yl) -glycine (compound 6):

n- (β -cyclodextrin-6-yl) glycine methyl ester (compound 5, about 2.0g, Fw ═ 1206,. about.1.6 mmol) was dissolved in 15mL of DMSO (dimethyl sulfoxide) and 15mL of DMF (dimethylformamide). The solution was cooled to 0 ℃ on an ice bath, followed by the addition of 10g of BaO and 10g of Ba (OH) under argon protection₂.H₂And O. To this white suspension was slowly added 20mL diethyl sulfate and the mixture was stirred at 0 ℃ for 1h, followed by stirring at room temperature for a further 24 h. 20mL of diethyl sulfate were slowly added over a period of 1h and then stirred at room temperature for a further 24 h. To the viscous reaction mixture was slowly added 60mL at 0 deg.C5N sodium hydroxide, and the mixture was stirred at room temperature for a further 1 h. This was extracted with 2X 200mL of chloroform. The combined organic phases are concentrated to a waxy product after drying over sodium sulfate. The crude product was dissolved in 20mL of methanol, followed by the addition of 20mL of distilled water. The mixture was filtered in vacuo to remove traces of precipitate. The clear filtrate was concentrated to give an orange foamy solid (about 1.8g) containing about 50% of the desired compound 6. The crude product is in P₂O₅After drying overnight in a desiccator, it was used directly in the next reaction.

PEG-SS-DECD(Compound 7):

crude compound 6(1.4g, 0.46mmol) was dried by co-evaporation with 2 × 20mL anhydrous DMF followed by re-dissolution in 20mL anhydrous DMF followed by addition of 0.19mL tributylamine (0.8mmol, Fw-185.36, d-0.778). The mixture was cooled to 0 ℃ on ice. To this cold solution was slowly added a solution of isobutyl chloroformate (60 μ L, 0.46mmol, Fw 136.6, d 1.053) in 2mL DMF. The mixture was stirred at 0 ℃ for 1.5h and then transferred at room temperature to a solution of PEG-SS-NH2 (compound 4, 0.4g) in 10mL of anhydrous DMF followed by the addition of DIPEA (28 μ L, 0.16mmol, Fw 129, d 0.724). The mixture was stirred at room temperature overnight and then concentrated to a slurry. The slurry was triturated with 30mL of ethyl ether, resulting in an orange precipitate. The precipitate was collected by filtration and washed with ethyl ether. The solid was further purified by 2 precipitations from ether in methanol. About 0.55g of a pale orange solid was obtained.¹H-NMR indicates that the product is the desired onePEG-SS-DECDThe product, but only about 1.5 CD moieties, were conjugated to a 20KD PEG molecule and had about 13 ethyl groups per cyclodextrin.

¹H-NMR (400MHz, D2O): δ, 5.1(7H, m, H1 'and H3'), 3.2-3.9(m, 1041H, 41H-CD, 1000H-PEG), 2.78(m, 30H, CH2-Et), 1.15(b, 45, CH3-Et).

Example 4

Synthesis of PEG-GFLG-DECD (Compound 11)

Mono-6- (N)³-Boc-3-amino-propylamino) -6-deoxy- β -cyclodextrin (compound 8):

mono-Boc-1, 3-diamino-propane (3.5g, 3.0mol, prepared as described in Jean FrancoisPons et al, eur.j.org.chem, 1998, 853-. The final dried oil was mixed with 100mL anhydrous DMF followed by the addition of DIPEA (2.1mL, 12mmol, Fw 129, d 0.742). To this solution was added 3.5g of 6-mono-tosyl-6-O- β -cyclodextrin. The mixture was stirred at room temperature until the solid was completely dissolved. Subsequently, the mixture was stirred in an oil bath at 70 ℃ overnight. The mixture was concentrated to about 10mL on a rotary evaporator at 45 ℃ and then precipitated with 100mL of acetone. The white precipitate was collected by filtration and washed with acetone. About 3.2g of product was obtained. It contains about 60% of the desired compound 8, according to TLC (thin-layer chromatography) (Rf ═ 0.12, silica gel, at 80: 10/AcOH: CHCl₃∶H₂Developed in O, stained with a solution of 5% phosphomolybdic acid in 95% ethanol). The product was sent directly to the next step for ethylation.

Mono- (hepta-2-O-ethyl-6)^B，6^C，6^D，6^E，6^F，6^G-hexa-O-ethyl-beta-cyclodextrin-6^A-yl) -1, 3-diamino-propane (compound 9):

mono-6- (N)³-Boc-3-amino-propylamino) -6-deoxy- β -cyclodextrin (compound 8, 3.0g) was dissolved in 40mL of anhydrous DMF and 40mL of DMSO at 0 ℃ and then mixed with 10g of barium oxide and 10mL of barium hydroxide-water under argon protection. The mixture was cooled to 0 ℃ and then 20mL diethyl sulfate was slowly added. The mixture was stirred at 0 ℃ for 6h, then at room temperature for a further 2 days. To the reaction mixture was added 25mL of cold ammonia, followed by stirring at room temperature for another 3 h. The final reaction mixture was diluted with 50mL of water and extracted with 3X 100mL of ethyl acetate. The organic phase is washed thoroughly with 2X 200mL of saturated sodium bicarbonate and 3X 200mL of water, then dried over sodium sulfate and concentrated. In thatAfter drying overnight under vacuum, about 2.8g of an orange solid was obtained. The product was dissolved in 10mL of trifluoroacetic acid. The clear solution was stirred at room temperature for 3h, followed by the addition of 15mL of water. The mixture was stirred at room temperature for a further 20min and subsequently dried at 45 ℃ on a rotary evaporator. The residue was dissolved in 150mL ethyl acetate and washed with 3X 100mL saturated sodium bicarbonate and 100mL brine. The organic phase is concentrated after drying over sodium sulfate. About 2.0g of crude compound 9 was obtained. The product was used directly in the next step of the coupling reaction.

PEG-GFLF-DECD(Compound 11):

PEG-GFLG (tetrapeptide Gly-Phe-Leu-Gly grafted PEG polymer, compound 10, approximately 4.5 GFLG peptides made from PEG-8PA and GFLG peptides in 20,000D PEG) (2.0g, approximately 0.4mmol-COOH, co-evaporated with 30mL DMF and dried) was dissolved under argon protection in 30mL anhydrous DMF and 0.17mL tributylamine (0.7mmol, Fw-185.36, D-1.053). After cooling to 0 ℃ a solution of 0.078mL (0.6mmol) of isobutyl chloroformate in 2mL of DMF was added. The mixture was stirred at 0 ℃ for 1.5h, then slowly added to a solution of 2.0g of Compound 9 in 20mL of DMF at room temperature followed by 0.087mL of DIPEA (0.5 mmol). The mixture was stirred at room temperature overnight, concentrated to about 10mL, and precipitated with 90mL cold ethyl ether. The orange precipitate was collected by filtration and further precipitated from methanol 3 times with ether. The final product was about 2.2 g. The retention time of the product (Rt 18.42min) was 0.67min longer than the starting PEG-GFLG polymer (Rt 17.76min) as determined by hplc (gpc) chromatography.¹H-NMR indicated that the product was the desired compound 11: each 20KD polymer contains about 4.5 tetrapeptide GFLG and 1.8 CD moieties and each CD moiety has about 13 ethyl groups.

¹H-NMR(400HMz，D₂O)：δ，7.20(5H，m，ArH-Phe)，5.1(2.8H，m，H1’-CD)，3.0-4.0(645H，m，41H-CD，574H-PEG，30H-Et)，1.1(15.6H，m，30H，CH3-Et)，0.9(6H，d，CH3-Leu).

Example 5

PEG-C3-AcCD，PEG-C3-DECDAndPEG-C3-BnCDsynthesis of (2)

Mono-6- (gamma-amino-propyl-amino) -6-deoxy-beta-cyclodextrin(Compound 12):

mono-6-tosyl-6-deoxy-cyclodextrin (6.5g, Fw ═ 1269) was dissolved in 200mL anhydrous DMF and 60mL diaminopropane at room temperature with vigorous stirring. The clear mixture was stirred at room temperature for 2h, followed by stirring at 65 ℃ for a further 20 h. The mixture was concentrated to about 20mL at 45 ℃. To this was added 200mL of cold isopropanol at room temperature. The white precipitate was collected by filtration. The solid was redissolved in 25mL of water and 25mL of TEA. 300mL of acetone was slowly added thereto at 0 ℃. The precipitate was collected by filtration, and the reprecipitation procedure was repeated 2 additional times. The final product was about 5.5 g. It contains about 80% of the desired compound 12 and about 20% free cyclodextrin. The product was used in the next reaction without further purification.

PEG-C3-CD(Compound 13):mono-6- (gamma-amino-propylamino) -6-deoxy-beta-cyclo Dextrin(Compound 12, 6.2g) was coupled to PEG-8PA (4.1g) using the same method as described above for the synthesis of PEG-SS-CD. About 4.3g of pure product is obtained after purification by GPC. The retention time of the product (17.87min) was 0.76min longer than that of the starting PEG-8PA (17.11 min).¹H-NMR indicated that the product was the desired compound 13, containing about 4.5 CD moieties per 20KDPEG molecule.

¹H-NMR(400HMz，D2O)：δ，5.0(7H，s，H1’-CD)，3.4-3.9(412H，m，41H-CD，371H-PEG).

PEG-C3-AcCD(Compound 15): PEG-C3-CD (1.0g, 4.5 CD/20KDPEG) was acetylated using the same method as described in the preparation of PEG-SS-AcCD. About 1.0g of product was obtained, the retention time (17.99min) of which was only about 7.2s longer than that of the base polymer (PEG-C3-CD, 17.87 min). However,¹H-NMR indicated that the product was the desired compound 15, the polymer contained about 4.5 CD moieties per 20kD PEG molecule and about 90% of the hydroxyl groups on the side chain CD were acetylated.

¹H-NMR (D2O): delta, 4.9-5.4(14H, m, H1 '-CD and H3' -CD), 3.2-4.5(m, 490H, 35H-CD, 455H-PEG), 2.03(d, 64H, CH)₃CO-).

PEG-C3-BnCD(Compound 16): PEG-C3-CD (Compound 13, 0.9g,. about.4.5 CD/20KD PEG) was dried by co-evaporation with 20mL of anhydrous pyridine, followed by re-dissolution in 30mL of pyridine under argon protection. At room temperature (the reaction temperature was increased and the reaction was cooled with ice), 3mL of butyryl chloride (Fw-106.55, d-1.026) was slowly added thereto. After the mixture was stirred at room temperature for 4 hours, methanol (5.0mL) was added, followed by further stirring at room temperature for 30 min. The mixture was concentrated on a rotary evaporator to a waxy solid. The solid was dissolved in 20mL methanol and diluted with 20mL water. The clear solution was dialyzed against 2X 5L 20% isopropanol/water. The opaque dialysis solution was concentrated in a Speed-Vac at room temperature. The particles were further precipitated 3 times from methanol with ether. The product is practically insoluble in water, but very soluble in methanol and chloroform. The yield was 90%.¹H-NMR indicated that the product was the desired compound 16: about 80% of the hydroxyl groups on the pendant cyclodextrin are butyrylated.

¹H-NMR(CDCl₃)：δ，4.6-5.3(14H，m，H1’and H3’)，3.2-4.5(m，541，35H-CD，486H-PEG)，2.30(m，36H，CH₃CH₂CH₂CO-，1.65(m，36H，CH₃CH₂CH₂CO-)，0.95(m，54H，CH₃CH₂CH₂CO-).

Example 6

PEG-L8-AcCD and PEG-L8-DECDSynthesis of (2)

Mono-6- (8-amino-3, 6-dioxy-octylamino) -6-deoxy-beta-cyclodextrin(Compound 17):

in a 500mL round bottom flask under argon protection was added 2, 2' - (ethylenedioxy) bis (ethylamine) (300mL, Fw ═ 148) and mono-6-tosyl- β -cyclodextrin (24.4g, Fw ═ 148)1269 at P₂O₅Dried in a desiccator overnight). The suspension was stirred at room temperature until all solids were completely dissolved (. about.1.0 h). The mixture was stirred at 75 ℃ for a further 4 h. The reaction mixture was poured slowly into 1.8L of cold isopropanol. The precipitate was collected by filtration and washed with isopropanol. The precipitate was dissolved in 200mL of warm water (50 ℃ C.), and then slowly poured into 1.8L of ice-cold isopropanol with stirring. After cooling to-20 ℃, the precipitate was collected by filtration. This isopropanol precipitation process was repeated 2 more times. About 24g of white powder was obtained. HPLC analysis (GPC, eluting with 0.1M sodium nitrate) showed the product to contain about 85% of the desired compound (Rt 39.21min) and about 15% unmodified β -CD (Rt 32.25min), with no detectable free diamine reactant. The product is then used directly in the next coupling step.

¹H-NMR (400HMz, D2O): δ, 4.97(7H, m, H1 '), 3.7-3.9(26H, m, 7H 3', 7H5 ', 6H 6', 6H6 "), 3.3-3.6(24H, m, 7H2/, 7H4 ', 1H 6', 1H 6", 8H-linker), 2.71(4H, m, CH, H-linker)₂An N-linker).

PEG-L8-CD(Compound 18):

PEG-8PA (4.0g, about 8-COOH/PEG-20K, about 1.7mmol COOH in P₂O₅Dried overnight in a desiccator and co-evaporated with 50mL dry DMF) was dissolved in 50mL dry DMF and 0.54mL tributylamine (TBA, Fw-185.36, d-0.778, 2.27 mmol). The clear mixture was cooled on ice and then 0.29mL of isobutyl chloroformate (IBCF, Fw 136.6, d 1.053, 2.2mmol) was added at 0 ℃. The mixture was stirred at 0 ℃ for a further 1h, then slowly added at room temperature to mono-6- (8-amino-3, 6-dioxy-octylamino) -6-deoxy- β -cyclodextrin (compound 17, 5.0g, Fw ═ 1336,. about.80% pure,. about.2, 6mmol, in P₂O₅Dried overnight in a desiccator) in 50mL of anhydrous DMF. After stirring overnight, the mixture was concentrated to about 20mL on a rotary evaporator at 50 ℃. The mixture was diluted with 60mL of water and purified on a Sephadex-G-50 column (2.5 × 80cm, eluted with 0.1MTEAA, pH 10.0, collected to 8 mL/mL). The fractions were analyzed by GPC-HPLC, and the polymer fractions were combined and divided into 2 fractionsDividing into: part A: fraction 9-30; and part B: fraction 31 to 35.

Both fractions were concentrated on a rotary evaporator as waxy solids and subsequently redissolved in 15mL of methanol. The product was precipitated with 5mL TEA and 120mL ethyl ether. The white precipitate was collected by filtration. Part A and part B weighed 4.7g and 0.55g, respectively.¹H-NMR analysis confirmed that both fractions were the desired product PEG-L8-CD, but differed in cyclodextrin loading: part a and part B contain an average of about 5.5 and 8.5 cyclodextrin moieties per 20KD-PEG polymer, respectively.

¹H-NMR (400MHz, D2O): δ, part a: 5.0(s, 7H, H1 '), 3.3-3.9(382H, m, 41H-CD, 12H linker, 329H-PEG; part B: 5.0(s, 7H, H1'), 3.3-3.9(256H, m, 41H-CD, 12H linker, 203H-PEG).

PEG-L8-AcCD(Compound 19):

PEG-L8-CD(Compound 18, 1.0g, about 5.5CD/20KD PEG at P₂O₅Dried overnight in a desiccator) was dried by co-evaporation with 40mL of anhydrous pyridine and subsequently redissolved in 40mL of anhydrous pyridine under argon. To this was added 3.0mL of acetic anhydride. The mixture was stirred at room temperature for 2 days and concentrated to about 10mL on a rotary evaporator at 45 ℃. To this was slowly added 90mL of ethyl ether. The precipitate was collected by filtration. The product was further purified by 3 more ether precipitations from methanol. The final white powder was dried under vacuum and weighed 1.07 g.¹H-NMR confirmed that the product was the desired product 19: PEG contains about 5.5CD moieties per 20KD, and about 90% of the hydroxyl groups on the side chain CD moieties of the polymer are acetylated.

¹H-NMR (D2O): δ, 4.9-5.4(14H, m, H1 '-CD and H3' -CD), 3.2-4.5(m, 422H, 34H-CD, 12H-linker, 376H-PEG), 2.03(d, 64H, CH3CO-).

PEG-L8-DECD(Compound 20):

PEG-L8-CD(Compound 18, 1.0g, about 5.5CD/20KD PEG at P₂O₅Dried overnight in a desiccator) was dissolved in 5mL of anhydrous DMSO and 5mL of anhydrous DMF and the solution was cooled to 0 ℃ on ice under argon. To this were added 0.75g of barium oxide and 0.75g of barium hydroxide-water powder, followed by 3mL of diethyl sulfate in 3 portions over a period of 1 hour. The suspension was stirred at 0 ℃ for 2h and subsequently at 4 ℃ for a further 2 days. 80mL of cold ethyl ether were then added at 0 ℃ and stirred for a further 30min at 0 ℃. The orange precipitate was collected by filtration and dissolved in 50mL 50% methanol/water. The mixture was dialyzed against 5L of 0.01N HCl followed by 2X 5L of water. The final dialyzed solution was concentrated to obtain about 1g of a waxy product. It was further purified by 2 ether precipitations from methanol.¹H-NMR analysis indicated about 4 CD's per 20KD-PEG, and each CD moiety carries about 11 ethyl groups. That is, about 30% of the CD moieties are removed from the PEG backbone during alkylation.

¹H-NMR (400HMz, D2O): δ, 4.9-5.3(7H, m, H1' -CD), 3.1-4.0(540H, m, 41H-CD, 469H-PEG, 8H-linker, 22H-CH 2-ethyl), 1.2(33H, m, CH 3-ethyl)

13 representative cyclodextrin-grafted PEG polymers (Table 1) were prepared according to examples 1-6 and FIGS. 4-8, where the linker was either biodegradable (-SS-or-GFLG-) or non-biodegradable (-C3-or-L8-). The pendant cyclodextrin moieties are either native β -CD (PEG-X-CD) or modified with hydrophobic groups, including ethyl (PEG-X-DECD), acetyl (PEG-X-AcCD), or butyryl (PEG-C3-BnCD). GPC-HPLC was used to monitor each step of the preparation process and found that all final products had longer retention times than the corresponding PEG precursors. All product polymer structures are used¹H-NMR analysis confirmed that their CD content varied from 1.5 CD to 8.5 CD on average per 20kD PEG backbone (Table 2). They are all highly soluble in most organic solvents (chloroform, methanol, ethanol, etc.). They are also highly soluble in water, except for PEG-C3-BnCD.

TABLE 2 structural characteristics of certain cyclodextrin grafted PEG copolymers

Name of Polymer	tR(min*)	CD number CDs/20Kd polymer	CD modification
				PEG-ss-CDPEG-ss-AcCDPEG-C3-CDPEG-C3-AcCDPEG-L8-CD(A)PEG-L8-AcCD(A)PEG-L8-CD(B)PEG-L8-AcCD(B)PEG-L8-CD(C)PEG-L8-AcCD(C)PEG-GFLG-DECDPEG-L8-DECDPEG-C3-BnCD	19.3419.2418.0717.8618.1217.9818.4318.0818.7118.5318.0	3.93.94.84.84.64.65.95.95.45.42.53.94.5	No-100% acetylation no-80% acetylation no-95% acetylation no-84% acetylation no-100% acetylation-67% ethylation-80% butyration

Waters Ultrahydrogel GPC columns (120 and 500), eluting with 0.1M sodium nitrate;

as recorded on Varian 400HMz¹H-NMR spectrum calculation;

example 7

Preparation of complexes of paclitaxel with CD polymers or CD monomers

(A) Co-dissolution method: the method is suitable for all compounds with water-soluble polymers

Polymer (or monomer control) in water (typically about 100mg/mL) and an equal volume (typically 40-2000. mu.L) of paclitaxel solution (C)_Paclitaxel0.1 to 8.0mg/ml in methanol). The mixture was incubated at room temperature for about half an hour. Subsequently, the solvent was removed in a centrifugal concentrator at room temperature. The concentrated slurry or wax solid is reconstituted to its original volume by the addition of water or PBS buffer. After 30min regeneration, the mixture is usually a clear or slightly turbid solution. Undissolved paclitaxel particles were removed by ultrafiltration (0.2 μm filter) or centrifugation (20min, 20,800g at room temperature). The paclitaxel concentration in the supernatant was quantified by calibrating the UV absorbance at 290nm against the corresponding cyclodextrin polymer solution as background.

(B) The dialysis method comprises the following steps: this method is suitable for preparing all paclitaxel/polymer complex solutions:

a solution of the polymer in methanol (typically about 100mg/mL) is mixed with an equal volume (typically 100. mu.L) of paclitaxel (1-3 mg/mL in methanol). The clear mixture was incubated at room temperature for about half an hour. Subsequently, dialysis (MWCO ═ 12,000) was performed at room temperature overnight into 2L of water. The dialysis solution is typically a clear solution. Trace paclitaxel particles were removed by ultrafiltration (0.2 μm filter) or centrifugation (20min, 20,800g and room temperature). The clear solution is stored at a temperature of 4 ℃ or less.

Example 8

Preparation of antisense oligonucleotide/CD-Polymer complexes

Cyclodextrin PEG polymer (50mg/ml) was mixed with a quantity of 21-mer-fluorescently labeled oligonucleotide in 20mM Tris-HCl buffer (pH 7.4). The solution was dried in a Speed-Vac and subsequently regenerated with the same amount of water. The DNA/polymer complexes in solution form were analyzed on a 1% agarose gel in TAE buffer at pH 7.4.

TABLE 3 comparison between paclitaxel or oligonucleotides loaded with certain of the copolymers and other existing CD derivatives

Polymer and method of making same	CD moiety/support polymer	Paclitaxel loading (mg/50mg polymer)	Oligonucleotides (mg/50mg polymer)
				PEG-ss-CDPEG-ss-AcCDPEG-C3-CDPEG-C3-AcCDPEG-L8-CD(A)PEG-L8-AcCD(A)PEG-L8-CD(B)PEG-L8-AcCD(B)PEG-ss-DECDPEG-GFLG-DECDPEG-C3-DECDPEG-L8-DECD	3.93.94.84.84.64.65.95.91.52.52.63.9	＜0.050.8＜0.05～2.0＜0.05～2.6＜0.05～2.90.43.0＜1.0＜1.0	ND**NDNDNDNDNDNDND～0.06～0.2～0.15～0.2
Control HP-CD (from Sigma) (SBE)7CD (from Cydex) DM-CD (from Sigma) EP-CD (from Sigma)		< 0.05 to.2 (day 1) < 0.05

Amount of drug in 1.0mL water or PBS in the presence of 50mg polymer or other CD derivative.

Not detected.

Example 9

Stabilization of paclitaxel/CD complexes in 50% serum or after 10-fold dilution with PBS Characterization of nature

(A) Stability in 50% fetal calf serum: paclitaxel/PEG-L8-AcCD (2.0mg/50mg in 1.0mL PBS buffer) or paclitaxel/DMCD (0.5mg/50mg in 1.0mL PBS buffer) complex solutions were prepared according to those described in method A of example 7. 50 μ L of each complex solution was diluted with an equal volume of fetal bovine serum. After incubation of the two mixtures at room temperature for 2h, 21h, 49h and 144h, respectively, centrifugation was carried out at 20,8000g at room temperature. The paclitaxel concentration in each supernatant was quantified by measuring the UV absorbance at 230 nm.

(B) Stability after 10-fold dilution with PBS: paclitaxel/PEG-L8-AcCD (2.0mg/50mg in 1.0mL PBS buffer) or paclitaxel/DMCD (0.5mg/50mg in 1.0mL PBS buffer) complex solutions were prepared according to those described in example 10. 50 μ L of the complex solution was diluted with 450 μ L of LPBS buffer, respectively. After incubation of the two mixtures at room temperature for 2h, 21h, 49h and 144h, respectively, centrifugation was carried out at 20,8000g at room temperature. The paclitaxel concentration in each supernatant was quantified by measuring the UV absorbance at 230 nm.

TABLE 4 stability test List of paclitaxel/PEG-L8-AcCD and paclitaxel/DMCD complexes in 50% serum or after dilution with PSBS

Time (h)	The remaining paclitaxel in the diluted solution%
					PEG-L8-AcCD 50% serum	PEG-L8-AcCD 10×PBS	DMCD 50% serum	DMCD 10×PBS
	022149144	100100979284	1001001009979	100100100100100					1008613

Example 10

Release of paclitaxel from paclitaxel/PEG-L8-AcCD and paclitaxel/DMCD complexes and cytotoxicity of free copolymers

The efficient release of free paclitaxel from the paclitaxel/PEG-L8-AcCD complex was confirmed by the cytotoxicity of the complex. Both the paclitaxel/PEG-L8-AcCD complex formulation (invention) and the currently commercially available paclitaxel/Cremophor formulation (paclitaxel, Bristol-Myers Squibb) were tested in all 3 cell lines tested according to the modified MTT test described below to obtain similar IC' s₅₀The value is obtained. However, PEG-L8-AcCD alone showed no detectable cytotoxicity at the highest concentration tested, whereas cremophor killed half of the cells at a concentration of about 0.5mg/ml (Table 5):

1. the cells were plated at about 5,000 cells/well in 0.1mL of medium per well on a 96-well plate and incubated at 37 ℃ for 24 h;

2. old medium was removed and 80. mu.L of fresh medium was added to each well;

3. add 20. mu.L of sample solution to each well (5-fold serial dilutions, at least 8 concentrations per sample);

4. cells were incubated for 3 or 4 days;

5. the medium was removed. Add 80. mu.L of fresh medium along with 20. mu.L of MTs Solution (Promega CellTiter 96 Aqueous One Solution Reagent # G358A). Incubating for 2-4 h at 37 ℃;

6. reading the absorbance at 490 nm;

7. computing IC₅₀The cell-free wells were used as blank controls and the drug-free wells were used as 100% survival controls.

TABLE 5 different paclitaxel formulations and vehicle controlsIC in 3 different cell lines₅₀Comparison

Preparation	IC50(ng/ml)
				Hela	HT1080	MCF7
	paclitaxel/PEG-L8-AcCD paclitaxel/Cremophor	3.03.0	2.04.0				2.02.0
CremophorPEG-L8-AcCD				500,000＞10,000,000	500,000＞10,000,000	500,000＞10,000,000

Concentration to assess 50% cell viability following the modified MTT assay:

example 11

Hemolytic activity of the copolymer and its possible biodegradation products:

to further investigate the cytotoxicity of the polymers according to the invention and their possible biodegradation products, the haemolysis of fresh human blood cells was tested in each case in comparison with the commercially available CD monomers. The degree of hemolysis is given as a percentage of the total output of hemoglobin in distilled water (table 6).

1. Red blood cells were isolated from whole blood by centrifugation at 1000g for 10 min.

2. Plasma was removed and red blood cells were resuspended in normal buffered saline (PBS, 0.154M sodium chloride and 0.01M phosphate, pH 7.4). The red blood cells were pelleted by centrifugation (1000g, 10 min).

3. Step 2 was repeated twice to remove heme released from damaged cells.

4. The final pellet was diluted with PBS to about 12 (or 5%) hematocrit by centrifugal sedimentation.

5.2 mL of a series of concentrations of polymer or cyclodextrin solutions in PBS buffer from 0 to 50mg/mL equilibrated in PBS buffer at 37 ℃ were equilibrated at 37 ℃. To this was added 100 μ L of the red blood cell suspension, followed by mixing the sample by gentle inversion. The samples were incubated at 37 ℃ for 30 min.

6. Intact cells and cell debris were pelleted by centrifugation at 1000g for 5 min. The amount of released hemoglobin in the supernatant was determined by analyzing the resultant value at 543nm with a spectrophotometer.

TABLE 6 comparison of hemolytic activity of different PEG-CD polymers and their precursor monomers with commercially available CD derivatives.

Component (b) or CD monomer	Hemolysis (HC)50mM)
		PEG-L8-AcCDPEG-L8-DECDPEG-L8-CDCD-L8-NH2(SBE)7-CDDM-CDβCDHPβCD	NDNDND25ND1.04.035

The above data show that the novel PEG-CD polymers of the present invention have great potential as safe drug carriers for paclitaxel (table 3, table 4, table 5 and table 6). Paclitaxel is soluble in water in the presence of 50mg/ml of polymer to a concentration of at least 2.2mg/ml, which corresponds to more than 10,000 times the solubility of free paclitaxel in water, and is at least 1000-fold and 20-fold better than hydroxypropyl- β -cyclodextrin (HPCD) and methyl- β -cyclodextrin (DMCD) under similar conditions [ Sharma et al, J. Med. Sci. 84(10), 1223-30(1995) ]. The sharp increase in solubility may be due to a combination of at least 3 of the following factors: 1) local concentration increase of CD moieties; 2) the binding constant is increased by synergy, the structure of paclitaxel has three phenyl groups surrounding a large fused, terpene ring system; and 3) additional hydrophobic interactions outside the CD cavity.

As expected, the toxicity of the β -cyclodextrin was significantly reduced after coupling to the PEG polymer. From all having cyclodextrinsNeither of the side-chain PEG polymers detected cytotoxicity as indicated by MTT and hemolytic assays (tables 5 and 6). Even monomers (building blocks) are much less toxic than native beta-cyclodextrin. On the other hand, since the proportion by weight of CD moieties in the copolymer of the invention is less than 25%, by weight¹The actual CD concentration in our experimental concentration (50mg copolymer/ml water) was determined by H-NMR to be less than 12.5 mg/ml. In other words, cyclodextrin: the weight ratio of paclitaxel is less than 6: 1 in the polymer composite of the present invention. Thus, this copolymer with non-biodegradable linker is a very safe drug carrier with very efficient drug release properties (table 5). In addition, biodegradable linkers are also acceptable if it is desired to accelerate drug release.

The above examples are given for the purpose of illustration only and should not be construed as limiting the invention in any way. Various modifications of the compounds and methods of the invention may be made without departing from the spirit and scope of the invention, it being understood that this invention is solely defined by the appended claims.

Claims (9)

1. A cyclodextrin grafted biocompatible polymer having the general formula 1:

general formula 1

Wherein P is a biocompatible hydrophilic polymer backbone, wherein the biocompatible hydrophilic polymer backbone is selected from the group consisting of polyethylene glycol, N- (2-hydroxypropyl) methacrylamide polymers, polyethylene imine, polylysine and derivatives thereof, having a molecular weight of 2,000 to 1,000,000 daltons; r' is hydrogen or a directing moiety; x is a linker having the formula:

-Q-Z-Q’-

wherein Q is covalently bonded to the biocompatible hydrophilic polymer backbone, either directly OR via alkyl side groups OR other functional groups, and Q' is covalently bonded to the cyclodextrin at the 2, 3 OR 6 position to replace OR, respectively₁、OR₂OR OR₃A group; q and Q' are independently selected from NR₄S, O, CO, CONH and COO; z is selected from the group consisting of alkylene disulfides, i.e. - (CH)₂)_aS-S(CH₂)_a-, alkylene, i.e. - (CH)₂)_a-, alkylene oxides, i.e. [ (CH)₂)_aO]_b(CH₂)_a-or a short-chain peptide, wherein a is an integer from 1 to 10, b is an integer from 1 to 20; r₁、R₂、R₃And R₄Independently selected from H, alkyl, i.e. C_n’H_2n’+1Alkenyl, i.e. C_n’+1H_2(n’+1)-1Or acyl radicals, i.e. C_n’H_2n’+1CO, wherein n' is an integer of 1 to 16; q is an integer of 5, 6 or 7; w is an integer such that each biocompatible hydrophilic polymer backbone contains 2-15 cyclodextrin moieties per 20KD biocompatible hydrophilic polymer backbone.

2. The cyclodextrin grafted biocompatible polymer of claim 1, wherein the biocompatible hydrophilic polymer backbone has a molecular weight of from 5,000 to 70,000.

3. The cyclodextrin grafted biocompatible polymer of claim 2,

wherein the biocompatible hydrophilic polymer backbone is polyethylene glycol and the cyclodextrin units grafted to the polyethylene glycol are randomly or uniformly distributed along the biocompatible hydrophilic polymer backbone.

4. The cyclodextrin grafted biocompatible polymer of claim 3,

wherein Q' is attached to the cyclodextrin at the 6-position and Q is covalently attached to the polyethylene glycol through a pendant alkyl group, wherein the pendant alkyl group is (CH)₂)_a。

5. The cyclodextrin grafted biocompatible polymer of any of claims 2-4, wherein Q is C (O) NH and Q' is NR₄。

6. The cyclodextrin grafted biocompatible polymer of claim 5, wherein Z is- (CH)₂)₂S-S(CH₂)₂-；R₄Is C₂H₅，R₁Is C₂H₅；R₂Is H and R₃Is C₂H₅。

7. A composition comprising the cyclodextrin grafted biocompatible polymer of any one of claims 1-6 and an active agent.

8. The composition of claim 7, wherein the active agent is a hydrophobic drug, a protein or peptide drug, a nucleic acid, or an oligonucleotide.

9. The composition of claim 8, wherein the active agent is paclitaxel.