Method for preparing linear cyclodextrin copolymers

0001The invention relates to methods of preparing water-soluble, linear cyclodextrin copolymers and water-soluble linear oxidized cyclodextrin copolymers.

0002Cyclodextrins are cyclic polysaccharides containing naturally occurring D(+)-glucopyranose units in an α-(1,4) linkage. The most common cyclodextrins are alpha (α)-cyclodextrins, beta (β)-cyclodextrins and gamma (γ)-cyclodextrins which contain, respectively, six, seven or eight glucopyranose units. Structurally, the cyclic nature of a cyclodextrin forms a torus or donut-like shape having an inner apolar or hydrophobic cavity, the secondary hydroxyl groups situated on one side of the cyclodextrin torus and the primary hydroxyl groups situated on the other. Thus, using (β)-cyclodextrin as an example, a cyclodextrin is often represented schematically as follows: <chemistry id="chem0001" num="0001"><img file="EP1764112B1_D0001.tif" /></chemistry>

0003The side on which the secondary hydroxyl groups are located has a wider diameter than the side on which the primary hydroxyl groups are located. The hydrophobic nature of the cyclodextrin inner cavity allows for the inclusion of a variety of compounds. (<nplcit id="ncit0001" npl-type="b"><text>Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996</text></nplcit>); <nplcit id="ncit0002" npl-type="s"><text>T. Cserhati, Analytical Biochemistry, 225:328-332 (1995</text></nplcit>); <nplcit id="ncit0003" npl-type="s"><text>Husain et al., Applied Spectroscopy, 46:652-658 (1992</text></nplcit>); <patcit id="pcit0001" dnum="FR2665169"><text>FR 2 665 169</text></patcit>).

0004Cyclodextrins have been used as a delivery vehicle of various therapeutic compounds by forming inclusion complexes with various drugs that can fit into the hydrophobic cavity of the cyclodextrin or by forming non-covalent association complexes with other biologically active molecules such as oligonucleotides and derivatives thereof. For example, <patcit id="pcit0002" dnum="US4727064A"><text>U.S. Patent 4,727,064</text></patcit> describes pharmaceutical preparations consisting of a drug with substantially low water solubility and an amorphous, water-soluble cyclodextrin-based mixture. The drug forms an inclusion complex with the cyclodextrins of the mixture. In <patcit id="pcit0003" dnum="US5691316A"><text>U.S. Patent 5,691,316</text></patcit>, a cyclodextrin cellular delivery system for oligonucleotides is described. In such a system, an oligonucleotide is noncovalently complexed with a cyclodextrin or, alternatively, the oligonucleotide may be covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.

0005Various cyclodextrin containing polymers and methods of their preparation are also known in the art. (<nplcit id="ncit0004" npl-type="b"><text>Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996</text></nplcit>)). A process for producing a polymer containing immobilized cyclodextrin is described in <patcit id="pcit0004" dnum="US5608015A"><text>U.S. Patent 5,608,015</text></patcit>. According to the process, a cyclodextrin derivative is reacted with either an acid halide monomer of an α,β-unsaturated acid or derivative thereof or with an α,β-unsaturated acid or derivative thereof having a terminal isocyanate group or a derivative thereof. The cyclodextrin derivative is obtained by reacting cyclodextrin with such compounds as carbonyl halides and acid anhydrides. The resulting polymer contains cyclodextrin units as side chains off a linear polymer main chain.

0006<patcit id="pcit0005" dnum="US5276088A"><text>U.S. Patent 5,276,088</text></patcit> describes a method of synthesizing cyclodextrin polymers by either reacting polyvinyl alcohol or cellulose or derivatives thereof with cyclodextrin derivatives or by copolymerization of a cyclodextrin derivative with vinyl acetate or methyl methacrylate. Again, the resulting cyclodextrin polymer contains a cyclodextrin moiety as a pendant moiety off the main chain of the polymer.

0007A biodegradable medicinal polymer assembly with supermolecular structure is described in <patcit id="pcit0006" dnum="WO9609073A1"><text>WO 96/09073 A1</text></patcit>. The assembly comprises a number of drug-carrying cyclic compounds prepared by binding a drug to an α, β, or γ-cyclodextrin and then stringing the drug/cyclodextrin compounds along a linear polymer with the biodegradable moieties bound to both ends of the polymer. Such an assembly is reportably capable of releasing a drug in response to a specific biodegradation occurring in a disease. These assemblies are commonly referred to as "necklace-type" cyclodextrin polymers.

0008Cyclodextrin polymers in which the cyclodextrin moiety is part of the main chain and not a pendant moiety off the main chain and a method for their preparation, are described in <patcit id="pcit0007" dnum="JP58167613B"><text>JP 58 167 613</text></patcit>, and are obtained by reacting a cyclodextrin with a diepoxy compound in a random monomer. However, there still exists a need for linear cyclodextrin-containing polymers.

0009This invention answers this need by providing a method for preparing a water-soluble, linear cyclodextrin copolymer comprising: <ul id="ul0001" list-style="none"><li>providing a diaminated cyclodextrin monomer precursor; and</li><li>copolymerizing the diaminated cyclodextrin monomer precursor with a linker precursor containing at least two functional groups through which a linkage to the cyclodextrin monomer precursor can be achieved, wherein the linker moieties comprise a polyethylene glycol group or a group having the following structure: -SCH₂CH₂NHC(NH₂⁺)(CH₂)_xC(NH₂⁺)NHCH₂CH₂S- wherein x = 1 to 50.</li></ul> The linear cyclodextrin copolymer may have a repeating unit of formula Ia, Ib, or a combination thereof: <chemistry id="chem0002" num="0002"><img file="EP1764112B1_D0002.tif" /></chemistry><chemistry id="chem0003" num="0003"><img file="EP1764112B1_D0003.tif" /></chemistry>

0010One possible method involves iodinating a cyclodextrin monomer precursor to form a diiodinated cyclodextrin monomer precursor, aminating the diiodinated cyclodextrin monomer precursor to form a diaminated cyclodextrin monomer precursor and then copolymerizing the diaminated cyclodextrin monomer precursor with said linker precursor to produce the linear cyclodextrin copolymer.

0011The linear oxidized cyclodextrin copolymer as defined above may be a linear cyclodextrin copolymer which contains at least one oxidized cyclodextrin moiety of formula VIa or VIb: <chemistry id="chem0004" num="0004"><img file="EP1764112B1_D0004.tif" /></chemistry><chemistry id="chem0005" num="0005"><img file="EP1764112B1_D0005.tif" /></chemistry> Each cyclodextrin moiety of a linear cyclodextrin copolymer may be oxidized so as to form a linear oxidized cyclodextrin copolymer having a repeating unit of formula VIa, VIb, or a combination thereof.

0012Also described is a method for the preparation of a linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer crosslinked to another polymer involving reacting a linear or linear oxidized cyclodextrin copolymer with a polymer in the presence of a crosslinking agent.

0013The invention further provides a method for bounding at least one ligand bound to the cyclodextrin copolymer. The ligand may be bound to either the cyclodextrin monomer moiety or the comonomer A moiety of the copolymer.

0014A linear cyclodextrin copolymer is a polymer containing cyclodextrin monomer moieties as an integral part of its polymer backbone. Previously, cyclodextrin moieties were not a part of the main polymer chain but rather attached off a polymer backbone as pendant moieties. According to the invention, a linear cyclodextrin copolymer may have a repeating unit of formula Ia, Ib, or a combination thereof. <chemistry id="chem0006" num="0006"><img file="EP1764112B1_D0006.tif" /></chemistry><chemistry id="chem0007" num="0007"><img file="EP1764112B1_D0007.tif" /></chemistry>

0015In formula Ia and Ib, C is a substituted or unsubstituted cyclodextrin monomer moiety and A is a linker moiety bound, <i>i.e</i>. covalently bound, to the cyclodextrin O monomer moiety. Polymerization of a cyclodextrin monomer precursor with a linker precursor results in a linear cyclodextrin copolymer of the invention. Within a single linear cyclodextrin copolymer of the invention, the cyclodextin monomer moiety may be the same or different and, likewise, the linker moiety may be the same or different.

0016A cyclodextrin monomer precursor may be any cyclodextrin or derivative thereof known in the art. As discussed above, a cyclodextrin is defined as a cyclic polysaccharide most commonly containing six to eight naturally occurring D(+)-glucopyranose units in an α-(1,4) linkage. Preferably, the cyclodextrin monomer precursor is a cyclodextrin having six, seven and eight glucose units, <i>i.e</i>., respectively, an alpha (α)-cyclodextrin, a beta (β)-cyclodextrin and a gamma (γ)-cyclodextrin. A cyclodextrin derivative may be any substituted cyclodextrin known in the art where the substituent does not interfere with copolymerization with the linker precursor as described below. For the purpose of the invention, a cyclodextrin derivative may be neutral, cationic or anionic. Examples of suitable substituents include, but are not limited to, hydroxyalkyl groups, such as, for example, hydroxypropyl, hydroxyethyl; ether groups, such as, for example, dihydroxypropyl ethers, methyl-hydroxyethyl ethers, ethyl-hydroxyethyl ethers, and ethyl-hydroxypropyl ethers; alkyl groups, such as, for example, methyl; saccharides, such as, for example, glucosyl and maltosyl; acid groups, such as, for example, carboxylic acids, phosphorous acids, phosphinous acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiophosphonic acid and sulfonic acids; imidazole groups; and sulfate groups.

0017A cyclodextrin monomer precursor may be further chemically modified (e.g. halogenated, laminated) to facilitate or affect copolymerization of the cyclodextrin monomer precursor with a linker precursor, as described below. Chemical modification of a cyclodextrin monomer precursor allows for polymerization at only two positions on each cyclodextrin monomer precursor, <i>i.e</i>. the creation of a bifunctional cyclodextrin monomer moiety. The numbering scheme for the C1-C6 positions of each glucopyranose ring is as follows: <chemistry id="chem0008" num="0008"><img file="EP1764112B1_D0008.tif" /></chemistry>

0018In a preferred embodiment, polymerization occurs at two of any C2, C3 and C6 position, including combinations thereof, of the cyclodextrin monomer precursor. For example, one cyclodextrin monomer precursor may be polymerized at two C6 positions while another cyclodextrin monomer precursor may be polymerized at a C2 and a C6 position of the cyclodextrin monomer precursor. Using β-cyclodextrin as an example, the lettering scheme for the relative position of each glucopyranose ring in a cyclodextrin is as follows: <chemistry id="chem0009" num="0009"><img file="EP1764112B1_D0009.tif" /></chemistry>

0019In a preferred embodiment of a linear cyclodextrin copolymer of the invention, the cyclodextrin monomer moiety has the following general formula (II). <chemistry id="chem0010" num="0010"><img file="EP1764112B1_D0010.tif" /></chemistry>

0020In formula (II), n and m represent integers which, along with the other two glucopyranose rings, define the total number of glucopyranose units in the cyclodextrin monomer moiety. Formula (II) represents a cyclodextrin monomer moiety which is capable of being polymerized at two C6 positions on the cyclodextrin monomer moiety. Examples of cyclodextrin monomer moieties monomer of formula (II) include, but are not limited to, 6^A,6^B-deoxy-α-cyclodextrin (n=0, m=4), 6^A,6^C-deoxy-α-cyclodextrin (n=1, m=3), 6^A,6^D-deoxy-α-cyclodextrin (n=2, m=2), 6^A,6^B-deoxy-β-cyclodextrin (n=0, m=5), 6^A,6^C-deoxy-β-cyclodextrin (n=1, m=4), 6^A6^D-deoxy-β-cyclodextrin (n=2, m=3), 6^A,6^B-deoxy-γ-cyclodextrin (n=0, m=6), 6^A,6^C-deoxy-γ-cyclodextrin (n=1, m=5), 6^A,6^D-deoxy-γ-cyclodextrin (n=2, m=4), and 6^A,6^E-deoxy-γ-cyclodextrin (n=3, m=3). In another preferred embodiment of the linear cyclodextrin copolymer of the invention, a cyclodextrin monomer moiety has the following general formula (III): <chemistry id="chem0011" num="0011"><img file="EP1764112B1_D0011.tif" /></chemistry> where p = 5-7. In formula (III), one of D(+)-glucopyranose units of a cyclodextrin monomer moiety has undergone ring opening to allow for polymerization at a C2 and a C3 position of the cyclodextrin monomer moiety. Cyclodextrin monomer moieties of formula (III) are commercially available from Carbomer of Westborough, MA. Examples of cyclodextrin monomer moieties of formula (III) include, but are not limited to, 2^A,3^A-deoxy-2^A,3^A-dihydro-α-cyclodextrin, 2^A,3^A-deoxy-2^A,3^A-dihydro-β-cyclodextrin, 2^A,3^A-deoxy-2^A,3^A-dihydro-γ-cyclodextrin, commonly referred to as, respectively, 2,3-deoxy-α-cyclodextrin, 2,3-deoxy-β-cyclodextrin, and 2,3-deoxy-γ-cyclodextrin.

0021A linker precursor may be any straight chain or branched, symmetric or asymmetric compound which upon reaction with a cyclodextrin monomer precursor, as described above, links two cyclodextrin monomer moieties together. Preferably, a linker precursor is a compound containing at least two functional groups through which reaction and thus linkage of the cyclodextrin monomer precursors can be achieved. Examples of possible functional groups, which may be the same or different, terminal or internal, of each linker precursor include, but are not limited to, amino, acid, ester, imidazole, and acyl halide groups and derivatives thereof. In a preferred embodiment, the two functional groups are the same and terminal. Upon copolymerization of a linker precursor with a cyclodextrin monomer precursor, two cyclodextrin monomer moieties may be linked together by joining the primary hydroxyl side of one cyclodextrin monomer moiety with the primary hydroxyl side of another cyclodextrin monomer moiety by joining the secondary hydroxyl side of one cyclodextrin monomer moiety with the secondary hydroxyl side of another cyclodextrin monomer or by joining the primary hydroxyl side of one cyclodextrin monomer moiety with the secondary hydroxyl side of another cyclodextrin monomer moiety. Accordingly, combinations of such linkages may exist in the final copolymer. Both the linker precursor and the linker moiety of the final copolymer may be neutral, cationic (<i>e.g</i>. by containing protonated groups such as, for example, quaternary ammonium groups) or anionic (<i>e.g</i>. by containing deprotonated groups, such as, for example, sulfate, phosphate or carboxylate anionic groups). The charge of the linker moiety of the copolymer may be adjusted by adjusting pH conditions. Examples of suitable linker precursors include. Copolymerization of a linker precursor with a cyclodextrin monomer precursor leads to the formation of a linear cyclodextrin copolymer of the invention containing a linker moieties of the following general formula:

0022In the above formula, x = 1-50. Preferably, x =1-30. More preferably, x = 1-20. In a preferred embodiment, the linker moiety is biodegradable or acid-labile. Also in a preferred embodiment, the linker precursor and hence the linker moiety may be selectively chosen in order to achieve a desired application. For example, to deliver small molecular therapeutic agents, a charged polymer may not be necessary and the linker moiety may be a polyethylene glycol group.

0023A linear cyclodextrin copolymer may be modified with at least one ligand attached to the cyclodextrin copolymer. The ligand may be attached to the cyclodextrin copolymer through the cyclodextrin monomer moiety or the linker moiety. Preferably, the ligand is attached to at least one cyclodextrin monomer moiety of the linear cyclodextrin copolymer. Preferably, the ligand allows a linear cyclodextrin copolymer to target and bind to a celL If more than one ligand, which may be the same or different, is attached to a linear cyclodextrin copolymer of the invention, the additional ligand or ligands may be bound to the same or different cyclodextrin monomer moiety or the same or different linker moiety of the copolymer. Examples of suitable ligands include, but are not limited to, vitamins (<i>e.g</i>. folic acid), proteins (<i>e.g</i>. transferrin, and monoclonal antibodies) and polysaccharides. The ligand will vary depending upon the type of delivery desired. For example, receptor-mediated delivery may be achieved by, but not limited to, the use of a folic acid ligand while antisense oligo delivery may be achieved by, but not limited to, use of a transferrin ligand. The ligand may be attached to a copolymer of the invention by means known in the art.

0024A method of preparing a linear cyclodextrin iodinates a cyclodextrin monomer precursor as described above to form a diiodinated cyclodextrin monomer precursor of formula IVa, IVb, IVc or a mixture thereof: <chemistry id="chem0012" num="0012"><img file="EP1764112B1_D0012.tif" /></chemistry>

0025The diiodinated cyclodextrin monomer precursor may be prepared by any means known in the art. (<nplcit id="ncit0005" npl-type="s"><text>Tabushi et al. J. Am. Chem. 106, 5267-5270 (1984</text></nplcit>); <nplcit id="ncit0006" npl-type="s"><text>Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984</text></nplcit>)). For example, β-cyclodextrin may be reacted with biphenyl-4,4'-disulfonyl chloride in the presence of anhydrous pyridine to form a biphenyl-4,4'-disulfony) chloride capped β-cyclodextrin which may then be reacted with potassium iodide to produce diiodo-β-cyclodextrin. The cyclodextrin monomer precursor is iodinated at only two positions. By copolymerizing the diiodinated cyclodextrin monomer precursor with a linkeR precursor, as described above, a linear cyclodextrin polymer having a repeating unit of formula Ia, Ib, or a combination thereof, also as described above, may be prepared. If appropriate, the iodine or iodo groups may be replaced with other known leaving groups.

0026The iodo groups or other appropriate leaving group may be displaced with a group that permits reaction with a linkeR precursor, as described above. For example, a diiodinated cyclodextrin monomer precursor of formula IVa, IVb, IVc or a mixture thereof may be aminated to form a diaminated cyclodextrin monomer precursor of formula Va, Vb, Vc or a mixture thereof: <chemistry id="chem0013" num="0013"><img file="EP1764112B1_D0013.tif" /></chemistry><chemistry id="chem0014" num="0014"><img file="EP1764112B1_D0014.tif" /></chemistry>

0027The diaminated cyclodextrin monomer precursor may be prepared by any means known in the art (<nplcit id="ncit0007" npl-type="s"><text>Tabushi et al. Tetrahedron Lett. 18:1527-1530 (1977</text></nplcit>); <nplcit id="ncit0008" npl-type="s"><text>Mungall et al., J. Org. Chem. 1659-1662 (1975</text></nplcit>)). For example, a diiodo-β-cyclodextrin may be reacted with sodium azide and then reduced to form a diamino-β-cyclodextrin. The cyclodextrin monomer precursor is aminated at only two positions. The diaminated cyclodextrin monomer precursor may then be copolymerized with a linkeR precursor, as described above, to produce a linear cyclodextrin copolymer having a repeating unit of formula Ia, Ib, or a combination thereof, also as described above. However, the amino functionality of a diaminated cyclodextrin monomer precursor need not be directly attached to the cyclodextrin moiety. Alternatively, the amino functionality may be introduced by displacement of the iodo or other appropriate leaving groups of a cyclodextrin monomer precursor with amino group containing moieties such as, for example, SCH₂CH₂NH₂, to form a diaminated cyclodextrin monomer precursor of formula Vd, Ve, Vf or a mixture thereof: <chemistry id="chem0015" num="0015"><img file="EP1764112B1_D0015.tif" /></chemistry><chemistry id="chem0016" num="0016"><img file="EP1764112B1_D0016.tif" /></chemistry>

0028A linear cyclodextrin copolymer may be oxidized so as to introduce at least one oxidized cyclodextrin monomer into the copolymer such that the oxidized cyclodextrin monomer is an integral part of the polymer backbone. A linear cyclodextrin copolymer which contains at least one oxidized cyclodextrin monomer is defined as a linear oxidized cyclodextrin copolymer. The cyclodextrin monomer may be oxidized on either the secondary or primary hydroxyl side of the cyclodextrin moiety. If more than one oxidized cyclodextrin monomer is present in a linear oxidized cyclodextrin copolymer, the same or different cyclodextrin monomers oxidized on either the primary hydroxyl side, the secondary hydroxyl side, or both may be present. For illustration purposes, a linear oxidized cyclodextrin copolymer with oxidized secondary hydroxyl groups has, for example, at least one unit of formula VIa or VIb: <chemistry id="chem0017" num="0017"><img file="EP1764112B1_D0017.tif" /></chemistry><chemistry id="chem0018" num="0018"><img file="EP1764112B1_D0018.tif" /></chemistry>

0029In formulae VIa and VIb, C is a substituted or unsubstituted oxidized cyclodextrin monomer moiety and A is a linker moiety bound, i.e. covalently bound, to the oxidized cyclodextrin monomer moiety C. Also in formulae VIa and VIb, oxidation of the secondary hydroxyl groups leads to ring opening of the cyclodextrin monomer moiety and the formation of aldehyde groups.

0030A linear oxidized cyclodextrin copolymer may be prepared by oxidation of a linear cyclodextrin copolymer as discussed above. Oxidation of a linear cyclodextrin copolymer may be accomplished by oxidation techniques known in the art (<nplcit id="ncit0009" npl-type="s"><text>Hisamatsu et al., Starch 44:188-191 (1992</text></nplcit>)). Preferably, an oxidant such as, for example, sodium periodate is used. It would be understood by one of ordinary skill in the art that under standard oxidation conditions the degree of oxidation may vary or be varied per copolymer. Thus in one embodiment of the invention, a linear oxidized copolymer may contain one oxidized cyclodextrin monomer. In another embodiment, substantially all to all cyclodextrin monomers of the copolymer would be oxidized.

0031Another method of preparing a linear oxidized cyclodextrin copolymer involves the oxidation of a diiodinated or diaminated cyclodextrin monomer precursor, as described above, to form an oxidized diiodinated or diaminated cyclodextrin monomer precursor and copolymerization of the oxidized, diaminated cyclodextrin monomer precursor with a linker precursor. In a preferred embodiment, an oxidized diiodinated cyclodextrin monomer precursor of formula VIIa, VIIb, VIIc, or a mixture thereof: <chemistry id="chem0019" num="0019"><img file="EP1764112B1_D0019.tif" /></chemistry><chemistry id="chem0020" num="0020"><img file="EP1764112B1_D0020.tif" /></chemistry> preferred. An oxidized diaminated cyclodextrin monomer precursor of formula VIIIa, VIIIb, VIIIc or a mixture thereof: <chemistry id="chem0021" num="0021"><img file="EP1764112B1_D0021.tif" /></chemistry><chemistry id="chem0022" num="0022"><img file="EP1764112B1_D0022.tif" /></chemistry> may be prepared by amination of an oxidized diiodinated cyclodextrin monomer precursor of formulae VIIa, VIIb, VIIc, or a mixture thereof, as described above. In still another preferred embodiment, an oxidized diaminated cyclodextrin monomer precursor of formula IXa, IXb, IXc or a mixture thereof: <chemistry id="chem0023" num="0023"><img file="EP1764112B1_D0023.tif" /></chemistry><chemistry id="chem0024" num="0024"><img file="EP1764112B1_D0024.tif" /></chemistry> may be prepared by displacement of the iodo or other appropriate leaving groups of an oxidized cyclodextrin monomer precursor disubstituted with an iodo or other appropriate leaving group with the amino group containing moiety -SCH₂CH₂NH₂.

0032Alternatively, an oxidized diiodinated or diaminated cyclodextrin monomer precursor, as described above, may be prepared by oxidizing a cyclodextrin monomer precursor to form an oxidized cyclodextrin monomer precursor and then diiodinating and/or diaminating the oxidized cyclodextrin monomer precursor, as described above. The oxidized diaminated cyclodextrin monomer precursor may then be copolymerized with said linker precursor, as described above, to form a linear oxidized cyclodextrin copolymer.

0033A linear oxidized cyclodextrin copolymer may also be further modified by attachment of at least one ligand to the copolymer. The ligand is as described above.

0034In a preferred embodiment of the invention, a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer terminates with at least one linker precursor or hydrolyzed product of the linker precursor, each as described above. As a result of termination of the cyclodextrin copolymer with at least one linker precursor, at least one free functional group, as described above, exists per linear cyclodextrin copolymer or per linear oxidized cyclodextrin copolymer. For example, the functional group may be an acid group or a functional group that may be hydrolyzed to an acid group. The functional group may be further chemically modified as desired to enhance the properties of the cyclodextrin copolymer, such as, for example, colloidal stability, and transfection efficiency. For example, the functional group may be modified by reaction with PEG to form a PEG terminated cyclodextrin copolymer to enhance colloidal stability or with histidine to form an imidazolyl terminated cyclodextrin copolymer to enhance intracellular and transfection efficiency.

0035Further chemistry may be performed on the cyclodextrin copolymer through the modified functional group. For example, the modified functional group may be used to extend a polymer chain by linking a linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer, as described herein, to the same or different cyclodextrin copolymer or to a non-cyclodextrin polymer. In a preferred embodiment of the invention, the polymer to be added on is the same or different linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer which may also be terminated with at least one linker precursor for further modification, each as described herein.

0036Alternatively, at least two of the same or different linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers containing a terminal functional group or a terminal modified functional group, as described above, may be reacted and linked together through the functional or modified functional group. Preferably, upon reaction of the functional or modified functional groups, a degradable moiety such as, for example, a disulfide linkage is formed. For example, modification of the terminal functional group with cysteine may be used to produce a linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer having at least one free thiol group. Reaction with the same or different cyclodextrin copolymer also containing at least one free thiol group wall form a disulfide linkage between the two copolymers. In a preferred embodiment of the invention, the functional or modified functional groups may be selected to offer linkages exhibiting different rates of degradation (<i>e.g</i>. via enzymatic degradation) and thereby provide, if desired, a time release system for a therapeutic agent The resulting polymer may be crosslinked, as described herein. A therapeutic agent, as described herein, may be added prior to or post crosslinking of the polymer. A ligand, as described herein, may also be bound through the modified functional group.

0037A linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer may be attached to or grafted onto a substrate. The substrate may be any substrate as recognized by those of ordinary skill in the art. In another preferred embodiment of the invention, a linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer may be crosslinked to a polymer to form, respectively, a crosslinked cyclodextrin copolymer or a crosslinked oxidized cyclodextrin copolymer. The polymer may be any polymer capable of crosslinking with a linear or linear oxidized cyclodextrin copolymer of the invention (<i>e.g</i>. polyethylene glycol (PEG) polymer, polyethylene polymer). The polymer may also be the same or different linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer. Thus, for example, a linear cyclodextrin copolymer may be crosslinked to any polymer including, but not limited to, itself, another linear cyclodextrin copolymer, and a linear oxidized cyclodextrin copolymer. A crosslinked linear cyclodextrin copolymer may be prepared by reacting a linear cyclodextrin copolymer with a polymer in the presence of a crosslinking agent. A crosslinked linear oxidized cyclodextrin copolymer may be prepared by reacting a linear oxidized cyclodextrin copolymer with a polymer in the presence of an appropriate crosslinking agent. The crosslinking agent may be any crosslinking agent known in the art. Examples of crosslinking agents include dihydrazides and disulfides. In a preferred embodiment, the crosslinking agent is a labile group such that a crosslinked copolymer may be uncrosslinked if desired.

0038A linear cyclodextrin copolymer and a linear oxidized cyclodextrin copolymer may be characterized by any means known in the art. Such characterization methods or techniques include, but are not limited to, gel permeation chromatography (GPC), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF Mass spec), ¹H and ¹³C NMR, light scattering and titration.

0039The following examples are given to illustrated the invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these example.

EXAMPLES

0040<b>Materials</b>. β-Cyclodextrin (Cerestar USA, Inc. of Hammond, IN) was dried in <i>vacuo</i> (&lt;0.1 mTorr) at 120 °C for 12 h before use. Biphenyl-4,4'-disulfonyl chloride (Aldrich Chemical Company, Inc. of Milwaukee, WI) was recrystallized from chloroform/hexanes. Potassium iodide was powdered with a mortar and pestle and dried in an oven at 200°C. All other reagents were obtained from commercial suppliers and were used as received without further purification. Polymer samples were analyzed on a Hitachi HPLC system equipped with an Anspec RI detector and a Progel-TSK G3000_PWXL column using water as eluant at a 1.0 mL min^-1 flow rate.

Preparation Example 1

: Biphenyl-4,4'-disulfonyl-A,D-capped-β-cyclodextrin, 1 (Tabushi et al.

J. Am. Chem. Soc.

106

, 5267-5270 (1984))

0041A 500 mL round bottom flask equipped with a magnetic stirbar, a Schlenk adapter and a septum was charged with 7.92 g (6.98 mmol) of dry β-cyclodextrin and 250 mL of anhydrous pyridine (Aldrich Chemical Company, Inc.). The resulting solution was stirred at 50°C under nitrogen while 2.204 g (6.28 mmol) of biphenyl-4,4'-disulfonyl chloride was added in four equal portions at 15 min intervals. After stirring at 50 °C for an additional 3 h, the solvent was removed in <i>vacuo</i> and the residue was subjected to reversed-phase column chromatography using a gradient elution of 0-40% acetonitrile in water. Fractions were analyzed by high performance liquid chromatography (HPLC) and the appropriate fractions were combined. After removing the bulk of the acetonitrile on a rotary evaporator, the requiting aqueous suspension was lyophilized to dryness. This afforded 3.39 g (38%) of <b>1</b> as a colorless solid.

Preparation Example 2

: 6

A

,6

D

-Diiodo-6

A

,6

D

-deoxy-β-cyclodextrin,

2

(Tabushi et al.

J

.

Am. Chem

. Soc.

106

, 4580-4584 (1984))

0042A 40 mL centrifuge tube equipped with a magnetic stirbar, a Schlenk adapter and a septum was charged with 1.02 g (7.2 mmol) of 1, 3.54 g (21.3 mmol) of dry, powdered potassium iodide (Aldrich) and 15 mL of anhydrous N,N-dimethylformamide (DMF) (Aldrich). The resulting suspension was stirred at 80°C under nitrogen for 2 h. After cooling to room temperature, the solids were separated by centrifugation and the supernatant was decanted. The solid precipitate was washed with a second portion of anhydrous DMF and the supernatants were combined and concentrated <i>in vacuo.</i> The residue was then dissolved in 14 mL of water and cooled in an ice bath before 0.75 mL (7.3 mmol) of tetrachloroethylene (Aldrich) was added with rapid stirring. The precipitated inclusion complex was filtered on a medium glass frit and washed with a small portion of acetone before it was dried under vacuum over P₂O₅ for 14 h. This afforded 0.90 g (92%) of <b>2</b> as a white solid.

Preparation Example 3

: 6

A

,6

D

-Diazido-6

A

,6

D

-deoxy-β-cyclodextrin, 3 (Tabushi et al.

Tetrahedron Lett

.

18

, 1527-1530 (1977))

0043A 100 mL round bottom flask equipped with a magnetic stirbar, a Schlenk adapter and a septum was charged with 1.704 g (1.25 mmol) of β-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 was stirred at 60°C under nitrogen for 14 h. The solvent was then removed <i>in vacuo</i>. The resulting residue was dissolved in enough water to make a 0.2 M solution in salt and then passed through 11.3 g of Biorad AG501-X8(D) resin to remove residual salts. The eluant was then lyophilized to dryness yielding 1.232 g (83%) of <b>3</b> as a white amorphous solid which was carried on to the next step without further purification.

Preparation Example 4:

6

A

,6

D

-Diamino-6

A

,6

D

-deoxy-β-cyclodextrin, 4 (Mungall et al.,

J. Org. Chem

. 1659-1662 (1975))

0044A 250 mL round bottom flask equipped with a magnetic stirbar and a septum was charged with 1.232 g (1.04 mmol) of β-cyclodextrin bisazide and 50 mL of anhydrous pyridine (Aldrich). To this stirring suspension was added 0.898 g (3.42 mmol) of triphenylphosphine. The resulting suspension was stirred for 1 h at ambient temperature before 10 mL of concentrated aqueous ammonia was added. The addition of ammonia was accompanied by a rapid gas evolution and the solution became homogeneous. After 14 h, the solvent was removed <i>in vacuo</i> and the residue was triturated with 50 mL of water. The solids were filtered off and the filtrate was made acidic (pH&lt;4) with 10% HCl before it was applied to an ion exchange column containing Toyopearl SP-650M (NH₄⁺ form) resin. The product 4 was eluted with a gradient of 0-0.5 M ammonium bicarbonate. Appropriate fractions were combined and lyophilized to yield 0.832 g (71%) of the product 4 as the bis(hydrogen carbonate) salt.

Comparative Example 5

: β-cyclodextrin-DSP copolymer,

5

0045A 20 mL scintillation vial was charged with a solution of 92.6 mg (7.65 x 10^-5 mol) of the bis(hydrogen carbonate) salt of <b>4</b> in 1 mL of water. The pH of the solution was adjusted to 10 with 1 M NaOH before a solution of 30.9 mg (7.65 x 10^-1 mol) of dithiobis(succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 mL of chloroform was added. The resulting biphasic mixture was agitated with a Vortex mixer for 0.5 h. The aqueous layer was then decanted and extracted with 3 x 1 mL of fresh chloroform. The aqueous polymer solution was then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin using water as eluant Fractions were analyzed by GPC and appropriate tractions were lyophilized to yield 85 mg (85%) as a colorless amorphous powder.

Comparative Example 6

: β-cyclodextrin-DSS copolymer,

6

0046A β-cyclodextrin-DSS copolymer, <b>6</b>, was synthesized in a manner analogous to the ASP polymer, 5, except that disuccinimidyl suberate (DSS, Pierce Chemical Co. of Rockford, IL) was substituted for the DSP reagent Compound 6 was obtained in 67% yield.

Comparative Example 7

: β-cyclodextrin-DTBP copolymer,

7

0047A 20 mL scintillation vial was charged with a solution of 91.2 mg (7.26 x 10^-5 mol) of the bis(hydrogen carbonate) salt of <b>4</b> in 1 mL of water. The pH of the solution was adjusted to 10 with 1 M NaOH before 22.4 mg (7.26 x 10^-5 mol) of dimethyl 3,3'-dithiobis(propionimidate) ·-2 HCl (DTBP, Pierce Chemical Co. of Rockford, IL) was added. The resulting homogeneous solution was agitated with a Vortex mixer for 0.5 h. The aqueous polymer solution was then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzed by GPC and appropriate fractions were lyophilized to yield 67 mg (67%) of a colorless amorphous powder.

Comparative Example 8

: β-cyclodextrin-cystamine copolymer,

8

0048To a solution of 166.2 mg (7.38 x 10^-5 mol) of cystamine dihydrochloride (Aldrich) in 15 mL of 0.1 N NaOH was added 100 mg (7.38 x 10^-5 mol) of 2 and 5 mL of acetonitrile. The resulting homogeneous solution was heated at 80°C for 2 h before it was subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzed by GPC and appropriate fractions were lyophilized to yield 17.2 mg (19%) of a colorless amorphous powder.

Preparation Example 9

: Polyethylene glycol 600 dihydrazide,

9

0049A 100 mL round bottom flask equipped with a magnetic stirbar and a reflux condenser was charged with 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 heated to reflux for 14 h. Solid sodium carbonate was added to quench the reaction and the solution of the PEG diester was transferred under nitrogen to an addition funnel. This solution was then added dropwise to a solution of 0.6 mL (9.0 mmol) of hydrazine hydrate (Aldrich) in 10 mL of absolute ethanol. A small amount of a cloudy precipitate formed. The resulting solution was heated to reflux for 1 h before it was filtered and concentrated. GPC analysis revealed a higher molecular weight impurity contaminating the product Gel permeation chromatography on Toyopearl HW-40 resin enabled a partial purification of this material to approximately 85% purity.

Comparative Example 10

: Oxidation of β-cyclodextrin-DSS copolymer 6 to obtain 10 (Hisamatsu et al.,

Starch

44

, 188-191 (1992))

0050The β-cyclodextrin-DSS copolymer <b>6</b> (92.8 mg, 7.3 x 10^-5 mol) was dissolved in 1.0 mL of water and cooled in an ice bath before 14.8 mg (7.3 x 10^-5 mol) of sodium periodate was added. The solution immediately turned bright yellow and was allowed to stir in the dark at 0°C for 14 h. The solution was then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40 resin using water as eluant. Fractions were analyzed by GPC. Appropriate fractions were combined and lyophilized to dryness to yield 84.2 mg (91%) of a light brown amorphous solid.

Preparation Example 11

: Polyethylene glycol (PEG) 600 diacid chloride,

11

0051<chemistry id="chem0025" num="0025"><img file="EP1764112B1_D0025.tif" /></chemistry>

0052A 50 mL round bottom flask equipped with a magnetic stirbar and a reflux condenser was charged with 5.07g (ca. 8.4 mmol) of polyethylene glycol 600 diacid (Fluka Chemical Corp of Milwaukee, WI) and 10 mL of anhydrous chloroform (Aldrich). To this stirring solution was added 3.9 mL (53.4 mmol) of thionyl chloride (Aldrich) and the resulting solution was heated to reflux for 1h, during which time gas evolution was evident. The resulting solution was allowed to cool to room temperature before the solvent and excess thionyl chloride were removed <i>in vacuo.</i> The resulting oil was stored in a dry box and used without purification.

Example 12

: β-cyclodextrin-PEG 600 copolymer,

12

0053<chemistry id="chem0026" num="0026"><img file="EP1764112B1_D0026.tif" /></chemistry><chemistry id="chem0027" num="0027"><img file="EP1764112B1_D0027.tif" /></chemistry>

0054A 20 mL scintillation vial was charged with a solution of 112.5 mg (8.95 x 10^-5 mol) of the bis(hydrogen carbonate) salt of 6^A,6^D-diamino-6^A,6^D-deoxy-ß-cyclodextrin, 50 µL (3.6 x 10^-4 mol) of triethylamine (Aldrich), and 5 mL of anhydrous <i>N,N</i>-dimethylacetamide (DMAc, Aldrich). The resulting suspension was then treated with 58 mg (9.1 x 10^-5 mol) of polyethylene glycol 600 diacid chloride, <b>11</b>. The resulting solution was agitated with a Vortex mixer for 5 minutes and then allowed to stand at 25 °C for 1h during which time it became homogeneous. The solvent was removed <i>in vacuo</i> and the residue was subjected to gel permeation chromatography on Toyopearl HW-40F resin using water as eluant. Fractions were analyzed by GPC and appropriate fractions were lyophilized to dryness to yield 115 mg (75%) of a colorless amorphous powder.

Comparative Example 13

: β-cyclodextrin-DSP copolymer,

13

.

0055<chemistry id="chem0028" num="0028"><img file="EP1764112B1_D0028.tif" /></chemistry>

0056A 8 mL vial was charged with a solution of 102.3 mg (8.80 x 10-5 mol) of 2^A,3^A-diamino-2^A,3^A-deoxy-β-cyclodextrin in 1 mL of water. The pH of the solution was adjusted to 10 with 1 M NaOH before a solution of 36.4 mg (8.80 x 10^-5 mol) of dithiobis(succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 mL of chloroform was added. The resulting biphasic mixture was agitated with a Vortex mixer for 0.5h. The aqueous layer was then decanted and extracted with 3 x 1 mL of fresh chloroform. The aqueous polymer solution was then subjected to gel permeation chromatography.

Preparation Example 14:

6

A

,6

D

-Bis-(2-aminoethylthio)-6

A

6

D

-deoxy-β-cyclodextrin,

14

(Tabushi, I: Shimokawa, K; Fugita, K.

Tetrahedron Lett.

1977

, 1527-1.530)

0057<chemistry id="chem0029" num="0029"><img file="EP1764112B1_D0029.tif" /></chemistry>

0058A 25 mL Schlenk flask equipped with a magnetic stirbar and a septum was charged with 0.91 mL (7.37 mmol) of a 0.81 M solution of sodium 2-aminoethylthiolate in ethanol. (<nplcit id="ncit0010" npl-type="b"><text>Fieser, L.F.; Fieser, M. Reagents for Organic Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266</text></nplcit>). The solution was evaporated to dryness and the solid was redissolved in 5 mL of anhydrous DMF (Aldrich). 6^A,6^D-Diiodo-6^A6^D-deoxy-B-cyclodextrin (100 mg, 7.38 x 10^-5 mol) was added and the resulting suspension was stirred at 60 °C under nitrogen for 2 h. After cooling to room temperature, the solution was concentrated <i>in vacuo</i> and the residue was redissolved in water. After acidifying with 0.1 N HCl, the solution was applied to a Toyopearl SP-650M ion-exchange column (NH₄⁺ form) and the product was eluted with a 0 to 0.4 M ammonium bicarbonate gradient. Appropriate fractions were combined and lyophilized to dryness. This afforded 80 mg (79%) of 14 as a white powder.

Comparative Example 15

: ß-cyclodextrin(cystamine)-DTBP copolymer,

15

0059<chemistry id="chem0030" num="0030"><img file="EP1764112B1_D0030.tif" /></chemistry><chemistry id="chem0031" num="0031"><img file="EP1764112B1_D0031.tif" /></chemistry><chemistry id="chem0032" num="0032"><img file="EP1764112B1_D0032.tif" /></chemistry>

0060A 4 mL vial was charged with a solution of 19.6 mg (1.42 x 10^-5 mol) of the bis(hydrogen carbonate) salt of 14 in 0.5 mL of 0.1 M NaHCO₃. The solution was cooled in an ice bath before 4.4 mg (1.4 x 10^-5 mol) of dimethyl 3,3'-dithiobispropionimidate-2 HCl (DTBP, Pierce) was added. The resulting solution was then agitated with a Vortex mixer and allowed to stand at 0°C for 1h. The reaction was quenched with 1M Tris-HCl before it was acidified to pH 4 with 0.1 N HCl. The aqueous polymer solution was then subjected to gel permeation chromatography on Toyopearl HW-40F resin. Fractions were analyzed by GPC and appropriate fractions were lyophilized to dryness. This afforded 21.3 mg (100%) of 15 as a white powder.

Example 16

: β-cyclodextrin(cystamine)-DMS copolymer,

16

0061<chemistry id="chem0033" num="0033"><img file="EP1764112B1_D0033.tif" /></chemistry><chemistry id="chem0034" num="0034"><img file="EP1764112B1_D0034.tif" /></chemistry>

0062A 10 mL Schlenk flask equipped with a magnetic stirbar and a septum was charged with 200 mg (1.60 x 10^-4 mol) of 14, 44 µL (3.2 x 10^-4 mol) of triethylamine (Aldrich Chemical Co., Milwaukee, WI), 43.6 mg (1.60 x 10^-4 mol) of dimethylsuberimidate-2HCl (DMS, Pierce), and 3 mL of anhydrous DMF (Aldrich Chemical Co., Milwaukee, WI). The resulting slurry was heated to 80°C for 18 hours under a steady stream of nitrogen during which time most of the solvent had evaporated. The residue which remained was redissolved in 10 mL of water and the resulting solution was then acidified with 10% HCl to pH 4. This solution was then passed through an Amicon Centricon Plus-20 5,000 NMWL centrifugal filter. After washing with 2 x 10 mL portions of water, the polymer solution was lyophilized to dryness yielding 41.4 mg (18%) of an off-white amorphous solid.

Comparative Example 17

: Folate Ligand Attachment to Cyclodextrin Polymer 1. Resin coupling:

006350 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers, Inc. of Huntsville, AL) is dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF) and is added to 10 equivalents of hydrazide 2-chlorotrityl resin (Novabiochem USA of La Jolla, CA) swelled in DMF. The mixture is stirred at 60°C until all the polymer is coupled to the resin, as determined by a GPC system equipped with a UV detector. The resin-polymer is then transferred to a sintered glass column for all further reactions.

2. Resin Capping:

0064The unreacted hydrazide groups on the resins are capped with acetic anhydride and the acetic acid products are neutralized by diisopropylethylamine.

3. Removal of protecting group:

0065The FMOC protecting group is removed by two washes with 20% piperidine in DMF (1 mL total volume). The resin is then washed 10 times with 1 mL DMF and 5 times with 1 mL H₂O.

4. Folic Acid coupling:

006610 equivalents of folic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is added to the resin along with 1.5 mL H₂O. 1N NaOH is added to the reaction mixture until the folic acid is dissolved (around pH 10). The glass column is then placed on a rotator and mixed overnight. The resin is then washed 10 times with 1 mL NaOH (1N), 10 times with 1 mL of 50 mM sodium bicarbonate, and then 5 times each with water, THF, and dichloromethane.

5. Cleavage from resin:

00671% trifluoroacetic acid (TFA) in 1 mL DCM is added to the resin twice for 1 minute each. The supernatant is collected and DCM evaporated. The resulting oily film is rehydrated in H₂O and lyophilized, resulting in a light yellow powder. An NMR is taken to confirm the presence of the PEG polymer.

6. Coupling to polymer:

0068Folic acid-linker is reacted with 6 equivalents of a cyclodextrin copolymer (oxidized as in Example 10) by mixing in 50 mmol borate (pH 8.5). The reaction mixture is analyzed and conjugation polymer confirmed by a GPC system with a UV detection at 285 nm. <chemistry id="chem0035" num="0035"><img file="EP1764112B1_D0035.tif" /></chemistry><chemistry id="chem0036" num="0036"><img file="EP1764112B1_D0036.tif" /></chemistry>

Comparative Example 18

: Folate Ligand Attachment to Cyclodextrin Polymer

1. Coupling:

006936 mg of t-butyl carbazate dissolved in 240 µL of DCM/ethyl acetate (1:1) was added to 260 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers) and mixed at room temperature for 2 hours. The product was precipitated two times from ethyl acetate/ether (1:1).

2. Removal of protecting group:

0070FMOC protecting group was removed with 20% piperidine in DMF. The solvent was removed <i>in vacuo</i> and product redissolved in 1.3 mL of DMSO.

3. Folic Acid Coupling:

00711.2 equivalents of folic acid and DCC and one drop of pyridine was then added and the resulting solution stirred in the dark at room temperature for 6 hours. DMSO was removed <i>in vacuo</i> and conjugation of folic acid was confirmed by GPC with UV monitoring at 285 nm.

4. Removal of Hydrazide Protecting Group:

0072Finally, the hydrazide was deprotected by stirring in 4M HCl in dioxane for 1 hour before removing the solvent <i>in vacuo</i>. The final product was purified by Toyopearl HW-40F column chromatography.

5. Coupling to Polymer.

0073Folic acid-linker is reacted with 6 equivalents of a cyclodextrin copolymer (oxidized as in Example 10) by mixing in 50 mmol borate (pH 8.5). The reaction mixture is analyzed and conjugation polymer confirmed by a GPC system with a UV detection at 285 nm. <chemistry id="chem0037" num="0037"><img file="EP1764112B1_D0037.tif" /></chemistry>

Comparative Example 19

: Transferrin Ligand Attachment to Cyclodextrin Polymer

1. Transferrin Oxidation:

0074500 mg of iron-free human transferrin (Sigma of St. Louis, MO) is dissolved in 30 mM sodium acetate buffer and cooled to 0°C. To this solution is added 20 mg of sodium periodate dissolved in 4 µL of 30 mM sodium acetate. The mixture is stirred at 0°C overnight. Next 1 g of AG501-X8 resin (Biorad) is added to remove salts before the solution is lyophilized.

2. Resin coupling

007520 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers, Inc. of Huntsville, AL) was 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) swelled in DMF. The mixture was stirred at 60°C until all the polymer was coupled to the resin, as determined by a GPC system equipped with an ultraviolet (UV) detector. The resin-polymer was then transferred to a sintered glass column for all further reactions.

3. Resin Capping:

0076The unreacted hydrazide groups on the resins were capped with acetic anhydride and the acetic acid products were neutralized by diisopropylethylamine.

4. Removal of protecting group:

0077The FMOC protecting group was removed by two washes with 20% piperidine in DMF (1 mL total volume). The resin was then washed 10 times with 1 mL DMF and 5 times with 1 mL H₂O.

5. Transferrin coupling:

0078To the resin is added 1.2 equivalents of transferrin dissolved in 0.05 M sodium carbonate and 0.1 M sodium citrate buffer, pH 9.5. 5 M cyanoborahydride in 1N NaOH is then added to the solution. The glass column is placed on a rotator and mixed for 2 hours. The resin is then washed 15 times with water and 5 times each with tetrahydrofuran (THF) and DCM.

6. Cleavage from resin:

00791% trifluoroacetic acid (TFA) in 1 mL DCM is added to the resin twice for 1 minute each. The supernatant is then collected and DCM evaporated. The resulting oily film is rehydrated in H₂O and lyophilized.

7. Coupling to polymer:

0080Transferrin linker is reacted with 6 equivalents of a cyclodextrin copolymer by reductive amination with sodium cyanoborohydride: first, the copolymer is added to transferrin linker dissolved in 0.05 M sodium carbonate and 0.1 M sodium citrate buffer. 5 M cyanoborohydride in 1N NaOH is added and the reaction is stirred for 2 hours at room temperature. Unreacted aldehyde sites are blocked by adding ethanolamine and reacting for 15 minuted at room temperature. The resulting conjugate is purified by dialysis. <chemistry id="chem0038" num="0038"><img file="EP1764112B1_D0038.tif" /></chemistry><chemistry id="chem0039" num="0039"><img file="EP1764112B1_D0039.tif" /></chemistry>

Comparative Example 20

: General Procedure for Cyclodextrin Copolymer Complexation with Small Molecules

0081Cyclodextrin-based copolymer (CD-polymer) is dissolved in water, buffer, or organic solvent at the appropriate concentration. The small molecule is dissolved in a solvent miscible with the solvent of the CD-polymer solution and is added to the CD-polymer solution. The mixture is then stirred for ½ hour and then allowed to come to equilibrium overnight.

Comparative Example 21

: Cyclodextrin Copolymer Complexation with Doxorubicin

0082Doxorubicin and CD-polymer were dissolved at various concentrations in PBS (phosphate buffered saline, pH 7.2). The association constant between the CD and doxorubicin was determined by measuring the extent of doxorubicin's fluorescence increase upon complexation with the CD. (The hydrophobic interaction between the CD and doxorubicin enhances the fluorescence intensity). Association constant was approximately 200 M^-1 at pH 7.1. Addition of β-CD consistently enhanced doxorubicin fluorescence, indicating complexation between the CD-polymer and doxorubicin. <nplcit id="ncit0011" npl-type="s"><text>Husain et al., Applied Spectroscopy Vol. 46, No. 4, 652-658 (1992</text></nplcit>) found the association constant between β-CD and doxorubicin to be 210 M^-1 at pH 7.1.

Example 22

: Small Molecule Delivery to Cultured Cells

0083Media containing doxorubicin and doxorubicin/CD-polymer complexes at various concentrations were applied to cultured cell lines. After 5 hours, the media was removed and replaced with fresh media. Doxorubicin effect on cell survival was determined by the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium) toxicity assay. (<nplcit id="ncit0012" npl-type="b"><text>R. Ian Feshney, "Culture of Animal Cells", 3rd ed., Wiley-Liss:New York (1994</text></nplcit>)). The results are illustrated in the table below. Copolymer <b>15</b> or <b>16</b> (138 µM equivalent of CD monomer) was not toxic to KB or KB-VI (a multidrug resistant derivative of KB) cell lines in the absence of doxorubicin. For receptor-mediated delivery, a ligand such a folate is covalently attached to the CD-polymer used for doxorubicin complexation. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="81mm" /><colspec colnum="3" colname="col3" colwidth="42mm" /><thead><row><entry align="center" valign="top"><b>Cell Line</b></entry><entry align="center" valign="top"><b>CD-polymer</b></entry><entry align="center" valign="top"><b>IC₅₀ (µM of doxorubicin)</b></entry></row></thead><tbody><row><entry align="center">KB</entry><entry align="center">none</entry><entry align="center">~0.1</entry></row><row><entry align="center">KB-VI (multidrug resistant)</entry><entry align="center">none</entry><entry align="center">~10</entry></row><row><entry align="center">KB-VI</entry><entry align="center">copolymer <b>15 or 16</b> (138 µM equivalent of CD monomer)</entry><entry align="center">-2-3</entry></row></tbody></tgroup></table></tables>

Comparative Example 23

: Fixed Permanent Charged Copolymer Complexation with Plasmid

0084In general, equal volumes of fixed charged CD-polymer and DNA plasmid solutions in water are mixed at appropriate polymer/plasmid charge ratios. The mixture is then allowed to equilibrate and self-assemble at room temperature overnight. Complexation success is monitored by transferring a small aliquot of the mixture to 0.6% agarose gel and checking for DNA mobility. Free DNA travels under an applied voltage, whereas complexed DNA is retarded at the well.

00851µg of DNA at a concentration of 0.2 µg/µL in distilled water was mixed with 10 µL of copolymer 15 at polymer amine: DNA phosphate charge ratios of 2.4, 6, 12, 24, 36, 60, and 120. The solution was mixed manually by a micropipette and then gently mixed overnight on a lab rotator. 1 µg/µL of loading buffer (40% sucrose, 0.25% bromophenol blue, and 200 mM Tris-Acetate buffer containing 5mM EDTA (Gao et al., <i>Biochemistry</i><b>35</b>:1027-1036 (1996)) was added to each solution the following morning. Each DNA/polymer sample was loaded on a 0.6% agarose electrophoresis gel containing 6 µg of EtBr/100 mL in 1 x TAE buffer (40mM Tris-acetate/1 mM EDTA) and 40V was applied to the gel for 1 hour. The extent of DNA/polymer complexation was indicated by DNA retardation in the gel migration pattern. The polymer <b>(15)</b> retarded DNA at charge ratios of 6 and above, indicating complexation under these conditions.

Example 24

: Crosslinking Copolymer Complexation with Plasmid

0086Copolymer <b>15</b> or copolymer <b>16</b> is oxidized as in Example 10. Oxidized copolymer <b>15</b> or <b>16</b> is then complexed with a DNA plasmid as in Examples 23 and 26. A crosslinking agent (for example, PEG₆₀₀-Dihydrazide) is then added to encapsulate the DNA. Encapsulation success is determined by light scattering and visualized by electron microscopy.

Comparative Example 25

: Variably Charged (pH-sensitive) Copolymer Complexation with Plasmid

0087Equal volumes of a CD-polymer and DNA plasmid solutions in water are mixed in appropriate polymer/plasmid charge ratios. The pH of the mixture is adjusted to form a charged CD-polymer. The mixture is then allowed to equilibrate and self-assemble at room temperature for 30 minutes. A crosslinking agent (for example, PEG₆₀₀-Dihydrazide) is then added to encapsulate the DNA. A concentrated buffer solution is then added to render the pH and thus the CD-polymer neutral. Encapsulation success is determined by light scattering and visualized by electron microscopy.

Comparative Example 26:

Transfection Studies with Plasmids Encoding

Luciferase reporter gene:

0088BHK-21 cells were plated in 24 well plates at a cell density of 60,000. cells/well 24 hours before transfection. Plasmids encoding the luciferase gene were encapsulated by the CD-polymer as in Examples 23 or 25 such that the DNA/polymer complexes were assembled at polymer amine: DNA phosphate charge ratios of 6, 12, 24, 36, and 60 as described in DNA <i>binding</i> studies of Example 23. Media solution containing the DNA/polymer complexes was added to cultured cells and replaced with fresh media after 5 hours of incubation at 37°C.

0089The cells were lysed 48 hours after transfection. Appropriate substrates for the luciferase light assay were added to the cell lysate. Luciferase activity, measured in terms of light units produced, was quantified by a luminometer. DNA/polymer complexes successfully transfected BHK-21 cells at a charge ratios of 6, 12, and 24. Cell lysate was also used to determine cell viability by the Lowry protein assay. (<nplcit id="ncit0013" npl-type="s"><text>Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951</text></nplcit>)). Maximum toxicity was seen at a polymer amine: DNA phosphate charge ratios of 36 and 60 with 91 % cell survival.

Example 27

: Transfection Studies with Plasmids Encoding

Luciferase reporter gene:

0090BHK-21 cells were plated in 24 well plates at a cell density of 60,000 cells/well 24 hours before transfection. Plasmids encoding the luciferase gene were encapsulated by the CD-polymer as in Example 23 except copolymer <b>15</b> was replaced with copolymer <b>16</b> and that the DNA/polymer complexes successfully transfected BHK-21 cells at charge ratios of 10, 20, 30, and 40 with maximum transfection at polymer amine:DNA phosphate charge ratio of 20. Media solution containing the DNA/polymer complexes was added to cultured cells and replaced with fresh media after 24 hours of incubation at 37 °C. The cells were lysed 48 hours after transfection. Appropriate substrates for the luciferase light assay were added to the cell lysate. Luciferase activity, measured in terms of light units produced, was quantified by a luminometer. The results are illustrated below. DNA/polymer complexes successfully transfected BHK-21 cells at a charge ratios of 6, 12, and 24. Cell lysate was also used to determine cell viability by the Lowry protein assay. (<nplcit id="ncit0014" npl-type="s"><text>Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951</text></nplcit>)). The results are illustrated below. Maximum toxicity was seen at a polymer amine: DNA phosphate charge ratios of 40 and 50 with 33% cell survival. <img file="EP1764112B1_D0040.tif" /><img file="EP1764112B1_D0041.tif" />

Example 28

: Transfection Studies with Plasmids Encoring

GFP reporter gene

0091Plasmids encoding the green fluorescent protein are encapsulated by the CD-polymer as in Examples 23 or 25. Media solution containing the DNA/polymer complexes is added to cultured cells and replaced with fresh media after 5 hours of incubation at 37°C. The cells are detached from the surface with trypsin, washed, and resuspended in Hanks Balanced Salt Solution with propidium iodide. The cells are then analyzed by fluorescence activated cell sorting (FACS). Cell viability is determined by cell size and propidium iodide exclusion, and transfection success by GFP protein fluorescence.

Comparative Example 29

: Polymer Complexation with Oligos

0092Complexation with antisense oligos is accomplished following the procedures for plasmid complexation of Examples 23 or 25.

Comparative Example 30

: Transfection studies with Oligos

0093Antisense oligos directed against the luciferase gene are encapsulated by the CD-polymer as described in Example 29. Media solution containing the oligo/polymer complexes is added to HeLa X1/5 cells (HeLa cells that constitutively express the luciferase gene, donated by CLONTECH) and replaced with fresh media after 5 hours of incubation at 37°C. Cells are lysed 48 hours after transfection and appropriate substrates for the luciferase assay are added to the lysates. Luciferase activity, measured in terms of light units produced, is quantified by a luminometer. Transfection success is determined by knockout of luciferase activity.

Comparative Example 31

: Toxicity of ß-cyclodextrin(cystamine)-DTBP copolymer,

15

0094The acute toxicity of copolymer <b>15</b> was investigated using Swiss-Webster "white mice." A total of 48 mice were used as described in the table below. Single intravenous (i.v.) or intraperitoneal (i.p.) injections of sterile saline solutions or of copolymer 15 were given to the mice. The animals were followed for five days after which they were sacrificed and gross necropsy was performed. No mortality and no toxicity was observed. <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="22mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="28mm" /><colspec colnum="5" colname="col5" colwidth="27mm" /><colspec colnum="6" colname="col6" colwidth="20mm" /><colspec colnum="7" colname="col7" colwidth="26mm" /><thead><row><entry align="center" valign="top">Group No.</entry><entry align="center" valign="top">#/Sex (M/F)</entry><entry align="center" valign="top">CoPolymer</entry><entry align="center" valign="top">Concentration (mg/mL)</entry><entry align="center" valign="top">Dose Volume (mL)</entry><entry align="center" valign="top">Dose (mg)</entry><entry align="center" valign="top">Treatment Regimen</entry></row></thead><tbody><row><entry align="center">1</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">0.5275</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.05</entry><entry align="center">i.v., once</entry></row><row><entry align="center">2</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">5.275</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.53</entry><entry align="center">i.v., once</entry></row><row><entry align="center">3</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">52.75</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">5.28</entry><entry align="center">i.v., once</entry></row><row><entry align="center">4</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">0.5275</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.05</entry><entry align="center">i.p., once</entry></row><row><entry align="center">5</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">5.275</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.53</entry><entry align="center">i.p., once</entry></row><row><entry align="center">6</entry><entry align="center">3/3</entry><entry align="center">CoPolymer 15</entry><entry align="center">52.75</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">5.28</entry><entry align="center">i.p., once</entry></row><row><entry align="center">7</entry><entry align="center">3/3</entry><entry align="center">0.9% saline</entry><entry align="center">0.000</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.00</entry><entry align="center">i.v., once</entry></row><row><entry align="center">8</entry><entry align="center">3/3</entry><entry align="center">0.9% saline</entry><entry align="center">0.000</entry><entry align="char" char=".">0.1</entry><entry align="char" char="." charoff="10">0.00</entry><entry align="center">i.p., once</entry></row></tbody></tgroup></table></tables>

Example 32

: Transfection Studies with Plasmids Encoding

Luciferase reporter gene

0095Plasmids encoding the luciferase gene were encapsulated by the CD-polymer as in Example 23 except copolymer <b>15</b> was replaced with copolymer <b>16.</b> The DNA/polymer complexes were used to successfully transfect BHK-21 or CHO-K1 cells, each plated in 24 well plates at a cell density of 60,000 cells/well 24 hours before transfection, at various charge ratios in 10% serum and serum-free conditions following the procedure outlined in Example 27. The cells were lysed 48 hours after transfection. Appropriate substrates for the luciferase light assay were added to the cell lysate. Luciferase activity, measured in terms of light units produced (<i>i.e</i>., relative light units (RLU)), was quantified by a luminometer. Cell lysate was also used to determine cell viability by the Lowry protein assay. (<nplcit id="ncit0015" npl-type="s"><text>Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951</text></nplcit>)). Toxicity was measured by determining total cellular protein in the wells 48 hours after transfection. The transfection and cell survival results in 10% serum and serum free media are illustrated below.

0096Luciferase protein activity in BHK-21 cells transfected in serum-free conditions reached a stable maximum at 30+/- with ~5 x 10⁷ RLUs. The presence of 10% serum in the transfection media decreased luciferase activity at all charge ratios except 70+/-. With CHO-K1 cells, increasing charge ratio also enhanced the transfection for all conditions tested. Additionally, transfection in serum decreased light units by an order of magnitude.

0097Copolymer <b>16</b> showed toxicity only to BHK-21 cells for transfections in the absence of serum. Toxicity was minimized with the presence of 10% serum during transfection. No noticeable toxicity was observed from transfections to CHO-K1 cells. <img file="EP1764112B1_D0042.tif" />

0098The effect of copolymer 16/DNA charge ratio and serum conditions on transfection efficiency (● and ■) and cell survival (▼ and ▲) in BHK-21 cells. Results from transfection in 10% serum and serum-free media are shown as, respectively, dotted and solid lines. Data are reported as the mean +/- S.D. of three samples. Toxicity data are presented as best fit lines. <img file="EP1764112B1_D0043.tif" />

0099The effect of copolymer 16/DNA charge ratio and serum conditions on transfection efficiency (● and ■) and cell survival (▼ and ▲) in CHO-K1 cells. Results from transfection in 10% serum and serum-free media are shown as, respectively, dotted and solid lines. Data are reported as the mean +/- S.D. of three samples. Toxicity data are presented as best fit lines.

Comparative Example 1

: Transfection Studies with Plasmids Encoding

Luciferase reporter gene

0100Following the procedure of Example 32, transfection efficiency and toxicity of various non-viral vectors with BHK-21 and CHO-K1 cells were studied and compared against those achieved with DNA/copolymer <b>16</b> complexes. The BHK-21 and CHO-K1 cells were transfected at a range of charge ratios and starting cell densities for all vectors in semm-free media. The results are illustrated below and illustrate the optimum transfection conditions found for each vector. <img file="EP1764112B1_D0044.tif" /><img file="EP1764112B1_D0045.tif" />