Linear cyclodextrin copolymers

 

(57) Abstract:

The invention relates to a linear cyclodextrin copolymers and oxidized cyclodextrin, which can be used as a carrier for the delivery of various therapeutic agents. Water-soluble, linear cyclodextrin copolymer, which is obtained by a process comprising: a) obtaining at least one predecessor cyclodextrines monomer to polymerization only in two positions; b) interaction predecessor cyclodextrines monomer with a compound containing two functional groups, which may be made binding predecessors cyclodextrines monomer, resulting in a gain of water-soluble linear cyclodextrines copolymer comprising repeating the elementary units of the formula Ia, Ib. The cyclodextrin copolymer, soderjali cyclodextrine fragments as an integral part of its main chain of a polymer comprising repeating the elementary units of the formula Ia, Ib. therapeutic composition includes the above-mentioned copolymer and an effective amount of therapeutic agent. The composition includes a) a first copolymer, where at least Icodextrin fragments, as a part of his main polymer chain. therapeutic composition includes the above cyclodextrins composition and an effective amount of therapeutic agent. The method of delivery of a therapeutic agent to a patient includes an introduction to the patient a therapeutically effective amount of the above-mentioned therapeutic compositions. The invention allows to obtain copolymers which can be used as a carrier for the delivery of a medicinal product and which can release the tool in response to specific biological decomposition. 14 C. and 36 C.p. f-crystals, 2 PL.

The scope of the invention

The invention relates to a linear cyclodextrin copolymers and linear copolymers of oxidized cyclodextrin. These copolymers contain, respectively, cyclodextrines fragment unoxidized or oxidized, as Monomeric elementary level, which is embedded in the main chain of the copolymer. The invention also relates to methods for linear cyclodextrin copolymers and linear copolymers of oxidized cyclodextrin. Such copolymers cyclodextrin can be used as a carrier for the delivery of various terapevticheskii polysaccharides, containing the elementary parts of the natural D(+)-glucopyranose b -(1,4)connection. The most common cyclodextrins are alpha ()-cyclodextrins, beta ()-cyclodextrins and gamma ()-cyclodextrins, which contain, respectively, six, seven or eight links glucopyranose. The cyclic structure of the cyclodextrin in the form similar to a torus or toroidal ring having an inner non-polar or hydrophobic cavity, and on one side cyclodextrines torus are secondary hydroxyl groups, and on the other side are primary hydroxyl groups. Thus, using as a model ()-cyclodextrin, the cyclodextrin often represent schematically as follows:

Side, where the secondary hydroxyl group has a larger diameter compared with the side on which the primary hydroxyl group. The hydrophobic nature of the internal cavity of the cyclodextrin gives the possibility to include different connection. Comprehensive Supramolecular Chemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996); T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al., Applied Spectroscopy, 46:652-658 (1992); FR 2665169).

Cyclodextrins were used previously as a researcher who with various drugs, which can fit into the hydrophobic cavity of the cyclodextrin, or through the formation of non-covalent associated complexes with other biologically active molecules such as oligonucleotides and their derivatives. For example, in U.S. patent 4727064 describes pharmaceutical preparations consisting of a medicinal product is essentially a low solubility in water and amorphous water-soluble mixtures based on cyclodextrin. The drug forms an inclusion complex with cyclodextrins above-mentioned mixture. In U.S. patent 5691316 describes cyclodextrine delivery system oligonucleotides in cells. In this system, the oligonucleotide using non-covalent connection associated with the cyclodextrin in the complex or, alternatively, the oligonucleotide via a covalent bond may connect with adamantium, which, in turn, ecovalence associated with the cyclodextrin.

Known also various polymers containing cyclodextrin, and methods for their preparation. Comprehensive Supramolecular Chemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996)). A method of obtaining a polymer containing immobilized cyclodextrin, as described in U.S. patent 5608015. According to this method, a derivative of cyclodextrin evaluation of isenay acid or its derivative, containing the isocyanate end group or a derivative isocyanato group. A derivative of cyclodextrin is produced by interaction of cyclodextrin with such compounds, such as halides of carboxylic acids and anhydrides of the acids. The resulting polymer contains cyclodextrine elementary links in the form of side chains extending from the main chain of the macromolecule a linear polymer.

In U.S. patent 5276088 describes a method of synthesis of polymers containing cyclodextrin, through the interaction of polyvinyl alcohol or cellulose or its derivatives with derivatives of cyclodextrin or co-polymerization of cyclodextrin derivative with vinyl acetate or methyl methacrylate. And again, the polymer contains cyclodextrines fragment in the lateral fragment, extending from the main chain of the polymer.

Able to decompose biologically polymer aggregates with supramolecular structure described in the publication WO 96/09073 A1. The unit consists of a series of cyclic compounds bearing drug, which is obtained by linking the medicinal product, or a cyclodextrin and the subsequent “stringing” of the compounds of the composition medicine/ is it ends. This unit is reportedly capable of releasing the drug in response to specific biological decomposition occurring in the disease. These units are usually called cyclodextrine polymers in the form of a necklace.”

However, there is still a need in the linear polycyclohexylene in which cyclodextrines segment is part of the main circuit, and not the lateral fragment, coming from the main circuit, and in the way they are received.

Brief description of the invention

This invention satisfies this need and provides a linear cyclodextrin copolymers. Such a linear cyclodextrin copolymers contain repeating elementary link of formulas Ia, Ib, or a combination of:

The invention also provides methods of obtaining a linear cyclodextrin copolymers. One such method involves the joint polymerization of the source cyclodextrines monomer, disubstituted by identical or different leaving groups, and the source of co monomer And, the ability to replace a leaving group. Another such method includes iodination source cyclodextrines monomer to obtain the original iodirovannoi cyclogest the Omer with the original co monomer And to obtain a linear cyclodextrin copolymer. Another method includes iodination source cyclodextrines monomer with the formation of the source iodirovannoi cyclodextrines monomer, amination of the original iodirovannoi monomer to obtain the source diaminononane cyclodextrines monomer and subsequent joint polymerization of the source diaminononane cyclodextrines monomer with the original co monomer And to obtain a linear cyclodextrin copolymer. Another method involves the restoration of a linear copolymer of oxidized cyclodextrin to a linear cyclodextrin copolymer.

The invention also provides a linear copolymer oxidized cyclodextrin. Linear copolymer oxidized cyclodextrin is a linear cyclodextrin copolymer that includes at least one piece of oxidized cyclodextrin of the formula VIa or VIb:

Each cyclodextrines fragment of linear cyclodextrin copolymer of the invention can be oxidized in such a way as to obtain a linear copolymer oxidized cyclodextrin containing repeating elementary link of the formula VIa, VIb or a combination thereof.

The invention also provides spnego cyclodextrin copolymer thus, to at least one cyclodextrines monomer was oxidized. Other methods include joint polymerization of the initial oxidized cyclodextrines monomer with the original co monomer A.

The invention also provides a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin grafted on the substrate, and a method thereof. The invention also provides a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin crosslinked with another polymer, and a method thereof. A method of obtaining a cross-linked cyclodextrin polymers involves the interaction of a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin to polymer in the presence of a crosslinking agent.

The invention provides a linear cyclodextrin copolymer or a linear copolymer oxidized dextrin containing at least one ligand associated with the cyclodextrin copolymer. The ligand can be associated either with cyclodextrines fragment or fragment of the co monomer A.

The invention also provides cyclodextrines composition comprising at least one linear copolymer of Tioga invention. The invention also provides a therapeutic composition comprising a therapeutic agent and a linear cyclodextrin copolymer and/or a linear copolymer oxidized cyclodextrin of the present invention. The method of treatment by administration of a therapeutically effective amount of a therapeutic composition of the invention are also described.

Detailed description of the invention

One embodiment of the invention is a linear cyclodextrin copolymer. Linear cyclodextrin copolymer is a polymer containing cyclodextrine fragments as an integral part of the main chain of the macromolecule polymer. Previously cyclodextrine fragments were not parts of the main chain of the macromolecule polymer, and depart from the main chain of the macromolecule polymer as the side pieces. According to this invention a linear cyclodextrin copolymer contains repeating elementary link of the formula Ia, Ib, or a combination of:

In formulas Ia and Ib represents a substituted or unsubstituted cyclodextrines monomer, And represents comonomer associated, for example, covalently linked, with the cyclodextrin C. Polymerization source the proper functioning of the invention. Within a linear cyclodextrin copolymer of the present invention elementary link cyclodextrines monomer can be the same or different, and similarly, comonomer And may be the same or different.

Predecessor cyclodextrines monomer may be any cyclodextrin or its derivative, known in this area. As described above, the cyclodextrin is defined as a cyclic polysaccharide, in most cases containing six to eight elementary units existing in the nature of D(+)-glucopyranose b -(1,4)linkages. Preferably predecessor cyclodextrines monomer is a cyclodextrin containing six, seven and eight glucose elementary units, i.e. respectively the alpha ()-cyclodextrin, beta ()-cyclodextrin and gamma ()-cyclodextrin. A derivative of cyclodextrin may be any known substituted cyclodextrin, in which the Deputy does not affect the joint polymerization precursor of the co monomer And, as described above. According to the invention a derivative of cyclodextrin can be neutral, cationic or anionic. Examples of suitable substituents include, but without limitation, the guide is, for example, group of simple dihydroxypropyl ether, simple methylhydroxyethylcellulose ether, simple ethylhydroxylamine ether and simple ethylhydrocupreine ether; alkyl groups, such as, for example, methyl; sugars, such as, for example, glucosyl and maltose; acid groups, such as, for example, the group of carboxylic acids, phosphoric acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiophosphorous acid and sulfonic acid; imidazole group and a sulfate group.

In addition, the predecessor cyclodextrines monomer may be chemically modified (e.g., halogenated, laminirovannyy) to promote or influence on the polymerization precursor cyclodextrines monomer with the predecessor of the co monomer And, as described below. Chemical modification of precursor cyclodextrines monomer allows the polymerization only in two positions on each cyclodextrines fragment, that is, to obtain bifunctional cyclodextrines fragment. The numbering scheme C1-C6 provisions of each ring glucopyranose is the following:

In the preferred embodiment the polymerization ASU the measures one predecessor cyclodextrines monomer can cure two C6 provisions, while the other predecessor cyclodextrines monomer may polymerization in positions C2 and C6 cyclodextrines fragment. In the case-cyclo-dextrin scheme record the relative position of each ring glucopyranose in the cyclodextrin will be as follows:

In the preferred embodiment of a linear cyclodextrin copolymer of the present invention cyclodextrines monomer C has the following General formula (II):

In the formula (II), n and m are integers which together with two glucopyranose rings determine the total number of elementary links glucopyranose in cyclodextrines monomer. Formula (II) is cyclodextrines monomer that can undergo polymerization in two C6 positions on cyclodextrines elementary level. Examples cyclodextrines monomers of formula (II) include, but without limitation, 6A,6B-deoxy-cyclodextrin (n=0, m=4), 6A,6C-deoxy-cyclodextrin (n=1, m=3), 6A,6D-deoxy-cyclodextrin (n=2, m=2), 6A,6B-deoxy-cyclodextrin (n=0, m=5), 6A,6CJn (n=0, m=6), 6A,6C-deoxy-cyclodextrin (n=1, m=5), 6A,6D-deoxy-cyclodextrin (n=2, m=4), 6A,6E-deoxy-cyclodextrin (n=3, m=3). In another preferred embodiment of a linear cyclodextrin copolymer of the present invention cyclodextrine elementary link of monomer C has the following General formula (III):

where R=5-7. In the formula (III) one of the elementary parts of D(+)-glucopyranose cyclodextrines monomer is subjected to disclosure cycle for carrying out the polymerization in position C2 and C3 cyclodextrines elementary level. Cyclodextrine monomers of formula (III) commercially available from Carbomer of Westborough, MA. Examples cyclodextrines monomers of formula (III) include, but without limitation, 2A,3A-deoxy-2A,3A-dihydro-cyclodextrin, 2A,3A-deoxy-2A,3A-dihydro-cyclodextrin, 2A,3A-deoxy-2A,3A-dihydro-cyclodextrin, commonly referred to as, respectively, 2,3-deoxy-cyclodextrin, 2,3-deoxy-cyclodextrin and 2,3-deoxy-cyclodextrin.

The predecessor of the co monomer And can represent any connection with a straight or branched chain, symmetric or asymmetric, which results in strinovic monomer. Preferably the precursor of the co monomer a is a compound containing at least two functional groups, which can be carried out the reaction and, therefore, binding cyclodextrines monomers. Examples of possible functional groups, which may be identical or different, located on the end or in the middle of molecules of each source of co monomer And include, but without limitation, an amino group, acid group, the group of ester, imidazoline group or a group of acylhomoserine and their derivatives. In the preferred embodiment of the two functional groups are the same and end. In the copolymerization of co monomer precursor And precursor cyclodextrines monomer can bind together two cyclodextrins monomer by connecting the primary hydroxyl groups of one cyclodextrines monomer with the primary hydroxyl groups of another cyclodextrines monomer, by connecting the secondary hydroxyl groups of one cyclodextrines monomer with the secondary hydroxyl groups of another cyclodextrines monomer or by connecting the primary hydroxyl anomer. Accordingly, a combination of chemical bonds can exist in the final copolymer.

As the predecessor of the co monomer a and comonomer And the final copolymer may be neutral, cationic (e.g., containing protonated group such as, for example, Quaternary ammonium groups) or anionic (for example, containing deprotonated groups, such as, for example, sulfate, phosphate or carboxylate anionic groups). The charge of the co monomer And copolymer can be regulated by regulating the pH. Examples of suitable precursors of co monomer And include, but without limitation, tsistamin, 1,6-diaminohexane, diimidazole, dithioketal, spermin, cityspire, digitalin, detioration, succinimide (for example, dithiobis(succinimidylester) (DSP) and disuccinimidyl (DSS)) and imidate (for example, dimethyl 3,3'-databasprojekt (DTBP)). The copolymerization of co monomer precursor And precursor cyclodextrines monomer yields a linear cyclodextrin copolymer of this invention containing the co monomer And the following General formulas:

-HNC(O)(CH2)xC(O)NH-, -HNC(O)(CH2)xSS(CH2)xC(O)NH-,

- 2CH2C(O)NH-,

-HNNHC(O)(CH2CH2O)xCH2CH2C(O)NHNH-,

-+H2NCH2(CH2CH2O)xCH2CH2CH2NH+2-,

-NC(O)(CH2CH2O)xCH2CH2SS(CH2CH2O)xCH2CH2C(O)NH-,

-HNC(NH+2)(CH2CH2O)xCH2CH2C(NH+2)NH-,

-SCH2CH2NHC(NH+2)(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)(CH2)xSS(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)CH2CH2(OCH2CH2)xC(NH+2)NHCH2CH2CH2S-

In the above formulas x=1-50 and y+z=x. Preferably x=1-30. More preferably x=1-20. In the preferred embodiment of comonomer And able to decompose biologically or is acid-labile. In addition, in the preferred embodiment the source comonomer and, therefore, comonomer And can be selectively chosen for the target application. For example, for DOS comonomer And can be polietilenglikoli group.

Linear cyclodextrin copolymer of the present invention can be modified at least one ligand attached to the cyclodextrin copolymer. The ligand can join the cyclodextrin copolymer through cyclodextrines monomer With or comonomer A. Preferably the ligand is attached to at least one cyclodextrines fragment of linear cyclodextrin copolymer. Preferably the ligand allows the linear cyclodextrin copolymer to reach the cells and connect with it. If a linear cyclodextrin copolymer of the invention attached several ligands, which may be the same or different, additional ligand or ligands can join the same or different cyclodextrins fragments or to the same or different comonomers And copolymer.

Examples of suitable ligands include, but are not limited to, vitamins (e.g. folic acid), proteins (eg, transferrin and monoclonal antibodies) and polysaccharides. The ligand will vary depending on the type of target delivery. For example, receptor-held delivery can be achieved through, but without limitation, the use of ligand folic acid, Bria, transferring ligand. The ligand can join the copolymer of the invention by known methods.

Another embodiment of the invention is a method of obtaining a linear cyclodextrin copolymer. According to the invention, the copolymer linear cyclodextrin invention can be obtained by the joint polymerization of the precursor cyclodextrines monomer, disubstituted suitable leaving groups, with the predecessor of the co monomer And which is able to replace the leaving group. Leaving group, which may be the same or different, can be any leaving group known in this field, which can be subjected to substitution in the process of joint polymerization precursor of the co monomer A. In the preferred embodiment of a linear cyclodextrin copolymer may be obtained by ladirovannye predecessor cyclodextrines monomer with getting predecessor iodirovannoi cyclodextrines monomer and joint polymerization of the precursor iodirovannoi cyclodextrines monomer with the predecessor of the co monomer And obtaining a linear cyclodextrin copolymer containing repeating elementary link formulier cyclodextrin of the present invention iodination predecessor cyclodextrines monomer, as described above, is carried out with obtaining iodirovannoi predecessor cyclodextrines monomer of formula IVa, IVb, IVc, or a mixture thereof:

Diadiajenia cyclodextrin can be obtained by any known methods. (Tabushi et al., J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al., J. Am. Chem. 106, 4580-4584 (1984)). For example, the cyclodextrin may be subjected to interaction with the biphenyl-4,4'-desulfonylation in the presence of anhydrous pyridine with education-cyclodextrin, blocked biphenyl-4,4'-desulfonylation, which can then be subjected to interaction with potassium iodide with getting dead--cyclodextrin. Predecessor cyclodextrines monomer audiruetsya only in two positions. Through copolymerization iodirovannoi predecessor cyclodextrines monomer with the predecessor of the co monomer And, as described above, can be obtained linear cyclodextrin polymer containing a repeating element of the formula Ia, Ib or their combination, which is also described above. If this is acceptable, then the iodine or iodine groups may be replaced by other known leaving groups.

In addition, according to the invention the iodine group or another suitable leaving group may(may) be replaced by groupthink iodirovannoi cyclodextrines monomer of formula IVa, IVb, IVc or their mixture may be subjected to amination reaction with the formation of the predecessor diaminononane cyclodextrines monomer of formula Va, Vb, Vc, or a mixture thereof:

Predecessor diaminononane cyclodextrines monomer can be obtained by any known method. (Tabushi et al. Tetrahedron Lett. 18:1527-1530 (1977); Mungall et al., J. Org. Chem. 1659-1662 (1975)). For example, did--cyclodextrin may be subjected to interaction with sodium azide and then recover with the formation diamino-cyclodextrin.

Predecessor cyclodextrines monomer mineralsa only in two positions. Predecessor diaminononane cyclodextrines monomer may then be copolymerized with a precursor monomer And, as described above, to obtain a linear cyclodextrin copolymer containing repeating elementary link of the formula Ia, Ib or a combination thereof, which is also described above. However, amidofunctional diaminononane predecessor cyclodextrines optional monomer is attached directly to cyclodextrines fragment. Alternatively, amidofunctional may be introduced by substitution of an atom of iodine or other suitable leaving the example, -SCH2CH2NH2with education predecessor diaminononane cyclodextrines monomer of formula Vd, Ve, Vf or mixtures thereof.

Linear cyclodextrin copolymer of the present invention can also be obtained by reduction of a linear oxidized cyclodextrin copolymer of the present invention, as described below. This method can be carried out, if comonomer And does not contain a fragment that can be recovered, or group, such as, for example, a disulfide bridge.

According to this invention a linear cyclodextrin copolymer according to this invention may be subjected to oxidation so as to introduce at least one monomer is oxidized cyclodextrin in the copolymer so that the monomer is oxidized cyclodextrin was an integral part of the main chain of the macromolecule polymer. Linear cyclodextrin copolymer, which contains at least one monomer is oxidized cyclodextrin, is called a linear copolymer oxidized cyclodextrin. Cyclodextrines monomer can be oxidized either side of the secondary hydroxyl groups either side of the primary hydroxyl groups cyclodextringlucosyl monomers oxidized cyclodextrin, it can be the same or different cyclodextrin monomers, oxidized on the side of the primary hydroxyl groups and secondary hydroxyl groups, or on both sides. For example, a linear copolymer oxidized cyclodextrin with oxidized secondary hydroxyl groups, for example, contains at least one elementary link of the formula VIa or VIb:

In formulas VIa and VIb is a substituted or unsubstituted monomer oxidized cyclodextrin, and represents comonomer connected, for example, covalently communication with oxidized cyclodextrin C. in Addition, in formulas VIa and VIb oxidation of secondary hydroxyl groups leads to ring opening cyclodextrines fragment and the formation of aldehyde groups.

Linear copolymer oxidized cyclodextrin can be obtained by oxidation of a linear cyclodextrin copolymer, as described above. Oxidation of linear cyclodextrin copolymer may be carried out using known methods of oxidation. (Hisamatsu et al., Starch 44:188-191 (1992)). Preferably used oxidant such as, for example, periodate sodium. Qualified it is clear that under standard conditions the oxide is oploschenii of the invention a linear oxidized copolymer of this invention can contain one monomer oxidized cyclodextrin. In another embodiment, essentially all, or all cyclodextrine monomers of the copolymer will be oxidized.

Another way to obtain a linear copolymer oxidized cyclodextrin invention involves the oxidation of the original iodirovannoi or diaminononane cyclodextrin monomer, as described above, with the formation of the initial oxidized iodirovannoi or diaminononane cyclodextrines monomer and copolymerization predecessor oxidized iodirovannoi or diaminononane cyclodextrines monomer with the predecessor of the co monomer A. In the preferred embodiment, the oxidized precursor iodirovannoi cyclodextrines monomer of formula VIIa, VIIb, VIIc, or a mixture of:

can be obtained(a) by oxidation of the precursor iodirovannoi cyclodextrines monomer of formula IVa, IVb, IVc, or a mixture thereof, as described above. In another preferred embodiment, the oxidized precursor diaminononane cyclodextrines monomer of formula VIIIa, VIIIb, VIIIc, or a mixture of:

can be obtained(a) aminating predecessor oxidized iodirovannoi cyclodextrines monomer of formula VIIa, VI Ib, VIIc, or a mixture thereof, as described above. In the Omer of formula IXa, IXb, IXc, or a mixture of:

can be obtained(a) by the substitution of iodine or other suitable leaving group precursor oxidized cyclodextrines monomer, disubstituted iodine or other suitable leaving group, a fragment containing the amino group, for example-SCH2CH2NH2.

Alternatively, the oxidized precursor iodirovannoi or diaminononane cyclodextrines monomer, as described above, can be obtained by oxidation of the precursor cyclodextrines monomer with the formation of the oxidized precursor cyclodextrines monomer and subsequent diadiajenia and/or diaminononane oxidized cyclodextrines monomer, as described above. As described above, cyclodextrines fragment can be modified by other leaving groups other than the group of iodine, and other functionalities containing the amino group. Predecessor oxidized iodirovannoi or diaminononane cyclodextrines monomer may then be subjected to copolymerization with the predecessor of the co monomer And, as described above, with the formation of a linear copolymer of oxidized cyclodextrin of the present invention.

is the your connection to a copolymer of at least one ligand. The ligand described above.

In the preferred embodiment of the invention, a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin contains at the end of at least one precursor of the co monomer a or hydrolyzed product of a precursor of the co monomer As described above. Due to the fact that the cyclodextrin copolymer ends of at least one precursor copolymer And at least one free functional group, as described above, is a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin. For example, the functional group may be an acid group or a group which can be subjected to hydrolysis to obtain the acid group. According to this invention, the functional group may be subjected to additional chemical modification, if necessary, to improve properties cyclodextrines copolymer, such as, for example, colloidal stability or efficiency of transfection. For example, the functional group can be modified by reaction with PEG with the formation of cyclodextrin copolymer with terminal PEG group to improve colloidal stabilnosti the effectiveness and efficiency of transfection.

Additional chemical modification of cyclodextrin copolymer may be carried out using a modified functional group. For example, the modified functional group can be used for building a polymer chain by incorporating a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin, as described in the invention, the same or different cyclodextrin copolymer or necklacessilver the polymer. In the preferred embodiment of the invention, the polymer to which should be joining, is the same or different linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin, which may also contain at the end of at least one precursor of the co monomer And further modifications, as described above.

Alternatively, at least two identical or different linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer containing on the end of a functional group or a modified functional group, as described above, can interact and connect together through functional or modificari functional groups formed fragment, which is able to decompose, such as, for example, a disulfide bridge. For example, modification of the terminal functional group in cysteine may be used to obtain a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin containing at least one free Tilney group. Interaction with the same or other cyclodextrin copolymer also contains at least one free Tilney group, will lead to the formation of a disulfide bridge between the two copolymers. In the preferred embodiment of the invention, or functional modified functional groups can be selected to obtain the bridges, exhibiting varying the speed of decomposition (e.g., enzymatic decomposition), and thus to provide the system with a long time of release of a therapeutic agent. The resulting polymer may be blended, as described in this invention. A therapeutic tool, which is described in this invention can be added before curing or after crosslinking of the polymer. The ligand, which is described above, may also join via a modified functional group.

Linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin of the present invention may be any known in the field of ways. Such methods or techniques characteristics include, but without limitation, helpanimals chromatography (GPC), auxiliary matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF Mass spec),1H and13With NMR, light scattering and titration.

The invention also relates to compositions cyclodextrin containing at least one linear cyclodextrin copolymer and at least one linear copolymer oxidized cyclodextrin of the present invention, as described above. Accordingly, either one of the linear cyclodextrin copolymer and linear copolymer oxidized cyclodextrin, or both of the copolymer can the but this invention contain a therapeutic agent and a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin, including cross-linked copolymers of the present invention. Linear cyclodextrin copolymer, a linear copolymer oxidized cyclodextrin and their derivatives described above. A therapeutic agent may be any synthetic or natural biologically active therapeutic agent, including the well-known therapeutic agent. Examples of suitable therapeutic agents include, but are not limited to, antibiotics, steroids, polynucleotide (e.g., genomic DNA, cDNA, mRNA and antisense oligonucleotides, plasmids, peptides, peptide fragments, small molecules (e.g., doxorubicin) and other biologically active macromolecules, such as proteins and enzymes.

A therapeutic composition of the invention can be obtained using known methods. In the preferred embodiment the copolymer of the invention is mixed with therapeutic agent, which is described above for spontaneous aggregation. According to the invention a therapeutic tool and a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin associated with each other so that the copolymer acts as a carrier for delivery of therapeutic tools.consistent professional reasons, for example, by electrostatic interaction and hydrophobic interaction. The degree of Association can be determined by known methods, including, for example, a study of the fluorescence, the study of the mobility of DNA, light scattering, electron microscopy, and will vary depending on therapeutic agent.

For example, a therapeutic composition of this invention containing the copolymer of the present invention and DNA, can be used as a delivery method for facilitating transfection, i.e. the introduction of DNA into the cell of a living organism (e.g. human). (Boussif, O. Proceedings of the National Academy of Sciences, 92:7297-7301 (1995); Zanta et al. Bloconjugate Chemistry, 8:839-844 (1997)).

A therapeutic composition of the invention may constitute, for example, solid, liquid, suspension or emulsion. Preferably, therapeutic composition of the present invention is a drug that can be injected intravenously. Other ways of introducing therapeutic composition of the present invention include, depending on the physical condition of therapeutic compositions known methods, such as, but without limitation, oral administration, topical application, ASS="ptx2">Depending on the type of therapeutic agent in therapeutic composition of this invention can be used in various therapeutic methods (e.g., DNA vaccines, antibiotics, anti-virus tools) for the treatment of inherited or acquired disorders, such as cystic fibrosis, Gaucher disease, muscular dystrophy, AIDS, cancers (e.g., multiple myeloma, leukemia, melanoma and ovarian carcinoma), cardiovascular disorders (e.g., progressive heart failure, restenosis and hemophilia) and neurological disorders (e.g. brain injury). According to this invention the method of treatment includes the introduction of a therapeutically effective amount of a therapeutic composition of the present invention. For professionals it is clear that therapeutically effective amount will be determined for each case. Factors to consider include, but without limitation, the disease to be treated, and the physical characteristics of a patient suffering from this disease.

Another embodiment of the invention is a composition containing at least one biologically active is oxidized cyclodextrin of the present invention. For example, suitable agricultural biologically active compounds include, but are not limited to, fungicides, herbicides, insecticides and tools to combat plant diseases.

The following examples are given to illustrate the invention. However, it should be understand that the invention is not limited to specific conditions or some details described in these examples.

EXAMPLES

Materials: -cyclodextrin (Cerestar USA, Inc. of Hammond, IN) is dried under vacuum (<a 0.1 mTorr) at 120°C before use. Diphenyl-4,4'-disulfonate (Aldrich Chemical Company, Inc. of Milwaukee, WI) is recrystallized from a mixture of chloroform/hexane. Potassium iodide ground to powder using mortar and pestle and dried in an oven at 200°C. All other reagents obtained from commercial sources and used without additional purification. Samples of the polymers analyzed by HPLC in the system Hitachi HPLC, equipped with a detector Anspec RI and column Progel-TSK G3000pwxLusing as eluent water volumetric flow rate 1.0 ml/min-1.

Example 1: -Cyclodextrin, locked in positions a and D-biphenyl-4,4'-desulfonema, 1 (Tabushi et al. J. Am. Chem. Soc. 106, 5267-5270 (1984))

In a round bottom flask volume of the dextrin and 250 ml of anhydrous pyridine (Aldrich Chemical Company, Inc). The resulting solution was stirred at 50°C under a nitrogen atmosphere, and add 2,204 g (6,28 mmol) of biphenyl-4,4'-disulfonated in four equal portions at intervals of 15 minutes. After stirring at 50°C for an additional 3 hours the solvent is removed in vacuo and the residue purified column chromatography with reversed phase, using a gradient elution (0-40% acetonitrile in water). Fractions analyzed by high-performance liquid chromatography (HPLC) and the appropriate fractions are combined. After removal of acetonitrile on a rotary evaporator, the resulting aqueous suspension lyophilizer dry. The result 3,39 g (38%) of 1 as a colorless solid.

Example 2: 6A, 6D-Diod-6A,6D-deoxy-cyclodextrin, 2 (Tabushi et al. J. Am. Chem. Soc. 106, 4580-4584 (1984))

In the centrifuge tube with a volume of 40 ml, equipped with a magnetic stirrer, adapter Slinka and membrane load of 1.02 g (7.2 mmol) of 1, of 3.54 g (of 21.3 mmol) of dry powdered potassium iodide (Aldrich) and 15 ml of anhydrous N,N-dimethylformamide (DMF) (Aldrich). The resulting suspension is stirred at 80°C under nitrogen atmosphere for 2 hours. After cooling to room temperatureare DMF and supernatant combined and concentrated in vacuo. After that, the residue is dissolved in 14 ml of water, cooled in an ice bath and then added with intensive mixing 0.75 ml (7,3 mmol) tetrachloroethylene (Aldrich). The precipitate containing the complex was filtered on a glass Frith environment, washed with a small amount of acetone and dried in vacuum over P2O5within 14 hours. The result of 0.90 g (92%) of 2 as a white solid.

Example 3: 6A, 6D-Diazo-6A,6D-diosi--cyclodextrin, 3 (Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))

In a round bottom flask of 100 ml, equipped with a magnetic stirrer, adapter Slinka and membrane, download 1,704 g (1.25 mmol) of cyclodextrin diiodide, 0,49 g (7,53 mmol) of sodium azide (EM Science of Gibbstown, NJ) and 10 ml of anhydrous N,N-dimethylformamide (DMF). The resulting suspension is stirred at 60°C under nitrogen atmosphere for 14 hours. After that, the solvent is removed in vacuum. The resulting residue is dissolved in sufficient water to obtain a 0.2 M solution of salt and then passed through 11.3 g of resin Biorad AG501-X8(D) to remove residual salts. Then the eluate lyophilizer dry, the result 1.232 metric g (83%) 3 in the form of a solid white amorphous substance, which is used in the next stage without the n, 4 (Mungall et al., J. Org. Chem. 1659-1662 (1975))

In a round bottom flask of 250 ml, equipped with a magnetic stirrer and a membrane, download 1.232 metric g (1.04 mmol) of cyclodextrin bis-azide and 50 ml of anhydrous pyridine (Aldrich). To this suspension with stirring 0,898 g (3,42 mmol) of triphenylphosphine. The resulting suspension is stirred for one hour at room temperature and then add 10 ml of concentrated aqueous ammonia. The addition of ammonia is accompanied by a rapid evolution of gas, and the solution becomes homogeneous. After 14 hours the solvent is removed in vacuum and the residue is ground to powder with 50 ml of water. The solid substance is filtered off, the filtrate is acidified (pH<4) 10% Hcl and then the resulting solution is loaded into the ion exchange column containing a resin Toyopearl SP-650M (NH+4form). Product 4 elute ammonium bicarbonate (eluent with a gradient of 0-0 .5 M ammonium bicarbonate). The appropriate fractions are combined and lyophilizers, the result 0,832 g (71%) of product 4 as bis(bicarbonate) salt.

Example 5: copolymer of b-cyclodextrin and DSP, 5

In a scintillation vial with a volume of 20 ml download solution of 92.6 mg (7,65×10-5mol) of bis(bicarbonate) salt 4 in 1 ml of water. The value of pH of the sludge propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 ml of chloroform. The obtained two-phase mixture is stirred by a Vortex mixer (Vortex mixer) for 0.5 hours. Then the aqueous layer was decanted and extracted with fresh chloroform. Then an aqueous solution of the polymer was analyzed by gel chromatography (GPC) on the resin Toypearl HW-40F, using as eluent water. Faction analyze the GPC and the appropriate fractions lyophilizer, the result is 85 mg (85%) of product as colorless amorphous powder.

Example 6: copolymer-cyclodextrin and DSS 6

Copolymer-cyclodextrin and DSS 6 synthesized in a manner analogous to obtaining a copolymer-cyclodextrin and DSP 5, with the difference that the DSP is replaced by disuccinimidyl (DSS, Pierce Chemical Co. of Rockford, IL). Connection 6 receive 67% yield.

Example 7: copolymer-cyclodextrin and DTBP, 7

In scintillation flask 20 ml upload the solution to 91.2 mg (7,26×10-5mol) of bis(bicarbonate) salt 4 in 1 ml of water. The pH value of the solution was adjusted to 10 using 1 M NaOH solution and add to 22.4 mg (7,26×10-5mol) of dimethyl-3,3'-dithiobis(propionamide) NS (DTBP, Prierce Chemical Co. Of Rockford, IL). The obtained homogeneous solution is stirred with a stirrer Vortex at t HW-40F, using as eluent water. Faction analyze the GPC and the appropriate fractions lyophilizer, the result is 67 mg (67%) of product as colorless amorphous powder.

Example 8: a Copolymer of b-cyclodextrin and applied, 8

The solution to 166.2 mg (7,38×10-5mol) of applied dihydrochloride (Aldrich) in 15 ml of 0.1 n NaOH solution was added 100 mg (7,26×10-5mol) of 2 and 5 ml of acetonitrile. The obtained homogeneous solution is heated to 80°C, kept at this temperature for 2 hours and chromatographic using gel chromatography (GPC) on the resin Toyopearl HW-40F (eluent: water). Faction analyze the GPC and the appropriate fractions lyophilizer, the result of 17.2 mg (19%) of product as colorless amorphous powder.

Example 9: polyethylene Glycol 6000 dihydrazide, 9

In a round bottom flask of 100, equipped with a magnetic stirrer and reflux condenser, download 1,82 g (3.0 mmol) of polyethylene glycol 600 (Fluka Chemical Corp. of Milwaukee, WI), 40 ml of absolute ethanol (Quantum Chemicals Pty Ltd of Tuscola, IL) and a few drops of sulfuric acid. The resulting solution was refluxed for 14 hours. Solid sodium carbonate is added to terminate the reaction, and under atmostphere 0.6 ml (9.0 mmol) of hydrazine hydrate is added (Aldrich) in 10 ml of absolute ethanol. A small amount of muddy sediment. The resulting solution was refluxed for 1 hour, filtered and concentrated. Civil identifies the product polluting its impurities with higher molecular weight. Helpanimals chromatography on resin Toyopearl HW-40F allows to partially clean the product with obtaining the target compound with a purity of 85%.

Example 10: the Oxidation of the copolymer-cyclodextrin and DSS, 10 (Husamatsu et al., Starch 44, 188-191 (1992))

Copolymer-cyclodextrin and DSS 6 (92,8 mg, 7,3×10-5mol) is dissolved in 1.0 ml of water, cooled in an ice bath and add to 14.8 mg (7,38×10-5mol) of periodate sodium. The solution immediately becomes bright yellow staining, and it is stirred in the dark at 0°C for 14 hours. Then the solution chromatographic using gel chromatography (GPC) on the resin Toyopearl HW-40F (eluent: water). Faction analyze GPC. The appropriate fractions are combined and lyophilizers dry, the result 84,2 mg (91%) of product as a bright yellow amorphous powder.

Example 11: polyethylene Glycol (PEG) 600 chloride decollate, 11

In a round bottom flask of 50 ml volume, equipped with a magnetic stir bar and a reflux holodilnikami (Aldrich). To this solution was added with stirring to 3.9 ml with 53.4 mmol) of thionyl chloride (Aldrich) and the resulting solution was refluxed for 1 hour, and see the evolution of gas. The resulting solution was allowed to cool to room temperature and the solvent and excess thionyl chloride removed in vacuo. The oil obtained is stored in a dry box and used without purification.

Example 12: copolymer-cyclodextrin and PEG 600, 12

In a scintillation vial with a volume of 20 ml upload the solution to 112.5 mg (8,95×10-5mol) of bis(bicarbonate) salt 6A,6D-diamino-6A,6D-deoxy-cyclodextrin, 50 ál (3,6×10-4mol) of triethylamine (Aldrich) and 5 ml of anhydrous N,N-dimethylacetamide (DMAc, Aldrich). The resulting suspension is treated with 58 mg (9,1×10-5mol) of polyethylene glycol 600, dichlorohydrin, 11. The resulting solution is stirred by a Vortex mixer for 5 minutes and then left at 25°C for 1 hour, during which time the solution becomes homogeneous. The solvent is removed in vacuo and the residue chromatographic gel chromatography resin Toyopearl HW-40F (eluent: water). Faction analyze the GPC and the appropriate fractions lyophilizer dry, the result p is a and DSP, 13

In a test tube with a volume of 8 ml download solution of 102.3 mg (8,80×10-5mol) 2A,2A-diamino-2A,3A-deoxy-cyclodextrin in 1 ml of water. The pH value of the solution was adjusted to 10 with 1M NaOH solution and add a solution of 36.4 mg (8,80×10-5mol) dithiobis(Succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, II) in 1 ml of chloroform. The obtained two-phase mixture is stirred by a Vortex mixer for 0.5 hours. The aqueous layer was decanted and extracted with fresh chloroform (3×1 ml). An aqueous solution of the polymer chromatographic using gel permeation chromatography.

Example 14: 6A,6DBis-(2-aminoacetic)-6A,6D-deoxy-cyclodextrin, 14 (Tabushi, I. Shimokawa, K; Fugita, K. Tetrahedron Lett. 1977, 1527-1530)

In the flask Slanka of 25 ml volume, equipped with a magnetic stirrer and a membrane, loads of 0.91 ml (7,37 mmol) 0.81 M solution of 2-aminoethylthiomethyl sodium in ethanol. (Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis, Wiley: New York, 1967; Vol. 3, pp. 265-266). The solution is evaporated to dryness and the solid residue re-dissolved in 5 ml of anhydrous DMF (ldrich). Add 6A,6D-Diod-6A,6D-deoxy-cyclodextrin (100 mg, 7,38×10-5mol) and the resulting suspension peremeshivayu in vacuum and the residue re-dissolved in water. After acidification with 0.1 n solution of Hcl solution is transferred into a column of ion-exchange resin Toyopearl SP-650M (NH+4form) and elute (sodium bicarbonate solution, eluent with a gradient of 0-0,4 M). The appropriate fractions are combined and lyophilizers dry. The result is 80 mg (79%) of 14 as a white powder.

Example 15: copolymer-cyclodextrin(applied) and DTBP, 15

In a test tube with a volume of 4 ml upload solution 19,67 mg (1,42·10-5mol) of bis(bicarbonate) salt 14 in 0.1 M solution of NaHCO3. The solution is cooled in an ice bath and add 4.4 mg (1,4·10-5mol) of dimethyl-3,3'-dithiobisbenzothiazole-2hcl (DTBP, Pierce). The resulting solution is stirred by a Vortex mixer and left for 1 hour at 0°C. the Reaction is quenched with a 1M solution of Tris-HCl, acidified to pH 4 with 0.1 n Hcl. The aqueous polymer solution is subjected to gel chromatography on resin Toyopearl HW-40F. Faction analyze the GPC and the appropriate fractions lyophilizer dry. The result of 21.3 mg (100%) of 15 as a white powder.

Example 16: copolymer-cyclodextrin (applied) and DSM 16

In the flask Slanka volume of 10 ml, equipped with a magnetic stirrer and a membrane, load 200 mg (1,60×10-4mol) 14,44 ál (3,2 se) and 3 ml of anhydrous DMF (Aldrich Chemical Co., Milwaukee, WI). The resulting suspension is heated to 80°C and maintained at this temperature for 18 hours under stationary flow of nitrogen, and a large part of the solvent evaporates. The resulting residue re-dissolved in 10 ml of water and the resulting solution was acidified with 10% Hcl to pH 4. Then the resulting solution was passed through an Amicon centrifugal filter Method Plus-20 5000 NMWL. After washing with water (2×10 ml) solution of polymer lyophilizer dry, the result is to 41.4 mg (18%) of product in the form of a solid amorphous material yellowish color.

Example 17: Attach poliarnogo ligand to cyclodextrines the polymer 1. Joining resin:

50 mg of FMOC-PEG3400-NHS (Shearwater Polymers, Inc. of Huntsville, AL) dissolved in 1 ml of anhydrous N,N-dimethylformamide (DMF) and added to 10 equivalents of hydrazide-2-chlorotrityl resin (Novobiochem USA of La Jolla, CA), swollen in DMF. The mixture was stirred at 60°C until all the polymer is not attached to the resin, which is determined by GPC system equipped with a UV detector. The connection of the composition of the resin-polymer is then transferred into the column sintered glass, sintered glass column) for further interaction.

2. Blocking the functional groups of the resin:

Aproriate neutralized with diisopropylethylamine.

3. Removing the protective groups:

The protective FMOC group is removed by two washes with 20% solution of piperidine in DMF (total volume 1 ml). Then the resin 10 times washed with DMF (each time 1 ml) and 5 times H2O (each time 1 ml).

4. Blocking of functional groups folic acid:

10 equivalents of folic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) are added to the resin together with 1.5 ml of N2Acting In the reaction mixture was added 1 n NaOH solution until such time as folic acid will not dissolve (approximately pH 10). Then a glass column was placed on a rotator and stirring is continued over night. The resin was washed with 10×1 ml NaOH (1 ad), 10×1 ml of 50 mm sodium bicarbonate and then 5 times with water, THF and dichloromethane.

5. The selection of resin.

1% triperoxonane acid (TFA) in 1 ml DCM is added twice to the resin every time within minutes. The supernatant is collected and DCM is evaporated. The obtained oily film again hydronaut in N2Oh and lyophilizers, the result is a light yellow powder. Analysis by NMR confirms the presence of PEG polymer.

6. Linking polymer:

The connection of the composition of the binding agent is a folic acid vzaimodeistvie borate (pH 8.5). The reaction mixture is analyzed, and the presence of a conjugated polymer is confirmed by GPC system with UV detector at 285 nm. (See diagram 1 at the end of the description).

Example 18: attaching foliat-ligand to cyclodextrines polymer

1. Binding:

36 mg of tert-BUTYLCARBAMATE dissolved in 240 μl of a mixture of DCM/ethyl acetate (1:1), add to 260 mg of FMOC-PEG3400-NHS (Shearwater Polymer) and stirred at room temperature for 2 hours. Product dropped twice from a mixture of ethyl acetate/simple ether (1:1).

2. Removing the protective groups:

FMOC protective group is removed using 20% piperidine in DMF. The solvent is removed in vacuum and the product re-dissolved in 1.3 ml DMSO.

3. Blocking of functional groups folic acid:

To 1.2 equivalent of folic acid and DCC add one drop of pyridine and the resulting solution was stirred in the dark at room temperature for 6 hours. DMSO is removed in vacuum and the presence of a pair of folic acid is confirmed by GPC with UV monitoring at 285 nm.

4. The removal of the protective group of the hydrazide:

Finally, hydrazide group is removed by stirring in 4M Hcl in dioxane for one hour with rl HW-40F.

5. Linking polymer:

The connection of the composition of the folic acid - binding agent interacts with 6 equivalents of cyclodextrin copolymer (oxidized as in example 10) by mixing 50 mmol borate (pH 8.5). The reaction mixture is analyzed and the presence of a conjugated polymer is confirmed by using a GPC system with UV detector at 285 nm. (See diagram 2 at the end of the description).

Example 19: attach transferring ligand to cyclodextrines polymer

1. Oxidation transferrin

50 mg of human transferrin, not containing iron (Sigma of St. Louis, MO), dissolved in 30 mm acetate-sodium buffer and cooled to 0°C. To this solution was added 20 mg of periodate sodium dissolved in 4 μl of 30 mm sodium acetate solution. The mixture was stirred at 0°C for the night. Then add 1 g of resin AG501-X8 (Biorad) to remove salts and the solution lyophilizer.

2. The binding resin:

20 mg of FMOC-PEG3400-NHS (Snearwater Polymers, Inc. of Huntsville, AL) dissolved in 0.5 ml of anhydrous N,N-dimethylformamide (DMF) and added to 10 equivalents of hydrazide 2-chlorotrityl resin (Novabiochem USA of La Jolla, CA), swollen in DMF. The mixture was stirred at 60°up until the entire polymer n is out of the composition of the resin-polymer is transferred into a column agglomerated glass for all additional reactions.

3. Blocking the functional groups of the resin:

Unreacted hydrazide group on the resin block acetic anhydride and products - derivatives of acetic acid is neutralized with diisopropylethylamine.

4. Removing the protective groups:

FMOC protective group is removed by twice washing with 20% piperidine in DMF (total volume 1 ml). The resin is then washed 10 times (each time 1 ml DMF and five times (each time 1 ml) H2O.

5. The binding of transferrin:

To the resin added 1.2 equivalent of transferrin dissolved in 0.05 M solution of sodium carbonate and 0.1 M citrate-sodium buffer, pH of 9.5. Then to the solution was added 5 M cyanoborohydride 1 N. NaOH solution. A glass column was placed on a rotator and stirred for 2 hours. Then the resin is washed 15 times with water and 5 times with tetrahydrofuran (THF) and DCM.

6. The selection of resin.

1% triperoxonane acid (TFA) in 1 ml DCM twice added to the resin, each time for one minute. The supernatant is collected and DCM is evaporated. The obtained oily film again hydronaut in N2Oh and lyophilizers.

7. Linking polymer:

The connection structure connecting aguinaga amination with cyanoborohydride sodium: first, the copolymer is added to atransferrinemia binding agent, dissolved in 0.05 M solution of sodium carbonate and 0.1 M sodium citrate buffer. Add 5 M cyanoborohydride 1 N. NaOH solution and the reaction mixture is stirred for two hours at room temperature. Unreacted aldehyde sites block the addition of ethanolamine and interaction within 15 minutes at room temperature. The resulting conjugate is purified by dialysis. (See scheme 3 in the end of the description).

Example 20: a General method of obtaining complexes of cyclodextrin with small molecules

Copolymer based on cyclodextrin (CD polymer) is dissolved in water, buffer or organic solvent to the appropriate concentration. Connection with small molecules dissolved in a solvent miscible with the solvent of the solution CD-polymer, and added to a solution of CD polymer. The mixture is then stirred for1/2hours and left to reach equilibrium overnight.

Example 21: the Formation of the complex of cyclodextrin copolymer with doxorubicin.

Doxorubicin and CD polymer dissolved in various concentrations in PBS (phosphate buffer saline, pH of 7.2). The constant Association between CD and doxorubicin determine metering the interaction of CD and doxorubicin increases the intensity of fluorescence). The constant Association of approximately 200 M-1at pH of 7.1. Adding-CD significantly increases the fluorescence of doxorubicin, which is an indicator of complexation between CD polymer and doxorubicin. In the publication Husain et al., Applied Spectroscopy Vol. 46, No. 4, 652-658 (1992) shows that the constant Association between CD and doxorubicin equal to 210 M-1at pH of 7.1.

Example 22: the delivery of small molecules to cultured cells

For cultured cell lines used medium containing doxorubicin and doxorubicin complexes/CD polymer at various concentrations. After 5 hours, the medium removed and replaced with fresh medium. The effect of doxorubicin on survival of cells determined using quantitative analysis of the toxicity of MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazole). (R. Ian Feshney, “Culture of animal cells”, 3rded., Wiley-Liss: New York (1994)). The results are presented in the table below. Copolymer of 15 or 16 (138 microns equivalent CD monomer) is not toxic to KB or KB-VI (a derivative of KB, multidrug-resistant) cell lines in the absence of doxorubicin. When the receptor-held delivery for complexation with doxorubicin is used ligand, such as foliate, covalent the output constant charge and plasmids

Typically, equal volumes of CD polymer with a fixed charge and a DNA plasmid are mixed in appropriate ratios charges polymer/plasmid. The mixture is then allowed to reach equilibrium and leave overnight for spontaneous aggregation at room temperature. The process of complex formation is controlled by transferring a small aliquot of the mixture in 0.6% agarose gel and check the mobility of the DNA. Free DNA is moved under the action of an applied voltage, while complex DNA slows down in the tank.

1 μg of DNA with a concentration of 0.2 μg/μl in distilled water mixed with 10 μl of the copolymer 15 when the charge ratio of polymeric amine : DNA phosphate, equal 2, 4, 6, 12, 24, 36, 60 and 120. The solution is mixed manually using a micropipette and gently mixed overnight on a laboratory rotator. The next morning in each solution add 1 µg/µl loading buffer (40% sucrose, and 0.25% bromophenol blue, 20 mm Ttis-acetate buffer containing 5 mm EDTA (Gao et al., Biochemistry 35: 1027-1036 (1996)). Each sample loaded on a 0.6% agarose gel electrophoresis containing 6 µg EtBr/100 ml in 1×TAE buffer (40 mm ris-acetate/1 mm EDTA) and within 1 hour the gel serves a voltage of 40 V. the Degree ket DNA at a ratio of charge 6 and above indicating that complexation in these conditions.

Example 24: the Formation of the complex cross-linked polymer with plasmid Copolymer of 15 or copolymer 16 are oxidized as in example 10. The oxidized copolymer of 15 or 16 then subjected to complexation with DNA plasmid, as shown in examples 23 and 26. Then add a crosslinking agent (for example, the ED600-dihydrazide) to encapsulate DNA. Implementation encapsulate determined by light scattering and scanning electron microscope.

Example 25: the Complexation of a copolymer with a variable (pH-sensitive) charge with plasmid

Equal volumes of solutions of CD polymer and DNA plasmids in the water are mixed in appropriate ratios, the charge of the polymer/charge plasmids. The mixture is left for 30 minutes to achieve equilibrium and spontaneous aggregation. Then add a crosslinking agent (for example, the ED600-dihydrazide) to encapsulate DNA. Then add concentrated buffer solution for pH regulation and thus receive a neutral CD polymer. Implementation encapsulate determined by light scattering and visually with the aid of an electron microscope.

Example 26: the Study of the density of 60,000 cells/cell for 24 hours before transfection. Plasmids encoding luciferase gene encapsulate using CD-polymer as described in examples 23 and 25, so that the formed complexes with the charge ratio of DNA/polymer equal to 6, 12, 24, 36 and 60, as described in example 23 the study of DNA binding. The medium containing the complexes of DNA/polymer, is added to cultured cells and replaced with fresh medium after 5 hours incubation at 37°C. the Cells are lysed after 48 hours after transfection. Suitable substrates for the light of the evaluation luciferase added to the cell lysate. Luciferase activity, measured with the received units of light, calculated using a luminometer. Complexes of DNA/polymer successfully transfection KSS-21 cells at the ratios of charges equal to 6, 12 and 24. The cell lysate is also used to determine cell viability by assessing protein by Lauri. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). Maximum toxicity observed when the ratio of the charges of the polymer amine : DNA phosphate, 36 and 60 at 91% survival of the cells.

Example 27: the Study of transfection with plasmids encoding Lusiferase reporter gene

KSS-21 cells are placed in a 24-cell tablet with a density of 60,000 cells/cell for 24 hours before Tr, with the difference that the polymer 15 is replaced by a polymer 16 and that complexes of DNA/polymer successfully transfection KSS-21 cells at the ratios of charges 10, 20, 30 and 40 with a maximum transfection when the charge ratio of polymeric amine : DNA phosphate equal to 20. The medium containing the complexes of DNA/polymer, is added to cultured cells and replaced with fresh medium after 24 hours incubation at 37°C. the Cells are lysed 48 hours after transfection. Suitable substrates for the light of the evaluation luciferase added to the cell lysate. Luciferase activity, measured with the received units of light, calculated using a luminometer. The results are presented below. Complexes of DNA/polymer successfully transfection KSS-21 cells at the ratios of charges equal to 6, 12 and 24. The cell lysate is also used to determine cell viability by assessing protein by Lauri. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). The results are presented below. Maximum toxicity observed when the ratio of the charges of the polymer amine : DNA phosphate equal to 40 and 50 at 33% survival of the cells.

Example 28: the Study of transfection with a plasmid encoding the GFP reporter gene

Plasmids encoding green fluoresc the complex DNA/polymer added to cultured cells and replaced with fresh medium after incubation at 37°C. the Cells are separated from the surface of trypsin, washed and re-suspended in balanced salt solution Hanks (Hanks) with propidium iodide. After that, cells are subjected to analysis by sorting fluorescently-activated cells (FACS). Cell viability is determined by the size of the cells and with the exception of propidium iodide, and the successful implementation of transfection by GFP protein fluorescence.

Example 29: the Formation of polymer complexes with origenae

Complexation with desensitizing Oligocene carried out in accordance with the methods for plasmid complexation of examples 23 and 25.

Example 30: the Study of transfection with Oligocene.

The antisense Oligocene against luciferase gene encapsulate using CD-polymer as described in example 29. Solution environment containing complexes of algogen/polymer added to HeLa X1/5 cells (HeLa cells that stably Express luciferase gene provided by CLONTECH) and replaced with fresh medium after incubation for 5 hours at 37°C. the Cells are lysed Spustit luciferase, measured in the form of units of light, calculated using a luminometer. Successful implementation transferase determine the capture (by knockout) activity luciferase.

Example 31: Toxicity copolymer-cyclodextrin(applied) and DTBP, 15

Active toxicity copolymer 15 examined using Swiss-Webster “white mouse”. Just use the 48 mice, as described in table 2 below. Each mouse is subjected to a single intravenous (i.v.) or intraperitoneally (i.p.) contamination of sterile saline solution or a solution of a copolymer of 15. Five days later, animals kill and perform a complete autopsy. Mortality and toxicity do not see.

Example 32: a Study of transfection with plasmids encoding Lucifer reporter gene

Plasmids encoding luciferase gene encapsulate using CD-polymer as described in example 23 with the difference that instead of the copolymer of 15 using the copolymer 16. Complexes of DNA/polymer used for successful transfection KSS-21 and Cho-K1 cells, each placed on a 24 cell tablets with cellular density of 60,000 cells/cell for 24 hours before transfection with various amounts of charges in the medium containing 10% serum or not steriade the work. Appropriate substrates for quantitative light definition luciferase added to the cell lysate. Activity luciferase, measured from the calculation of derived units of light (i.e. the relative light units relative light units (RLU)), calculated using a luminometer. The cell lysate is also used to determine the viability of cells by quantitative determination of protein by Lauri. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). Toxicity is measured by determination of the total cellular protein in cells after 48 hours after transfection. Results transfection and survival of cells in 10% serum in the medium containing no serum, below.

Activity luciferase protein KSS-21 cells, transfection in the medium containing no serum, reaches a stable maximum at 30+/- 5×107RLU. The presence of 10% serum in transactional environment reduces the activity luciferase when the ratio of the charges, except 70+/-. For Cho-K1 cells increase the ratio of charge also leads to increased transfection for all test conditions. Additionally, tranfaglia serum reduces the number of units of light.

The copolymer 16 shows toxichem in the presence of 10% serum during transfection. Notable toxicity not observed when transfected on Cho-K1 cells.

The influence of the ratio of the charges of copolymer 16/DNA and the presence of serum on transfection efficiency (a ) and survival of juvenile ( ) KSS-21 cells. The results of transfection in 10% serum and the medium does not contain serum, presented by the dashed and solid lines. Data are presented as mean values of three samples +/- Art. deviation. Toxicity data presented in the form of lines of best fit (best fit lines).

The influence of the ratio of the charges of copolymer 16/DNA and the presence of serum on transfection efficiency (a ) and survival of cells ( ) in Cho-K1 cells. The results of transfection in 10% serum in the medium containing no serum, presented by the dashed and solid lines. Data are presented as mean values of three samples +/- Art. deviation. Toxicity data presented in the form of lines of best fit (best fit lines).

Comparative example 1: the Study of transfection with a plasmid encoding the Luciferase reporter gene

In accordance with the method of example 32 examine the transfection efficiency and toxicity of different non-viral vectors with CL is O-K1 cells transfection in the interval between charges and the source densities for all vectors in the environment, does not contain serum. The results are shown below and they represent the optimal transfection conditions set for each vector.

You should imagine that the above discussion and examples represent only a detailed description of certain preferred embodiments. For a qualified professional clear that various modifications and equivalents can be made without releasing the volume and scope of the invention. All the patents, journal articles and other documents discussed or cited above, is entered in the description by reference.

1. Water-soluble, linear cyclodextrin copolymer obtained by the process comprising (a) obtaining at least one predecessor cyclodextrines monomer to polymerization only in two positions; b) interaction predecessor cyclodextrines monomer with a compound containing two functional groups, which may be made binding predecessors cyclodextrines monomer, resulting in a gain of water-soluble linear cyclodextrines copolymer comprising repeating the elementary units of the formula Ia, Ib monomer;

And represents comonomer associated with the cyclodextrin C.

2. Water-soluble, linear cyclodextrin copolymer containing cyclodextrine fragments as an integral part of its main chain of a copolymer comprising repeating the elementary units of the formula Ia, Ib, or both-level:

where C represents a substituted or unsubstituted cyclodextrines monomer;

And represents comonomer associated with the cyclodextrin C.

3. The copolymer according to any one of paragraphs.1-2, where cyclodextrine fragments or cyclodextrine monomers are the same throughout the polymer.

4. The copolymer according to any one of paragraphs.1-2, where cyclodextrine fragments or cyclodextrine monomers contain at least two different cyclodextrins fragment.

5. The copolymer under item 4, where at least a few cyclodextrines fragments or cyclodextrines monomers are substituted.

6. The copolymer under item 5, where at least one cyclodextrines monomer or fragment substituted ligand, capable of binding the copolymer with the cell.

7. The copolymer under item 1 or 2, where cyclodextrine fragments or cyclodextrine monomer is extrenely

fragments or cyclodextrine monomers independently selected from the group comprising 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclodextrin, 6AND,6D-deoxy-cyclodextrin, 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclo-dextrin 6AND,6D-deoxy-cyclodextrin, 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclodextrin, 6AND,6D-deoxy-cyclodextrin, 6AND,6E-deoxy-cyclodextrin.

9. The copolymer under item 1 or 2, where these cyclodextrine fragments or cyclodextrine monomers contain oxidized cyclodextrine the monomers having the General formula (III)

where R = 5-7.

10. The cyclodextrin copolymer under item 9, where these oxidized cyclodextrine fragments or cyclodextrine monomers include fragments selected from 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin, 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin, 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin.

11. The copolymer under item 1, where comonomer choose from:

-HNC(O)(CH2)xCB>xNH+2-, -HNC(O)(CH2CH2O)xCH2CH2C(O)NH-,

-HNNHC(O)(CH2CH2O)xCH2CH2C(O)NHNH-,

-+H2NCH2(CH2CH2O)xCH2CH2CH2NH+2-,

-HNC(O)(CH2CH2O)xCH2CH2SS(CH2CH2O)xCH2CH2C(O)NH-,

-HNC(NH+2)(CH2CH2O)xCH2CH2C(NH+2)NH-,

-SCH2CH2NHC(NH+2)(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)(CH2)xSS(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)CH2CH2(OCH2CH2)xC(NH+2)NHCH2CH2S-,

and

where x = 1-50 and y+z=x.

12. The copolymer under item 1, where the comonomers contain biodegradable or acid-labile binding.

13. The copolymer under item 1 or 2, where the crosslinked copolymer with another polymer.

14. The copolymer under item 13, where at m is n with the copolymer.

16. The copolymer under item 1 or 2, where at least one cyclodextrines fragment or the monomer is oxidized.

17. The copolymer according to p. 16, where cyclodextrines fragment represents -, -, -cyclodextrin, or a combination thereof.

18. The copolymer according to p. 16, where the crosslinked copolymer with another polymer.

19. The copolymer under item 18, where at least one ligand is associated with oxidized copolymer.

20. The copolymer according to p. 16, where at least one ligand is associated with the copolymer.

21. The copolymer under item 1 or 2, where basically all cyclodextrine the oxidized fragments.

22. The copolymer under item 1 or 2, where all cyclodextrine the oxidized fragments.

23. A therapeutic composition comprising the copolymer according to any one of the preceding paragraphs and an effective amount of therapeutic agent.

24. Composition comprising a) a first copolymer according to p. 16 and (b) a second water-soluble, linear cyclodextrin copolymer comprising cyclodextrine fragments, as a part of his main copolymer chain.

25. The composition according to p. 24, where at least one of the specified first copolymer and the second copolymer is crosslinked with another polymer.

the first and second copolymers.

27. The composition according to p. 24, where at least one ligand associated with at least one of these first and second copolymers.

28. Therapeutic composition comprising cyclodextrins composition according to any one of paragraphs.24-27 and an effective amount of therapeutic agent.

29. The method of obtaining water-soluble, linear cyclodextrin copolymer comprising the following stages: a) obtaining at least one predecessor cyclodextrines monomer, which allows the polymerization only in two positions; b) interaction predecessor cyclodextrines monomer with a compound containing two functional groups through which the communication predecessors cyclodextrines monomer, resulting in a gain of water-soluble linear cyclodextrines copolymer comprising repeating the elementary units of the formula Ia, Ib, or both-level:

where C represents a substituted or unsubstituted cyclodextrines monomer;

And represents comonomer associated with the cyclodextrin C.

30. The method according to p. 29, where the specified predecessor cyclodextrines monomer is

31. The method according to p. 29, where the specified predecessor cyclodextrines monomer is a -, -, -cyclodextrin, or a combination thereof.

32. The method according to p. 29, where the specified predecessor cyclodextrines monomer selected from the group including: 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclodextrin, 6AND,6D-deoxy-cyclodextrin, 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclodextrin, 6AND,6D-deoxy-cyclodextrin, 6AND,6IN-deoxy-cyclodextrin, 6AND,6WITH-deoxy-cyclodextrin, 6AND,6D-deoxy-cyclodextrin, 6AND,6E-deoxy-cyclodextrin.

33. The method according to p. 29, where the specified predecessor cyclodextrines monomer has the General formula (III)

where p = 5.

34. The method according to p. 33, where the specified predecessor cyclodextrines monomer selected from the group comprising 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin, 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin, 2AND,3AND-deoxy-2AND,3AND-dihydro-cyclodextrin.

35. The method according to p. 29, where the compound containing two functional UB>xC(O)NH-, -+H2N(CH2)xSS(CH2)xNH+2, -HNC(O)(CH2CH2O)xCH2CH2C(O)NH-, -HNNHC(O)(CH2CH2O)xCH2CH2C(O)NHNH-,

-+H2NCH2(CH2CH2O)xCH2CH2CH2NH+2-,

-NC(O)(CH2CH2O)xCH2CH2SS(CH2CH2O)xCH2CH2C(O)NH-, -HNC(NH+2)(CH2CH2O)xCH2CH2C(NH+2)NH-, -SCH2CH2NHC(NH+2)(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)(CH2)xSS(CH2)xC(NH+2)NHCH2CH2S-,

-SCH2CH2NHC(NH+2)CH2CH2(OCH2CH2)xC(NH+2)NHCH2CH2CH2S-

or

where x = 1-50 and y+z=x.

36. The method according to p. 29, comprising the additional step of interaction the specified copolymer with ligand with formation of a linear copolymer containing at least one ligand that is associated with the copolymer.

37. The method according to p. 30, vkluchaushsih with the formation of the predecessor diaminononane cyclodextrines monomer and the polymerization of the specified predecessor diaminononane cyclodextrines monomer with the formation of the specified copolymer.

38. The method according to p. 37, where the specified predecessor diaminononane cyclodextrines monomer is a monomer having formula Va, Vb or Vc, or mixtures thereof:

39. The method according to p. 37, where the specified predecessor diaminononane cyclodextrines monomer is a monomer of formula Vd, Ve or Vf or mixtures thereof:

40. A method of producing a copolymer comprising a stage of recovery of the linear copolymer oxidized cyclodextrin, provided that the specified linear copolymer oxidized cyclodextrin does not contain able to restore comonomer A.

41. A method of producing a copolymer comprising a stage of oxidation of the copolymer under item 1 or 2.

42. The method according to p. 41 comprising the additional step of interaction the specified copolymer with ligand with formation of a copolymer containing at least one ligand that is associated with the copolymer.

43. A method of producing a copolymer comprising the following stages:

(a) iodination predecessor oxidized cyclodextrines monomer with the formation of the oxidized precursor iodirovannoi cyclodextrines monomer of formula VIIa, VIIb, VIIc is bathing cyclodextrines monomer with a co monomer precursor And the formation of linear copolymer oxidized cyclodextrin.

44. The method according to p. 43, comprising the additional step of interaction specified linear copolymer oxidized cyclodextrin with ligand with formation of a linear copolymer oxidized cyclodextrin containing at least one ligand that is associated with the copolymer.

45. A method of obtaining a copolymer, which includes stages (a) amination of oxidized precursor iodirovannoi cyclodextrines monomer with the formation of the oxidized precursor diaminononane cyclodextrines monomer of formula VIIIa, VIIIb, VIIIc, or a mixture thereof:

(b) interaction of the specified predecessor oxidized diaminononane cyclodextrines monomer with a co monomer precursor And the formation of linear copolymer oxidized cyclodextrin.

46. A method of obtaining a copolymer, which includes stages (a) amination of oxidized precursor iodirovannoi cyclodextrines monomer with getting predecessor oxidized diaminononane cyclodextrines monomer of formula IXa, Ib, CHD, or a mixture thereof:

(b) interaction of the specified predecessor oxidized diaminononane Chicago cyclodextrin.

47. A method of obtaining a cross-linked copolymer cyclodextrin, including the interaction of the copolymer under item 1 or 2 with the second copolymer in the presence of a crosslinking agent.

48. The method according to p. 47, where the specified second copolymer is a linear cyclodextrin copolymer or a linear copolymer oxidized cyclodextrin.

49. The method of delivery of a therapeutic agent to a patient comprising the administration to a patient a therapeutically effective amount of a therapeutic composition for p. 23.

50. The method of delivery of a therapeutic agent to a patient comprising the administration to a patient a therapeutically effective amount of a therapeutic composition for p. 28.



 

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