RU2288921C2 - Inclusion complexes-containing compositions - Google Patents

Abstract

FIELD: pharmaceutical dosage forms.

SUBSTANCE: invention relates to compositions and methods for delivery of drugs. Composition according to invention represents powdered composite containing polymer, therapeutical agent, and complexing agent, said polymer containing one or more cyclodextrin fragments. Polymer interacts with complexing agent in a host/guest or guest/host manner resulting in formation of inclusion complex. Both polymer of composite and complexing agent may be used in a way to introduce functionality into therapeutical composition. Invention also discloses a method for preparing composition and to a method for delivering therapeutical agent.

EFFECT: widened choice of drug delivery methods.

26 cl, 31 dwg, 1 tbl, 65 ex

Description

FIELD OF THE INVENTION

The invention relates to compositions and methods used for the delivery of therapeutic agents. More specifically, the present invention relates to a composition comprising a polymer, a therapeutic agent and a complexing agent, in which a host-guest or guest-host interaction of the polymer with a complexing agent is observed to form an inclusion complex. The composition of the invention can be used to deliver a therapeutic agent in the treatment of various disorders.

BACKGROUND OF THE INVENTION

Cyclodextrins are cyclic polysaccharides containing natural D (+) - glucopyranose residues connected by an α- (1,4) -bridge. Alpha (α) -cyclodextrins, beta (β) -cyclodextrins and gamma (γ) -cyclodextrins, which contain, respectively, six, seven or eight glucopyranose residues, are most widely distributed. The structurally cyclic nature of cyclodextrin is expressed in the form of a torus or toroid with an internal non-polar or hydrophobic cavity, secondary hydroxyl groups located on one side of the cyclodextrin torus, and primary hydroxyl groups located on the other side of the torus. So, in the example below, (β) -cyclodextrin shows a schematic representation of cyclodextrin:

The side on which the secondary hydroxyl groups are located has a larger diameter than the side on which the primary hydroxyl groups are located. The hydrophobic nature of the internal cavity of cyclodextrin makes it possible to include many compounds (Comprehensive Supramolecular Chemistry, Volume 3, JL Atwood et al., Eds., Pergamon Press (1996); T. Cserhati, Analytical Biochemistry, 225: 328-332 (1995); Husain et al., Applied Spectroscopy, 46: 652-658 (1992); French Patent 2665169).

Cyclodextrins were used as carriers of various therapeutic compounds due to their ability to form inclusion complexes with various medicinal substances that can adhere to the hydrophobic cavity of cyclodextrin, or due to the formation of non-covalently associated complexes with other biologically active molecules, such as oligonucleotides and their derivatives. For example, US Pat. No. 4,727,064 discloses pharmaceutical preparations consisting of a drug substance with a very low solubility in water and an amorphous water-soluble cyclodextrin-based mixture. The drug substance forms an inclusion complex with cyclodextrin from this mixture. US Pat. No. 5,691,316 describes a cellular cyclodextrin-based oligonucleotide delivery system. In such a system, the oligonucleotide binds to the cyclodextrin non-covalently to form a complex, or the oligonucleotide can covalently bind to adamantane, which in turn is non-covalently associated with cyclodextrin.

Various cyclodextrin-containing polymers and methods for their preparation are also known in the art (Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., Eds., Pergamon Press (1996)). A method for producing polymers containing immobilized cyclodextrin is described in US Pat. No. 5,608,015. According to this method, a cyclodextrin derivative is reacted with either a monomeric α, β-unsaturated acid halide or a derivative thereof, or an α, β-unsaturated acid or a terminal derivative thereof isocyanate group or its derivative. A cyclodextrin derivative is prepared by reacting a cyclodextrin with compounds such as acid halides or anhydrides. The resulting polymer contains cyclodextrin fragments (residues) in the form of side chains, outside the main linear polymer chain.

US Pat. No. 5,276,088 describes a method for synthesizing cyclodextrin polymers, either by reacting polyvinyl alcohol or cellulose or derivatives thereof with cyclodextrin derivatives, or by copolymerizing a cyclodextrin derivative with vinyl acetate or methyl methacrylate. The resulting cyclodextrin-containing polymer also contains a cyclodextrin fragment as an additional fragment located outside the main polymer chain.

An ensemble (assembly) of a biodegradable polymer for medical use with a supramolecular structure is described in International Application WO 96 / 09073A1 and in US Pat. No. 5,855,900. The ensemble contains a number of cyclic compounds carrying a drug substance obtained by binding the drug substance to α, β or γ-cyclodextrin followed by folding strands of drug / cyclodextrin compounds along a linear polymer with biodegradable fragments bonded to both ends of the polymer. It is reported that such an ensemble is capable of releasing a drug substance in response to specific biodegradation during the course of the disease. Such ensembles (self-assemblies) are usually called “molecular necklaces” based on cyclodextrin polymers.

However, there is a need in the art for more effective non-viral delivery systems exhibiting, for example, properties such as increased stability (for example, under physiological conditions) and effective targeting (targeting) capabilities. This invention solves these problems ("meets such needs").

SUMMARY OF THE INVENTION

This invention primarily includes a compound of the formula

Where

J is —NH—, —C (= O) NH— (CH ₂ ) _d -, —NH — C (= O) - (CH ₂ ) _d -, —CH ₂ SS—, —C (= O) O - (CH ₂ ) _e -O-P (= O) (O- (CH ₂ ) _e -Y) O-,

peptide or polypeptide residue, or

-NH- (C = O) -CH (R ¹ ) -NH- (C = O) -CH (R ¹ ) -NH-;

Y denotes additional host / guest functionality;

R ¹ is - (CH ₂ ) _a —CO ₂ H, its ester or salt; or - (CH ₂ ) _a —CONH ₂ ;

PEG (PEG) means —O (CH ₂ CH ₂ O) _z -, where z varies from 2 to 500;

L is H, -NH ₂ , -NH- (C = O) - (CH ₂ ) _e - (C = O) -CH ₂ -, -S (= O) ₂ -HC = CH ₂ -, - SS- , —C (═O) O— or a carbohydrate residue;

the host is selected from adamantyl, naphthyl, cholesterol, cyclodextrin, or any combination thereof;

a is 0 or 1;

b is 0 or 1;

d has a value in the range from 0 to 6;

e matters in the range from 1 to 6;

n has a value in the range from 0 to 6;

q has a value in the range of 1 to 5;

w has a value in the range from 1 to 5;

y is 1;

x is 1; and

z has a value in the range of 1 to 5.

The invention also includes a composition containing a powdered composite of a polymer and a therapeutic agent, and a complex for including said polymer and a complexing agent, the complexing agent containing a functional group and the polymer containing one or more cyclodextrin fragments.

The present invention also relates to methods for preparing a therapeutic composition. According to the invention, this method combines a therapeutic agent, a polymer with host or guest functionality and a complexing agent with guest or host functionality to form a therapeutic composition. The complexing agent forms an inclusion complex with a polymer.

The invention also relates to a method for delivering a therapeutic agent. According to this method, a therapeutically effective amount of a composition of the invention is administered to a mammal (eg, a human or animal) recognized as in need of a therapeutic agent. Thus, the invention encompasses the treatment of a disease using the composition of the invention to deliver an appropriate therapeutic agent.

Short Description of Shapes

In the Figures depicting various embodiments of the invention, compound 12 is also designated as βCDP6. Composites containing nucleic acid and a cationic polymer in a particular composite are defined as polyplexes. Below is a brief description of the figures.

Figure 1. The structure of various molecules of adamantane - PEG

Figure 2 The hydrodynamic diameter of GALA and GALA-Ad modified compositions. Example 30

Figure 3. Zeta potential of compositions modified using GALA and GALA-Ad, Example 32.

Figure 4. Absorption by cells of BHK-21 compositions modified using GALA and GALA-Ad, Example 31.

Figure 5. Absorption by HUH-7 cells of polyplex-polyplex compositions modified using GALA and GALA-Ad, Example 33.

Figure 6. Transfection of VEK-21 cells with luciferase using β-cyclodextrin-DMS copolymer 12 based compositions modified with GALA and GALA-Ad, Example 34.

Figure 7. The toxicity of polyplexes modified with the participation of GALA and GALA-Ad, in relation to BHK-21 cells, Example 35.

Figure 8. Scheme of pegylation using grafted copolymerization after complexing with DNA, Example 39.

Figure 9. Particle sizes of specific PEI and 12 composites and polyplex-polyplex compositions during post-DNA complexation, Example 39.

Figure 10. Stabilization of polyplex compositions by pegylation, Example 40.

Figure 11. Joint delivery of 12 using PEG _3400- FITC, Example 42.

Figure 12. The structure of the compound Lactose-12, Example 37.

Figure 13. Transfection of 12 and LAC-CDP6 polyplexes into HUH-7 cells, Example 43.

Figure 14. Schematic representation of the experimental protocol, Example 43.

Figure 15. The diameters of the particles, Example 47.

Figure 16. Loss of DNA due to precipitation of the complex. Example 47

Figure 17. Inclusion complexes for the modification of composite 12 / DNA, Example 48.

Figure 18 Transfection of modified polyplexes into HepG2 cells, Example 49.

Figure 19. Competitive substitution experiments. Example 52

Figure 20. Synthesis of Adamantan-PEG Transferrin (Ad-PEG-Tf), Example 55.

Figure 21. The load of iron on transferrin, Example 55.

Figure 22. The binding affinity of transferrin-PEG-Ad, Example 55.

Figure 23. Linking transferrin through lysine groups, Example 56.

Figure 24. The binding affinity of transferrin-PEG-Ad with transferrin receptors on RSZ cells, Example 57.

Figure 25. Change in zeta potential and particle size as a function of particle modification in polyplexes modified with transferrin and PEG, Example 58.

Figure 26. Measurement of the zeta potential, Ad-anionic-PEG, Example 62.

Figure 27. Measurement of stability, Example 62.

Figure 28. Attachment of an increasing complexing agent for transferrin, Example 62.

Figure 29. Synthesis of histidylated 12.

Figure 30. pH-sensitive polymers for endosomal isolation (Synthesis of polymers containing secondary amines).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions in which inclusion complexes are used to deliver therapeutic agents. Inclusion complexes are molecular compounds having a characteristic adduct structure in which a molecule of one of the compounds (the host molecule) spatially limits at least a portion of the molecule of the other compound. The molecule of the compound enclosed in this volume (the guest molecule) is located in the cavity of the host molecule in such a way that it does not affect the core structure of the host. A characteristic feature of the inclusion complex is that the size and configuration of the corresponding cavity most often remain unchanged, apart from slight deformation, the “host” can be any host compound or any host molecule known in the art. Examples of suitable “hosts” include, but are not limited to, cyclodextrins, carcerands, cavitands, crown ethers, cryptands, cucurbiturils, calixarenes, spherands, and the like. Examples of “guests” useful as complexing agents include compounds such as adamantane, diadamantane, naphthalene and cholesterol (cholesterol).

Cyclodextrins are preferred hosts capable of interacting with many ionic and molecular compounds and resulting in inclusion compounds belonging to the class of guest-host complexes. In order for the host-guest interaction to take place, the compounds must meet several requirements: one of them is that the binding sites of the host and guest molecules must be complementary in terms of stereo-electronic effects. Cyclodextrins are capable of forming inclusion complexes with compounds whose molecular sizes are comparable to those of the cavity. However, the degree of complex formation also depends on the polarity of the guest molecule. The formation of a complex with molecules that are significantly larger in volume than the cavity is also possible provided that only some groups or side chains penetrate the carbohydrate channel. See J. Szejtly, Akademiai Kiado, Cyclodextrins and their inclusion complexes, Budapest, 1982.

The composition according to the invention contains at least one polymer and at least one therapeutic agent, mainly in the form of a powdery composite consisting of a polymer and a therapeutic agent. The therapeutic composition also contains one or more complexing agents. At least one polymer of the powdered composite interacts with the complexing agent according to the "host-guest" or "guest-host" scheme with the formation of an inclusion complex consisting of a polymer and a complexing agent. The polymer and in particular the complexing agent can be used to introduce functional groups into the composition of the invention. In one embodiment of the invention, at least one polymer of the powdered composite carries host functionality and forms an inclusion complex with a complexing agent with guest functionality. In another embodiment of the present invention, at least one polymer of the powdered composite has guest functionality and forms an inclusion complex with a complexing agent with host functionality. In yet another embodiment of the invention, the polymer powder composite may contain both host and guest functionality and may form inclusion complexes with complexing agents both as a “guest” and as a “host”.

1. Powdered composite

A powdered composite of a therapeutic agent and a polymer is a combination or integration of a therapeutic agent and a polymer. A powder composite is an associated structure containing one or more therapeutic agents within a multidimensional polymer network. You can use a single polymer or a mixture of polymers. In addition to the ability to form a polymer multidimensional network of a powdered composite, as discussed below, guest and / or host functionality carriers can form inclusion complexes with one or more complexing agents.

A. Polymer

Any type of polymer capable of forming a powdery composite with a therapeutic agent and having host and / or guest functionality may be used in the composition of the invention. The polymer may be linear or branched. The polymer may be a homopolymer or a copolymer. If a copolymer is used, this copolymer may be a random copolymer or a branched copolymer. Preferably, the polymer is dispersible in water, and more preferably, the polymer is water soluble. For example, suitable polymers include, but are not limited to, polysaccharides, polyesters, polyamides, polyethers, polycarbonates, polyacrylates, etc. For therapeutic pharmaceutical purposes, the polymer should be low toxic and, preferably, it should be non-toxic or cytotoxic. As discussed below, a preferred polymer for use in the composition of the invention is a cyclodextrin-based polymer. The water-soluble linear cyclodextrin copolymers described below having molecular weights in the range of 3000-100000 are preferred, and such copolymers with a molecular weight of 3000-50000 are particularly preferred.

In accordance with the invention, the polymer in the powder composite may be a single polymer or a mixture of two or more polymers, which may be the same or different polymers. Each polymer of the powdered composite may additionally contain or may be modified so as to contain a crosslinking group with which it is possible to associate the polymers with the formation of the powdered composite.

At least one polymer of the powdered composite is a polymer capable of forming an inclusion complex. A “polymer capable of forming an inclusion complex” can be any polymer capable of forming one or more guest-host associates through non-binding interactions (eg, due to van der Waals forces, hydrogen bonds, dipole-dipole interactions , interactions of the type of ion pairs, “soluophobic” interactions) with another compound (complexing agent) or a substituent in this compound. In other words, at least one polymer has guest or host functionality in the formation of an inclusion complex with a complexing agent or with a substituent in the complexing agent. The functionality of the host or guest may be part of the backbone of the polymer or may be present as a substituent in either the side or branched chains. An example of a polymer with host functionality in the backbone is the linear cyclodextrin polymer described below. An example of a polymer in which guest functionality is not part of the polymer backbone is a polymer with adamantane groups in the side chain. Other examples of suitable “hosts” that can be used with the polymer include, but are not limited to, carcerands, cavitands, crown ethers, cryptands, cucurbiturils, calixarenes, spherands, and the like. Examples of “guests” for inclusion complexes suitable as complexing agents for such hosts include, but are not limited to, compounds known in the art, such as adamantane, diadamantane, naphthalene and cholesterol (cholesterol).

In a preferred embodiment of the invention, the polymer may contain various types of functionalities of the host or guest, or the polymer may contain the functionality of both the guest and the host. This makes it possible to obtain more diverse inclusion complexes based on this polymer. The presence of multiple host functionalities, multiple guest functionalities, or both host and guest functionalities of the same polymer increases the variety of functionality that can be introduced into the therapeutic composition of the invention using an inclusion complex.

Due to the host-guest association, the polymer interacts with the complexing agent to form an inclusion complex. Preferably, the binding constants resulting from the non-binding interaction or association of the inclusion complex are about> 10 ² , preferably about> 10 ³ , and more preferably about> 10 ⁴ . As a rule, the binding constants are in the range of about 10 ² -10 ⁶ .

The polymer powder composite can be modified with one or more ligands. The ligand can be introduced during or after the formation of the powder composite by modifying the ligand of the therapeutic agent and / or the polymer of the powder composite. A ligand can be any ligand that makes it possible to target and / or bind to a given cell. One of skill in the art will recognize that targeting and binding to a cell may include attachment to a cell receptor, which in turn can lead to receptor-mediated endocytosis. If two or more ligands are attached, the ligands may be the same or different. Examples of suitable ligands include, but are not limited to, vitamins (e.g., folic acid), proteins (e.g., transferrin and monoclonal antibodies), monosaccharides (e.g., galactose), peptides and polysaccharides. The choice of ligand, as understood by an ordinary specialist in this field, may vary depending on the specified type of delivery. As another example, the ligand may be a membrane-permeabilizing or membrane-permeable agent, such as HIV-1 TAT protein. The TAT protein is a viral transcription activation protein that is actively imported into the cell nucleus. Torchilin, V.P. et al, PNAS. 98, 8786-8791 (2001).

In a preferred embodiment of the invention, at least one of the polymers of the powdered composite is a substantially linear polymer that has host and / or guest functionality capable of forming an inclusion complex. Almost a linear polymer can be obtained by any method known in the art. The polymer can be obtained from the corresponding monomer capable of forming an inclusion complex, or from a mixture of monomers, of which at least one has the functionality of a host or guest. The functionality of the host or guest may be located within the polymer chain, in the side (or branched) chain with respect to the polymer chain, or may be present as an end group. Or, after the polymer is obtained, it can be further modified by adding (adding) the guest or host functionality, as discussed above, with the formation of an almost linear polymer capable of forming an inclusion complex. A substantially linear polymer may be a block copolymer in which the blocks introduce properties such as host functionality, dispersibility in water and / or solubility in water. Examples of such blocks include, for example, linear polyethyleneimine (PEI), linear cyclodextrin-containing polymer, bis (2-aminoethyl) -1,3-propanediamine (AEPD) and N ₂ , N ₂ , N ₃ , N ₃ - (3'-PEG ₅₀₀₀ aminopropane) bis (2-aminoethyl) -1,3-propanediammonium difluoroacetate (AEPD-PEG).

In another preferred embodiment of the invention, the polymer used to prepare the powder composite is a cyclodextrin-containing polymer, more preferably a substantially linear cyclodextrin polymer, as described below. The polymer may also be polyethyleneimine (PEI) or a polymer containing cyclodextrin side chains. A linear cyclodextrin polymer is a polymer containing cyclodextrin fragments as an integral part of the polymer chain. Polymers containing lateral cyclodextrin fragments rather than as part of the main polymer chain, but as fragments attached outside the polymer chain, can also be used in the compositions of the invention. The linear cyclodextrin-containing polymer may be any linear polymer containing at least one cyclodextrin fragment as part of the main polymer chain. The cyclodextrin-containing polymer is preferably water soluble. More preferably, the linear cyclodextrin-containing polymer is a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer, each of which is described below. Cyclodextrin groups within the polymer give the polymer the functionality of the host, allowing it to form inclusion complexes. A substantially linear polymer capable of forming an inclusion complex may further comprise, or may be further modified to contain an additional functional group (e.g., a thiol group).

Linear cyclodextrin-containing polymers

A linear cyclodextrin copolymer that can be used to form a powdery composite contains substituted or unsubstituted cyclodextrin fragments bifunctionally linked to the main linear chain of the copolymer by linking at 2, 3, or 6 position of at least one cyclodextrin glucopyranose cycle with divalent binding fragments cyclodextrin molecules of a linear cyclodextrin polymer. As described in International Patent Application WO 00/01734, such a linear cyclodextrin polymer contains a repeating element of formula Ia, Ib (see below), or a combination thereof. Linear cyclodextrin copolymers, their preparation and properties are also described in Gonzalez, H., Hwang, S. and Davis, M (1999) New class of polymers for the delivery of macromolecular therapeutics. Bioconjugate Chem., 10, 1068-1074 and Hwang, S., Bellock, N. and Davis, M (2001) Effects of Structure of Beta-Cyclodextrin-Containing Polymers on Gene Delivery. Bioconjugate Chem., 12 (2), 280-290.

In formulas Ia and Ib, C denotes a substituted or unsubstituted cyclodextrin monomer, and A denotes a comonomer bound, i.e. covalently linked to cyclodextrin C. Polymerization of the precursor of cyclodextrin monomer C with the precursor of comonomer A gives a linear cyclodextrin copolymer. In a separate linear cyclodextrin copolymer, the monomeric element of cyclodextrin C may be the same or different and, likewise, comonomer A may be the same or different.

The cyclodextrin monomer precursor may be any cyclodextrin known in the art or any derivative known in the art. As discussed above, cyclodextrin is defined as a cyclic polysaccharide, usually containing from six to eight residues (elements) of the natural D (+) glucopyranose, connected by an α- (1,4) bond. Preferably, the cyclodextrin monomer precursor is cyclodextrin containing six, seven and eight glucose moieties, i.e., respectively, alpha (α) cyclodextrin, beta (β) cyclodextrin and gamma (γ) cyclodextrin. The cyclodextrin derivative may be any substituted cyclodextrin known in the art in which the substituent does not interfere with copolymerization with the precursor of comonomer A, as described below. The 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, glycosyl and maltosyl; acid groups, for example, carboxylic acids, phosphoric acids, thiophosphonic acids and sulfonic acids; imidazole groups; sulfate groups; and protected thiol groups.

The cyclodextrin monomer precursor can be further chemically modified (e.g., halogenated, aminated) to facilitate the copolymerization of the cyclodextrin monomer precursor with the comonomer A precursor or to influence this copolymerization as described below. Chemical modification of the cyclodextrin monomer precursor makes it possible to polymerize only in two positions of each cyclodextrin fragment, i.e. as a result of the modification, a bifunctional cyclodextrin fragment is formed. The following is the numbering for the C1-C6 positions of each glucopyranose ring:

In a preferred embodiment, the polymerization proceeds at any two positions C2, C3 and C6, including combinations thereof, of a cyclodextrin fragment. For example, one cyclodextrin monomer precursor can polymerize at two C6 positions, while another cyclodextrin monomer precursor can polymerize at positions C2 and C6 of a cyclodextrin fragment. Below, as an example of β-cyclodextrin, the letter designations of the relative position of each cycle of glucopyranose in cyclodextrin are shown schematically

In a preferred embodiment, the cyclodextrin C monomer of the linear cyclodextrin copolymer of the invention has the following general formula (II):

In formula (II), n and m are integers which, together with two other glucopyranose cycles, determine the total number of glucopyranose elements in the cyclodextrin monomer. Formula (II) shows a cyclodextrin monomer that is capable of polymerizing at two positions C6 of a cyclodextrin element. Examples of cyclodextrin monomers of formula (II) include, but are not limited to, 6 ^A , 6 ^B dideoxy-α-cyclodextrin (n = 0, m = 4), 6 ^A , 6 ^C- dideoxy-α-cyclodextrin (n = 1, m = 3), 6 ^A , 6 ^D- dideoxy-α-cyclodextrin (n = 2, m = 2), 6 ^A , 6 ^B- dideoxy-β-cyclodextrin (n = 0, m = 5), 6 ^A , 6 ^C dideoxy-β-cyclodextrin (n = 1, m = 4), 6 ^A , 6 ^D dideoxy-β-cyclodextrin (n = 2, m = 3), 6 ^A , 6 ^B- dideoxy-γ-cyclodextrin (n = 0, m = 6), 6,6-dideoxy-γ-cyclodextrin (n = 1, m = 5), 6 ^A , 6 ^D- dideoxy-γ-cyclodextrin (n = 2, m = 4) and 6 ^A , 6 ^E- dideoxy-γ-cyclodextrin (n = 3, m = 3).

In another preferred embodiment, the linear cyclodextrin copolymer of the invention may comprise an open glucose monomer cyclodextrin element C, wherein the glucopyranose cyclodextrin cycles are opened while maintaining the cyclodextrin cyclic system. The general formula (III) shows a cyclodextrin with an open glucopyranose ring opening at positions C2 and C3.

In formula (III), p varies from 5 to 7. In formula (III), at least in one of the elements D (+) - glucopyranose of the cyclodextrin monomer, a cycle is opened, which allows polymerization at the positions of C2 and C3 of the cyclodextrin element. Cyclodextrin monomers of formula (III), such as, for example, 2 ^A , 3 ^A- diamino-2 ^A , 3 ^A- dideoxy-β-cyclodextrin and 2 ^A , 3 ^A- dialdehyde-2 ^A , 3 ^A- dideoxy-β- cyclodextrin is manufactured by Carbomer in Westborough, MA. Examples of cyclodextrin monomers of formula (III) include, but are not limited to, 2 ^A , 3 ^A- dideoxy-2 ^A , 3 ^A- dihydro-α-cyclodextrin, 2 ^A , 3 ^A- dideoxy-2 ^A , 3 ^A- dihydro-β β-cyclodextrin, 2 ^A , 3 ^A -dideoxy-2 ^A , 3 ^A- dihydro-γ-cyclodextrin, commonly called, respectively, 2,3-dideoxy-α-cyclodextrin, 2,3-dideoxy-β-cyclodextrin and 2, 3-dideoxy-β-cyclodextrin.

The comonomer A precursor may be a linear or branched, symmetric or asymmetric compound, which, when reacted with the cyclodextrin monomer precursor described above, binds two monomers together. Preferably, the comonomer A precursor is a compound containing at least two crosslinking groups, through which a reaction can be carried out and, therefore, cyclodextrin monomers can be combined. Examples of possible linking groups, which may be the same or different, terminal or internal, of each comonomer A precursor include, but are not limited to, an amino group, an acid group, an ester group, an imidazole group and an acid chloride group and derivatives thereof. In a preferred embodiment, the two crosslinking groups are the same and are terminal. When copolymerizing the precursor of comonomer A with the precursor of a cyclodextrin monomer, two cyclodextrin molecules can bind by connecting the side with the primary hydroxyl groups of one cyclodextrin monomer to the side with the primary hydroxyl groups of another cyclodextrin monomer, by connecting the side with the secondary hydroxyl groups of one cyclodextrin monomer with the side hydroxyl groups of another cyclodextrin monomer or by bonding the side with the first chnymi hydroxyl groups of one cyclodextrin monomer with the side with the secondary hydroxyl groups of another cyclodextrin monomer. Thus, in the resulting copolymer can be all combinations resulting from such bonds.

Both the precursor of comonomer A and comonomer A in the final copolymer can be neutral, cationic (for example, due to the presence of protonated groups, such as, for example, quaternary ammonium groups) or anionic (for example, due to the presence of deprotonated groups, such as, for example as a sulfate, phosphate or carboxyl anionic group). The counterion of the charged precursor comonomer A or comonomer A can be any suitable counteranion or countercation (for example, the counterion of the cationic precursor of comonomer A can be a halogen anion (for example, a chlorine anion). The charge of the comonomer A of the copolymer can be adjusted by adjusting the pH.

Examples of suitable comonomer A precursors include, but are not limited to, cystamine, 1,6-diaminohexane, diimidazole, dithioimidazole, spermine, dithiospermine, digistidine, dithiohistidine, succinimide (e.g., dithiobis (succinimidyl propionate) DSP) and disuccinimide (D) succinimide for example, dimethyl-3,3'-dithiobispropionimidate (DTBP)).

Copolymerization of the comonomer A precursor with a cyclodextrin monomer leads to the formation of a linear cyclodextrin polymer containing comonomer A bridges (bonds) having the following general formulas:

-HNC (O) (CH ₂ ) _x C (O) NH-, -HNC (O) (CH ₂ ) _x SS (CH ₂ ) _x C (O) NH-, - ⁺ H ₂ N (CH ₂ ) _x SS (CH ₂ ) _x NH ₂ ⁺ -, -HNC (O) (CH ₂ CH ₂ O) _x CH ₂ CH ₂ C (O) NH-, = NNHC (O) (CH ₂ CH ₂ O) _x CH ₂ CH ₂ C (O) NH =, - ⁺ H ₂ NCH ₂ (CH ₂ CH ₂ O) _x CH ₂ CH ₂ CH ₂ , NH ₂ ⁺ -, -HNC (O) (CH ₂ CH ₂ O) _x CH ₂ CH ₂ SS (CH ₂ CH ₂ O) _x CH ₂ CH ₂ C (O) NH-, -HNC (NH ₂ ⁺ ) (CH ₂ CH ₂ O) _x CH ₂ CH ₂ C (NH ₂ ⁺ ) NH-, -SCH ₂ CH ₂ NHC (NH ₂ ⁺ ) (CH ₂ ) _x C (NH ₂ ⁺ ) NHCH ₂ CH ₂ S-, -SCH ₂ CH ₂ NHC (NH ₂ ⁺ ) (CH ₂ ) _x SS (CH ₂ ) _x C (NH ₂ ⁺ ) NHCH ₂ CH ₂ S-, -SCH ₂ CH ₂ NHC (NH ₂ ⁺ ) CH ₂ CH ₂ (OCH ₂ CH ₂ ) _x C (NH ₂ ⁺ ) NHCH ₂ CH ₂ S-,

and

In the above formulas, x = 1-50, a y + z = x. Preferably, x = 1-30. More preferably, x = 1-20. In a preferred embodiment, comonomer A contains a biodegradable bridge (bond). Comonomer A may also include a functional group unstable in an acidic environment, such as ester groups and other acid-unstable groups known to those skilled in the art.

In another preferred embodiment of the invention, the precursor of comonomer A and, therefore, comonomer A can be selectively selected to suit the intended application. For example, in order to deliver a low molecular weight therapeutic agent, a charged polymer may not be required, and comonomer A may or may contain a hydrophilic group, such as polyethylene glycol, which further increases water solubility. In the case of polypeptide therapeutic agents, such as DNA or proteins, comonomer A preferably carries a cationic charge (plus), increasing the ability of the linear cyclodextrin polymer to form a powdery composite with a polypeptide therapeutic agent. It is also understood that the linear cyclodextrin polymer may contain a mixture of comonomer groups A.

A linear cyclodextrin copolymer can be prepared by copolymerizing a cyclodextrin monomer precursor containing two leaving groups as substituents with a comonomer A precursor capable of replacing the leaving groups. Leaving groups, which may be the same or different, can be any leaving groups known in the art that can be substituted during the copolymerization with the precursor of comonomer A.

A linear cyclodextrin polymer can be prepared by iodinating a cyclodextrin monomer precursor to form a di-substituted cyclodextrin monomer precursor and copolymerizing the di-substituted cyclodextrin monomer precursor with comonomer A precursor to form a copolymer containing a repeating element of Formula Ia, Ib, or a combination thereof.

Another method of preparation: linear cyclodextrin iodinates the cyclodextrin monomer precursor described above to form a di-substituted monomeric cyclodextrin precursor of the formula IVa, IVb, IVc, or a mixture thereof:

Diiodinated cyclodextrin can be prepared by any method known in the art (see, for example, Tabushi et al., J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al., J. Am. Chem. 106, 4580- 4584 (1984)). For example, β-cyclodextrin can react with biphenyl-4,4′-disulfonyl chloride in the presence of anhydrous pyridine to form β-cyclodextrin “coated” with biphenyl-4,4'-disulfonyl chloride, which can react with potassium iodide to form diiodo-β- cyclodextrin. The precursor of cyclodextrin monomer is iodinated in only two positions. By copolymerizing a diiodinated precursor of a cyclodextrin monomer with a precursor of comonomer A, as described above, a linear cyclodextrin polymer containing a repeating element of formula Ia, Ib, or a combination thereof can be obtained. If desired, iodine or iodine-containing groups can be replaced with other known leaving groups.

Iodine-containing groups or other suitable leaving groups can be replaced with a group that is capable of reacting with the precursor of comonomer A, as described above. For example, a di-substituted cyclodextrin monomer precursor of the formula IVa, IVb, IVc or a mixture thereof can be aminated to form a diamino substituted cyclodextrin monomer precursor of the formula Va, Vb, Vc, or a mixture thereof:

A diamine substituted cyclodextrin monomer precursor can be prepared by any methods known in the art (see, for example, Tabushi et al., Tetrahedron Lett. 18: 1527-1530 (1977); Mungall et al., J. Org. Chem. 1659-1662 (1975 )). For example, diiodo-β-cyclodextrin can react with sodium azide, and then the product can be reduced to form diamino-β-cyclodextrin. The monomeric cyclodextrin precursor is aminated at only two positions. The diaminated cyclodextrin monomer precursor can then undergo a copolymerization reaction with the comonomer A precursor described above to give a linear cyclodextrin copolymer containing a repeating element of formula Ia, Ib, or a combination thereof, also described above. However, the functional amino group of the diamino-substituted cyclodextrin monomer precursor does not have to be directly connected to the cyclodextrin fragment. Or the functional group can be introduced, replacing the iodo or other suitable leaving group monomer cyclodextrin precursor amine-containing moieties, such as ^- SCH ₂ CH ₂ NH _2, to form diaminosubstituted precursor monomer cyclodextrin of formula Vd, Ve, Vf, Vg, Vh and Vi or mixtures thereof:

A linear cyclodextrin copolymer can also be prepared by reduction of a linear oxidized cyclodextrin copolymer, as described below. This method can be carried out if comonomer A does not contain a fragment capable of being restored, for example, a disulfide bridge.

The linear cyclodextrin polymer can be oxidized so as to include at least one oxidized cyclodextrin monomer in the polymer so that the oxidized cyclodextrin monomer is part of a linear polymer chain. A linear cyclodextrin copolymer that contains at least one oxidized cyclodextrin monomer is defined as a linear oxidized cyclodextrin copolymer. The linear oxidized cyclodextrin, therefore, contains substituted and unsubstituted cyclodextrin fragments bifunctionally linked to the linear main chain of the copolymer due to the bonds of substituents at position 2, 3 or 6 of at least one glucopyranose cycle of cyclodextrin, with bifunctional fragments, comonomer A fragments that bind cyclodextrin is a linear cyclodextrin copolymer, and wherein the glucopyranose cycle of the cyclodextrin fragment is oxidized. The cyclodextrin monomer can be oxidized either at the primary or secondary hydroxyl groups of the cyclodextrin fragment. If more than one oxidized cyclodextrin monomer is present in a linear oxidized cyclodextrin copolymer, then these oxidized cyclodextrin monomers can be oxidized either by primary or secondary hydroxyl groups, or both. As an illustration, a linear copolymer of cyclodextrin with oxidized secondary hydroxyl groups is shown, which contains, for example, at least one element (unit) of formula VIa or VIb.

In formulas VIa and VIb, C is a substituted or unsubstituted oxidized cyclodextrin monomer, and A is a comonomer bound, i.e. covalently linked to oxidized cyclodextrin C. Also in formulas VIa and VIb, the oxidation of secondary hydroxyl groups leads to the opening of the cyclodextrin fragment cycle and the formation of an aldehyde group.

A linear oxidized cyclodextrin copolymer can be prepared by oxidizing a linear cyclodextrin copolymer, as discussed above. The oxidation of a linear cyclodextrin copolymer can be carried out by oxidation methods known in the art (Hisamatsu et al., Starch 44: 188-191 (1992)). Preferably, an oxidizing agent such as, for example, sodium periodate is used. Those skilled in the art will recognize that under standard oxidation conditions, the degree of oxidation may vary in the polymer or depending on the polymer. Thus, in one embodiment of the invention, the linear oxidized copolymer may contain one oxidized cyclodextrin monomer. In another embodiment, practically all cyclodextrin monomers in the copolymer are oxidized.

Another method for preparing a linear oxidized cyclodextrin copolymer involves the oxidation of a di-substituted or diamino substituted cyclodextrin monomer precursor described above to form an oxidized di-substituted or diamine substituted cyclodextrin monomer precursor and the copolymerization of an oxidized diiodo substituted or diamino substituted monomer substituted precursor of A. invention nickname of cyclodextrin monomer of formula VIIa, VIIb, VIIc or a mixture thereof:

The oxidized cyclodextrin monomer can be prepared by oxidizing a diiodosubstituted precursor of a cyclodextrin monomer of formula IVa, IVb, IVc, or a mixture thereof, as described above. In another embodiment of the invention, the precursor of the oxidized diamino substituted cyclodextrin monomer of formula VIIIa, VIIIb, VIIIc, or a mixture thereof

can be obtained by amination of an oxidized diiodosubstituted cyclodextrin monomer precursor of formulas VIIa, VIIb, VIIc, or a mixture thereof, as described above.

In yet another embodiment of the invention, the oxidized diamine-containing precursor of a cyclodextrin monomer of formula IXa, IXb, IXc, IXd, IXe, IXf, or a mixture thereof

can be obtained by substituting iodine or other corresponding leaving groups of the disubstituted oxidized precursor of cyclodextrin monomer having iodine or another corresponding leaving group as substituents, on an amino group containing the ^- SCH ₂ CH ₂ NH ₂ fragment.

Alternatively, the oxidized diiodated, dicarboxylated or diaminated cyclodextrin monomer precursor described above can be prepared by oxidizing the cyclodextrin monomer precursor to form an oxidized monomer precursor, and then diiodating and / or diaminating the oxidized cyclodextrin monomer as described above. The amino groups of any diaminated oxidized cyclodextrin monomers may have protecting groups to avoid unwanted side reactions. As discussed above, the cyclodextrin fragment can be modified using other leaving groups, other than iodine and other amino groups containing functional groups. The oxidized diiodinated or diaminated cyclodextrin monomer precursor can then be copolymerized with the comonomer A precursor to form a linear oxidized cyclodextrin copolymer.

A linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer terminates with at least one comonomer A precursor or a product of hydrolysis of a comonomer A precursor due to the fact that at least one comonomer A precursor is at the end of the cyclodextrin copolymer, on a linear cyclodextrin copolymer or on The linear oxidized cyclodextrin copolymer accounts for the free derivatizing group described above. For example, a derivatizing group that can be hydrolyzed to an acid group. According to the invention, the derivatizing group can be further modified in such a way as to improve the properties of the cyclodextrin copolymer, such as, for example, colloidal stability and transfection efficiency. For example, a derivatizing group can be derivatized by reaction with PEG to obtain a cyclodextrin copolymer with PEG at the end to increase the stability of the colloidal system, or by reaction with histidine or imidazoleacetic acid to obtain a cyclodextrin copolymer with an imidazolyl group at the end to increase intracellular activity (e.g. , endosomal excretion) and transfection activity. See Figures 29 and 30.

Further chemical transformations of the cyclodextrin copolymer can be carried out with the participation of a modified derivatizing group. For example, a modified derivatizing group can be used to increase the polymer chain by linking a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer with the same or a different cyclodextrin copolymer or non-cyclodextrin polymer. The polymer to be attached may be the same or a different linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer, which may also have a comonomer A precursor at the end for further modification.

Or, at least two identical or different linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers containing a terminal derivatizing group or a terminal modified derivatizing group described above can be reacted and linked together by a functional or modified derivatizing group. Preferably, a cleavable moiety, such as, for example, a disulfide bridge, is formed during the reaction of a functional or modified derivatizing group. For example, modification of the terminal derivatizing group with cysteine can be used to produce a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer containing a free thiol group. Reaction with the same or with a different thiol group-containing cyclodextrin copolymer leads to the formation of a disulfide bond between the two copolymers. Functional or modified derivatizing groups can be selected so that bonds are formed with different cleavage rates (e.g., enzymatic cleavage), and thereby provide, if necessary, a sustained release system for the therapeutic agent. The resulting polymer may be crosslinked as described herein. The therapeutic agent as described herein may be added before or after crosslinking of the polymer. The ligand may also be coupled to a cyclodextrin copolymer due to a modified derivatizing group. For example, a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer can be modified with a ligand adjacent to the cyclodextrin copolymer. The ligand may be attached to the cyclodextrin copolymer by the cyclodextrin monomer C or by the comonomer A. Preferably, the ligand is attached to the cyclodextrin fragment of the cyclodextrin copolymer. See International Application WO 00/01734, incorporated herein by reference.

Branched Cyclodextrin-Containing Polymers

A powder composite polymer having host and / or guest functionality may also be a substantially branched polymer, such as, for example, branched polyethyleneimine (PEI) or branched cyclodextrin-containing polymer, preferably branched cyclodextrin-containing polymer. The branched cyclodextrin-containing polymer may be any water-soluble polymer containing at least one cyclodextrin fragment, which may be part of the main polymer chain and / or branching from the main polymer chain. The branched cyclodextrin-containing polymer is a branched cyclodextrin copolymer or a branched oxidized cyclodextrin copolymer. A branched cyclodextrin copolymer or a branched oxidized cyclodextrin copolymer is, respectively, a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer described above, from which the side chain departs. The branched side chain may be any saturated or unsaturated, linear or branched hydrocarbon chain. The branched side chain may further comprise various derivatizing groups or substituents, such as, for example, hydroxyl, amino, acidic, ester, amide, keto, formyl and nitro. The branched side chain may also contain a cyclodextrin or other fragment with guest and / or host functionality. The branched side chain can also be modified using a ligand. Such a modification with the participation of the ligand includes, but is not limited to, the attachment of the ligand to the cyclodextrin fragment in the branched side chain.

Preferably, the branched cyclodextrin-containing polymer is a branched cyclodextrin copolymer or a branched oxidized cyclodextrin copolymer in which the branched side chain contains a cyclodextrin fragment. If the branched side chain contains a cyclodextrin fragment, the cyclodextrin fragment can facilitate the formation of an inclusion complex as well as the encapsulation of a therapeutic agent. Preferably, the cyclodextrin fragment of the branched side chain facilitates the formation of an inclusion complex and the encapsulation of a therapeutic agent in combination with the cyclodextrin fragment in the polymer backbone. A branched cyclodextrin-containing polymer can be prepared by any methods known in the art, including but not limited to derivatization (e.g., substitution) of a polymer (e.g., linear or branched PEI) using a cyclodextrin monomer precursor. Examples of polymers containing cyclodextrins in the side chain are described in Tojima, et al., J. Polym. Sci. Part A: Polym. Chem. 36, 1965 (1998), Crini, et al., Eur. Polym. J. 33, 1143, (1997), Weickenmeier et al., Maromol. Rapid Commun. 17, 731 (1996), and Bachmann, et al., J. Carbohydrate Chemistry 17, 1359 (1998); each of these sources is incorporated into this description by reference. (The Weickenmeier article describes polyesters with cyclodextrin in the side chain, their synthesis and incorporation (inclusion) of adamantane derivatives). The branched cyclodextrin-containing polymer may be crosslinked as discussed above.

Poly (ethyleneimine) (PEI) for use according to the invention has an average molecular weight between 800 and 800,000 daltons, preferably between 2000 and 100000 daltons, more preferably between 2000 and about 25000 daltons. PEI can be linear or branched. Suitable PEI compounds are commercially available from many companies, including polyethyleneimine from Aldrich Chemical Company, polyethyleneimine from Polysciences and POLYMIN poly (ethyleneimine) and LUPASOL ™ poly (ethyleneimine) from BASF Corporation.

Other polymers with host functionality

As discussed above, at least one polymer of the powdered composite is a polymer capable of forming inclusion complexes. Polymers in which cyclodextrin has a preferred host functionality are described above. In the same way, any polymer, linear or branched, having host functionality, can be used in the practice of this invention. Other examples of suitable “hosts” that can be used with polymers include, but are not limited to, cavitands, crown ethers, cryptands, cucurbiturils, calixarenes, spherands, and the like. The polymers of these other hosts can be obtained in the same manner as described above for cyclodextrin-containing polymers. The host of interest can be derivatized due to a functional group, such as, for example, a hydroxyl group, by addition of a leaving group, such as iodine, tosylate, etc., and can react with the corresponding comonomer A, which replaces the leaving group and forms a copolymer "master". Alternatively, the host may contain or may be derivatized in such a way as to contain a functional group, such as an amino or carboxy group, which makes possible the reaction of the host with comonomer A to form a "copolymer". The "host" copolymers, therefore, can be obtained by having a mixture of the host functionalities in the polymer backbone, and also, if the polymer is branched, in the side chains.

Polymers with Guest Functionality

A polymer with guest functionality may be any polymer capable of forming an inclusion complex with a complexing agent with host functionality. Typically, guest functionality is present in the side chain or in the end group. An example of a polymer having guest functionality not as part of the polymer backbone is a polymer having adamantane side groups. Examples of “inclusion” functionality that can be incorporated into a polymer include those known in the art, for example, but not limited to, adamantane, diadamantane, naphthalene and cholesterol.

B. Therapeutic agent

According to the invention, at least one therapeutic agent is encapsulated in a polymer to form a powdery composite, as described above. The term “therapeutic agent” is intended to encompass any active agent that has pharmacological or therapeutic uses, as discussed below, as active compounds or agents having a microbicidal effect. Examples of such therapeutic agents (or active agents) are discussed below. Encapsulation is defined as any method of associating a therapeutic agent (e.g., electrostatic interaction, hydrophobic interaction, actual encapsulation) with a polymer. The degree of association can be determined by methods known in the art, including, for example, fluorescence studies, DNA motility studies, light scattering, electron microscopy; and the degree of association varies greatly depending on the therapeutic agent. As a delivery method, for example, a therapeutic composition comprising a multidimensional polymer network created from a polymer of a powdery composite as described above, and DNA can be used to facilitate transfection, i.e. DNA entering an animal (e.g., human) cell (Boussif, O. Proceedings of the National Academy of Sciences, 92: 7297-7301 (1995); Zanta et al. Bioconjugate Chemistry, 8: 839-844 (1997); Gosselin et al. "Efficient Gene Transfer Using Reversibly Cross-Linked Low Molecular Weight Polyethilenimine", College of Pharmacy, The Ohio Statue University, published on the Internet (web), revised manuscript July 5, 2001). When the therapeutic agent is a polymer based on a nucleic acid (eg, DNA), the therapeutic agent forming the composite may be in the form of a “polyplex”. Polyplex is a composite of nucleic acid and "accountable" polymers. See Felgner, et al. "Nomenclature for Synthetic Gene Delivery Systems". Hum. Gene Ther. 8, 511-512 (1997).

Any mixture of therapeutic agents can be used with the composition of the invention. When a powdery composite is formed, the therapeutic agent may or may not retain its biological or therapeutic activity. When released from a therapeutic composition, namely, from a polymer of a powdery composite, the activity of the therapeutic agent is restored. Or a powdery composite favorably affects the protection of the therapeutic agent from loss of activity due to, for example, destruction and provides increased bioavailability. Therefore, the composition of the invention can be used to provide stability, in particular when stored and in solution, of a therapeutic agent or any active chemical compound. Encapsulating a lipophilic therapeutic agent provides increased, if not complete, solubility of the lipophilic therapeutic agent. The therapeutic agent can be further modified with a ligand before or after the formation of the powdered composite or therapeutic composition.

The therapeutic agent may be any lipophilic or hydrophilic, synthetic or natural biologically active therapeutic agent, including agents known in the art. The Merck Index, An Encyclopedia of Chemicals , Drugs, and Biologicals, 13 ^th Edition, 2001, Merck and Co., Inc., Whitehouse Station, NJ. Examples of such therapeutic agents include, but are not limited to, low molecular weight pharmaceuticals, antibiotics, steroids, polynucleotides (e.g., genomic DNA, cDNA, mRNA, antisense polynucleotides, viruses and chimeric polynucleotides), plasmids, peptides, peptide fragments, small molecules (e.g. doxorubicin), chelating agents (e.g. deforoxamine (DESFERAL), ethylenediaminetetraacetic acid (EDTA), natural products (e.g. Taxol, Amphotericin), and other biologically active macromolecules, such as proteins and f See also US Pat. No. 6,048,736, which lists active agents (therapeutic agents) used as a guest to prepare inclusion compounds with cyclodextrin polymers. US Pat. No. 6,048,736 is incorporated herein by reference. Low molecular weight therapeutic agents may not only be therapeutic agents in the particle (component) of the composite, but, in a further embodiment of the invention, may be covalently bound to the polymer in the composite. Preferably, the covalent bond is “reversible” (for example, a prodrug or biodegradable bond, such as a disulfide bridge) and creates a different delivery route for the therapeutic agent.

2. Complexing agent

According to the invention, the complexing agent is a compound having host or guest functionality, which is capable of forming an inclusion complex with a polymer in a powder composite having, respectively, guest or host functionality. As described above, the guest complexing agent can be used to modify a polymer of a powdery composite having host functionality or a monomer of a polymer having host functionality in order to obtain an inclusion complex. Also, as described above, the guest-forming agent can form an inclusion complex with at least one polymer of the powdery composite, acting as a guest with respect to the polymer with host functionality. The complexing agent may have two or more inclusion functionality. For example, a complexing agent having two inclusion functionality may be a complexing guest, a guest; host, host; host, guest. The complexing agent may also contain a mixture or several functionalities of the host and / or guest. The complexing agent also contains a functional group that confers the beneficial properties of the composition of the invention. This functional group may, for example, be a ligand, a hydrophilic or hydrophobic group, an additional therapeutic agent, etc. The complexing agent may include a spacer between the guest or inclusion host and the functional group.

Preferably, the binding constants of the complexing agent are about> 10 ² , preferably about> 10 ³ , and more preferably, about> 10 ⁴ . Typically, the values of the binding constant lies in the approximate range of 1 ² -10 ⁶ . Examples of inclusion guests useful as complexing agents include complexing agents known in the art, for example, but not limited to, such as adamantane, diadamantane, naphthalene, cholesterol (-ol). and their derivatives. Adamantane or diadamantane are preferably used. Amiel et al., Int. J. Polymer Analysis & Characterization, Vol. 1, 289-300 (1995); Amiel et a Journal of Inclusion Phenomena and Molecular Recognition in Chemistry, 25: 61-67 (1996); Amiel et al., Advances in Colloid and Interface Science 105-122 (1999); and Sandier et al., Langmuir, 16, 1634-1642 (2000).

The complexing agent contains a functional group that has a beneficial effect on the composition of the invention. The simple attachment of a hydroxyl or amino functional group is one way of introducing functionality. In a preferred embodiment of the invention, the complexing agent can form an inclusion complex with the polymer of the powdered composite, and also modify the composite, for example, facilitate cell contact, intercellular directed migration and / or cell entry gates and release from the cell. Any such group known in the art may be used. Examples of suitable “functional” groups include, but are not limited to, ligands, nuclear localization signals (see Zanta et al., Proc. Natl. Acad. Sci. USA, 96, pp. 91-96 (1999), endosomal release peptides, endosomal release polymers, membrane permeabilization agents, or mixtures thereof. The nuclear localization signal (NLS) may be any nuclear localization signal known in the art. The endosomal release peptide or polymer may be any peptide or endosomal release polymer known in the art (for example, HA-2 and GALA). See "Gene delivery by negatively charged terna ry complexes of DNA, cationic liposomes and transferrin or fusigenic peptides "Simoes S, Slepushkin V, Gaspar R, de Lima MCP, Duzgunes N, GENE THERAPY 5: (7) 955-964 JUL 1998. An example of a cell membrane permeabilization agent (or agent cell membrane permeability) is the TAT HIV-1 protein. The TAT protein is a viral transcription activator actively imported into the cell nucleus. Torchilin, V. P. et al, PNAS. 98, 8786-8791, (2001).

The complexing agent can also be functionalized using a polymer that increases solubility and / or resistance to impacts, in particular under biological conditions. Stabilization of the composition can be achieved or improved using complexing agents having hydrophilic groups or lipophilic groups. Preferred hydrophilic groups are polyethylene glycol or a polyethylene glycol-containing copolymer (PEG). Preferred polyethylene glycols have the formula OH (CH ₂ CH ₂ O) _z H, where z varies from 2 to 500, preferably 10-300. PEG 600, PEG 34000 and PEG 5000 are representatives of polyethylene glycols that can be used according to the invention. In general, the higher the molecular weight of PEG in the complexing agent, the higher the stabilization of the composition. Generally, a higher molecular weight is preferred. A preferred complexing agent is pegylated adamantane or pegylated diadamantane. The structures of some Adamantan-PEG molecules suitable as complexing agents are shown in Figure 1. To increase lipophilicity (hydrophobicity), the complexing agent may contain lipophilic groups such as long chain alkyls, fatty acids, etc. The choice of lipophilic group depends on the desired degree of lipophilicity. As can be seen from this discussion, the complexing agent can be modified using any type of functionality to introduce a given property into the composition. The complexing agent can be obtained by conventional methods of organic chemistry. The use of mixtures of various complexing agents allows to obtain a greater variety and greater specificity of the desired properties of the composition.

To connect a functional group with a complexing agent, a spacer group can be used. The spacer group may be any spacer group known in the art that does not adversely affect the properties of the guest complexing agent or functional group. For example, a spacer group may be a simple bond, so that the functional group is directly linked to the complexing agent. Or the spacer group may be a fragment that is soluble in water, is a high charge anion at physiological pH, or has fusogenic properties in an acidic environment. Preferably, the spacer group increases the binding affinity of the complexing agent to the polymer in the inclusion complex (for example, the anionic spacer group may also contain a reducing bond (eg, a disulfide bridge), the reduction of which releases the functional group of the complexing agent. Examples of suitable spacer groups include, but are not limited to, single bond, polyglutamic acid, GALA and polyethylene glycols (PEG).

The functional group may also be an additional therapeutic agent. A therapeutic agent may be reversibly bound to a complexing agent (for example, due to a prodrug form or biodegradable bonds). This provides a method of delivering additional therapeutic agents using a complexing agent.

As can be understood from the discussion above, the complexing agent used in the composition of the invention is a compound of the formula:

Where

J is NH-, -C (= O) NH- (CH ₂ ) _d -, -NH-C (= O) - (CH ₂ ) _d -, -CH ₂ SS-, -C (= O) O- (CH ₂ ) _e -O-P (= O) (O- (CH ₂ ) _e -Y) O-,

peptide or polypeptide residue or

-NH- (C = O) -CH (R ¹ ) -NH- (C = O) -CH (R ¹ ) -NH-;

Y denotes additional host / guest functionality;

R ¹ is - (CH ₂ ) _a —CO ₂ H, an ester or salt thereof; or - (CH ₂ ) _a —CONH ₂ ;

PEG (PEG) means —O (CH ₂ CH ₂ O) _z -, where z varies from 2 to 500;

L is H, -NH ₂ , -NH- (C = O) - (CH ₂ ) _e - (C = O) -CH ₂ -, -S (= O) ₂ -HC-CH ₂ -, - SS- , C (= O) O or carbohydrate residue;

a is 0 or 1;

b is 0 or 1;

d has a value in the range from 0 to 6;

e matters in the range from 1 to 6;

n has a value in the range from 0 to 6;

y is 0 or 1; and

x is 0 or 1.

The complexing agent may also be a compound of the formula:

where the variables have the same value as above, with z having values in the range of 1 to 5, q having values in the range of 1 to 5, and w having values in the range of 1 to 5.

As discussed above, examples of guest functionality include, but are not limited to, adamantyl, naphthyl, cholesterol, and cyclodextrin is a preferred guest functionality. Mixtures of host and guest functionalities, as indicated in the formula, may be present in the complexing agent.

A preferred class of complexing agents having adamantane guest functionality are compounds of the formula:

J is NH-, -C (= O) NH- (CH ₂ ) _d -, -NH-C (= O) - (CH ₂ ) _d -, -CH ₂ SS-, -C (= O) O - (CH ₂ ) _e -O-P (= O) (O- (CH ₂ ) _e -Ad) O-,

peptide or polypeptide residue or

-NH- (C = O) -CH (R ¹ ) -NH- (C = O) -CH (R ¹ ) -NH-;

Ad is adamantyl;

R ¹ is - (CH ₂ ) _a —CO ₂ H, its ester or salt; or - (CH ₂ ) —CONH ₂ ;

PEG means —O (CH ₂ CH ₂ O) _z -, where z varies from 2 to 500;

L is H, -NH ₂ , -NH- (C = O) - (CH ₂ ) _e - (C = O) -CH ₂ -, -S (= O) ₂ -HC = CH ₂ -, - SS- , C (= O) O or carbohydrate residue;

a is 0 or 1;

b is 0 or 1;

d has a value in the range from 0 to 6;

e matters in the range from 1 to 6;

y is 0 or 1; and

x is 0 or 1.

When using functionalized complexing agents, the therapeutic composition of the invention can be modified or functionalized so as to facilitate cell contact and / or cell entry gates. In order to obtain several functions and / or advantages, the composition may form two or three types of inclusion complexes using a complexing agent having different functionalities. As described above, a ligand can be used to modify the polymer of the powdered composite or complexing agent. Thus, in accordance with the invention, the composition of the invention may, through the inclusion complex, contain more than one ligand and, therefore, carry more than one site for targeting the cell and / or delivery. A powdery composite containing complex complexing agents functionalized with a ligand or otherwise can be stabilized by the addition of complexing agents with stabilization or solubility functionality, such as, for example, pegylated complexing agents.

Since the polymer can form several inclusion complexes with a mixture of various functionalized complexing agents, the therapeutic composition of the invention may contain, for example, several therapeutic agents, various ligands and / or different polymers for stabilization. If the complexing agent is functionalized with a therapeutic agent or prodrug, the formation of inclusion complexes allows the delivery of many therapeutic agents using the same therapeutic composition. If a ligand is present, the entire combination (cocktail) of therapeutic agents can be directed to a specific cell type, a specific disease, or other therapeutic use.

A functionalized guest - complexing agent can be obtained by any method known in the art. See Amiel et al., Int. J. Polymer Analysis & Characterization, Vol. 1, 289-300 (1995); Amiel et al., Journal of Inclusion Phenomena and Molecular Recognition in Chemistry, 25, 61-67 (1996); Sandier et al., Langmuir, 16, 1634-1642 (2000).

3. Obtaining a composition according to the invention

The invention also relates to a method for producing a composition. The method comprises combining a therapeutic agent, a polymer having host or guest functionality, and a complexing agent to form a therapeutic composition. A complexing agent playing the role of a guest or host forms an inclusion complex with a polymer. In another embodiment of the invention, the polymer and the therapeutic agent are first combined to form a powdery composite. The powder composite is then combined with a complexing agent to form an inclusion complex of the therapeutic composition. The composition can also be obtained by first mixing the polymer with a complexing agent, and then combining the mixture with a therapeutic agent to form the composite and, accordingly, the composition of the invention.

A. Obtaining a powdery composite polymer-agent

The powdered composite of the therapeutic agent and polymer can be prepared by any suitable method known in the art. For example, a powdery composite can be prepared by simply contacting, mixing, or dispersing a therapeutic agent with a polymer. For example, the polymer and therapeutic agent can be mixed in a solvent in which they are both soluble, in which the polymer is soluble and the therapeutic agent is dispersed, or in a solvent in which the polymer and therapeutic agent are dispersed, but in which the powder composite is solubilized. For use in pharmacy, the solvent may be any physiologically acceptable aqueous solution. A powdery composite may be formed by association of a polymer and a therapeutic agent, by self-association of a polymer, or by chemical means. Prior to the formation of the powdered composite, the polymer of the powdered composite, as a rule, does not exist as a practically associated structure, for example, a polymer gel. However, the polymer, as part of the powdery composite, depending on the nature of the polymers and the therapeutic agent, can form a substantially associated structure, such as a gel. A powdery composite may also be prepared by polymerizing monomers, which may be the same or different, in the presence of a therapeutic agent, to form a linear or branched polymer. A powdered composite can also be obtained by polymerizing monomers, which may be the same or different, capable of forming a linear or branched polymer, in the presence of a therapeutic agent, provided that the agent behaves like a matrix for polymerization. Troubetskoy et al., Nucleic Acids Research, Vol. 26, No. 18, pp. 4178-4185 (1998).

The amount of polymer and therapeutic agent used may be any amount that makes assembly possible of the composite. Typically, the polymer is taken in excess with respect to the therapeutic agent. When the polymer used carries a charge of a cation or anion, for example, such as in the case of a positively (“cationically”) charged comonomer A or in the case of polyalkyleneimine, for example, such as PEI, and when the therapeutic agent carries such a charge as, for example, in an anionic polynucleotide , the ratio of polymer to therapeutic agent can be expressed as the ratio of charges. The charge ratio is an expression of the ratio of the charge of the polymer to the charge of the therapeutic agent. As shown in the examples, powdered composites of cationic polymers of cyclodextrin and anionic DNA, as a rule, are prepared in the ratio of charges 5 +/-, that is, five cationic charges of the polymer cyclodextrin per one anionic charge of DNA. The charge ratio can be any ratio that makes possible the formation of a composite and which can exceed the minimum required ratio. If the polymer and / or therapeutic agent is not charged, the amount or ratio of polymer to therapeutic agent can be expressed in weight units, in moles, or as concentration, as is known in the art.

According to the invention, the polymer composite powder can also be processed under conditions sufficient to form a powder composite containing a therapeutic agent and a multidimensional polymer network. Such multidimensional polymer networks are described in International Application WO 00/33885, which is incorporated herein by reference. As described in International Application WO 00/33885, processing a polymer of a powdery composite under conditions sufficient to form a multidimensional polymer network can be carried out under any suitable reaction conditions, including the addition of additional agents or reagents that promote the association of the polymer and therapeutic agent of the powdery composite. A polymer may be associated due to interpolymer covalent bonds, non-covalent bonds (e.g., ionic bonds), or non-covalent bonds (e.g., Van der Waals interactions). Association due to intrapolymer covalent binding, non-covalent binding, or non-covalent polymer interactions may also occur. As a result of this association, the polymer powder composite interacts with the formation of a multidimensional polymer network.

In one embodiment of the invention, the formation of a powdery composite containing a therapeutic agent and a multidimensional polymer network includes crosslinking (crosslinking) reactions. For example, if the polymer of the powdery composite is a single polymer molecule, the polymer may react with the molecule (s), oligomer (s) or other polymer (s) that promote crosslinking or crosslink (s), such as crosslinking within a polymer molecule (intramolecular) or real crosslinking with a single polymer molecule of a powdered composite. Similarly, if the polymer of the powder composite is a mixture of two or more polymers, the polymer or polymers can react with the molecule (s), oligomer (s) or other polymer (s) that promote crosslinking or crosslink (stitching). The resulting crosslinking can be inside the polymer molecule (intramolecular) and / or between the polymer molecules (intermolecular), preferably, they are intermolecular crosslinking of the polymer or polymers of the powdered composite.

The crosslinking agent may be any crosslinking agent known in the art. The crosslinking agent may be any oligomer or polymer (for example, a polyethylene glycol (PEG) polymer, a polyethylene) capable of initiating internal crosslinking or actual crosslinking with the polymer of the powdered composite. The crosslinking oligomer or polymer may be the same as or different from the polymer powder composite. Similarly, a crosslinking agent may be any suitable molecule capable of crosslinking a polymer of a powdery composite. The crosslinking agent itself may have a ligand.

As described in International Application WO 00/33885, the degree of association of a polymer of a powdery composite forming a multidimensional polymer network can vary from partial association to full association. By varying the degree of association of the polymer, it is possible for the short-chain polymer to give the characteristics of the long-chain polymer, while dissociation preserves the desired characteristics of the short-chain polymer. For example, a long chain polymer promotes overall stability, i.e. resistance to destruction, until the target cell is reached, while a feature of the short-chain polymer is the promotion of DNA release inside the target cell. This duality gives the therapeutic composition containing a therapeutic agent and a multidimensional polymer network, increased stability in both non-physiological and physiological conditions and provides stability during longer storage. Changing the degree of association of the polymer of the therapeutic composition also allows you to control the release of the therapeutic agent.

The particle size of the powder composite depends on the polymer and therapeutic agent used to form the composition of the invention. As shown in the examples below, particle sizes can vary in the range of 50-1000 nm, preferably 50-500 nm. Usually, with the formation of an inclusion complex, the particle size increases slightly. Compositions remain in the form of discrete particles. As discussed below, compositions containing pegylated complexing agents exhibit excellent stability in salt solutions. The advantage is that the compositions are stable under physiological conditions, which makes it possible to use them as carriers for the delivery of therapeutic agents and in the treatment of various diseases and disorders.

B. Preparation of inclusion complex

The inclusion complex can be obtained by any method known in the art. For example, an inclusion complex can be formed by simply contacting, mixing or dispersing a powdery composite and a complexing agent. For example, a powdery composite and a complexing agent can be mixed in a solvent in which they are both soluble, in which a powdery composite or a complexing agent is soluble and the other component is dispersed, or in a solvent in which a powdery composite and a complexing agent are dispersed, but the inclusion complex is solubilized. Preferably, an inclusion complex is prepared by adding a complexing agent to the powdery composite in the same vessel that is used to mix the polymer and therapeutic agent to form an inclusion complex. For use in pharmacy, the solvent may be any physiologically acceptable aqueous solution.

The complexing agent can be added to the composite fraction in any molar ratio to the number of moles of host and / or guest functionality (in moles) in the composite polymer that forms the inclusion complex. Typically, the complexing agent is added in a 1: 1 molar ratio to the number of moles of host and / or guest functionality. Lower molar ratios (excess functionality of the host and / or guest in the polymer) can be used when the composition contains at least one complexing agent and at least one functionality of the host and / or guest in the polymer to form an inclusion complex . An excess of complexing agent may also be used. Thus, as a rule, the range of the molar ratio of the complexing agent to the functionality of the host and / or guest (in moles) in the polymer is from 0.01: 1 to 1: 0.01, preferably from 0.5: 1 to 1: 0.5. When several complexing agents are used, the molar ratio of the individual complexing agents can be selected based on the functionality to be introduced into the composition. For example, in this composition it may be that the pegylated stabilizing complexing agent is present in a ratio of 0.9: 1, and the complexing agent containing the ligand is present only in minor amounts, for example, 1-2% of the complexing agent. The total amount of complexing agent in such a composition is typically within the above ranges.

4. Compositions and methods of treatment

The therapeutic composition of the invention can be prepared in dry form, in the form of a liquid, suspension or emulsion. Preferably, the therapeutic composition of the invention is in an intravenous injection form. Other methods of administering the therapeutic composition of the invention include methods known in the art, for example, but not limited to, such as oral administration, inhalation, topical administration, parenteral, intravenous, intranasal, intraocular, intracranial or intraperitoneal injection and pulmonary administration. The route of administration often depends on the formulation of the therapeutic composition. Prior to administration, the therapeutic composition can be isolated and purified by any methods known in the art, including, for example, centrifugation, dialysis and / or lyophilization.

The invention relates to pharmaceutical compositions that contain an effective amount of a therapeutic composition according to the invention and a pharmaceutically and physiologically acceptable carrier. Suitable dry or liquid galenic preparations are, for example, granules, powders, coated tablets, microcapsules, suppositories, syrups, elixirs, suspensions, emulsions, drops or injectable solutions. Common additives in pharmaceutical compositions include, but are not limited to, excipients, comminuting agents, binders, coating agents, swelling, swallowing, or lubricating agents, flavoring agents, sweeteners, or solubilizing agents. More specifically, additives are often used, such as magnesium carbonate, titanium dioxide, mannitol and other sugars, talc, lactalbumin, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents. Solvents include sterile water and monohydric or polyhydric alcohols, such as glycerin.

Depending on the type of therapeutic agent used, the therapeutic composition of the invention can be used in various therapeutic methods (e.g. DNA vaccines, antibiotics, antiviral agents) for treating congenital or acquired disorders such as cystic fibrosis, Gaucher disease, muscular dystrophy, AIDS, various types of cancer (e.g., multiple myeloma, leukemia, melanoma, and ovarian cancer), cardiovascular disorders (e.g., progressive heart failure, restenosis, and hemophilia) and neurological disorders (e.g., head injury). According to the invention, the treatment method comprises administering to a human or mammal in need of treatment a therapeutically effective amount of a therapeutic composition of the invention. A therapeutically effective amount, as recognized by those skilled in the art, is determined empirically. Factors to be considered include, but are not limited to, the disorder to be treated and the physical characteristics of the sufferer.

6. Other applications (utility)

The inclusion complexes of the invention may also find application in the delivery of chemicals in agricultural production. In another embodiment of the invention, the “therapeutic agent” is a biologically active compound with bactericidal and agricultural utility. These biologically active compounds include compounds known in the art. For example, agriculturally acceptable biologically active compounds include, but are not limited to, fertilizers, fungicides, herbicides, insecticides, and mildew. Bactericidal compounds are also used for water treatment in the processing of municipal water supply systems and industrial water supply systems, for example, cooling water supply systems, aerated flow systems in the pulp and paper industry. Aquatic systems that are sensitive to microbiological effects or microbiological degradation are also available in the leather, textile, and coating or paint industries. Examples of such bactericidal compounds and their use, individually or in combination, are described in US Pat. Nos. 5,693,631, 6,034,081 and 6,060,466. Compositions containing, for example, the active agents discussed above, can be used in a manner known per se. It should be noted, since these fields of application are not related to pharmacology, the polymer of the composite does not have to meet the toxicity profile required for pharmaceutical use.

7. Examples

The following are examples to illustrate the invention. It should be understood, however, that the invention should not be limited to the specific conditions and details described in these examples.

The materials, β-cyclodextrin (Cerestar USA, Inc. of Hammond, IN) are dried in vacuum (<0.1 mTorr) at 120 ° C for 12 hours before use. Biphenyl-4,4'-disulfonyl chloride (Aldrich Chemical Company, Inc. of Milwaukee, WI) is recrystallized from a chloroform / hexanes mixture. Potassium iodide is ground with a pestle in a mortar and dried in an oven at 200 ° C. All other starting materials are industrial reagents and are used without further purification. Polymer samples were analyzed using a Hitachi HPLC system equipped with Anspec RI detector, Precision Detectors DLS and a Progel-TSK G3000 _{HWXL column} with 0.3 M NaCI or water as eluent at a flow rate of 1.0 ml min ^-1 .

Example 1: β-Cyclodextrin connected by A, D (capped) with biphenyl-4,4'-disulfonyl, 1 (Tabushi et al. J. Am. Chem. Soc. 106, 5267-5270 (1984))

In a 500 ml round bottom flask equipped with a magnetic stirrer, a Schlenk nozzle and a membrane, 7.92 g (6.98 mmol) of dry β-cyclodextrin in 250 ml of anhydrous pyridine (Aldrich Chemical Company, Inc.) were placed. The resulting solution was stirred under nitrogen at 50 ° C. while 2.204 g (6.28 mmol) of biphenyl-4,4'-disulfonyl chloride were added in four equal portions at 15-minute intervals. After stirring at 50 ° C for another 3 hours, the solvent was distilled off in vacuo, and the residue was subjected to reverse phase chromatography, eluting with a gradient of 0-40% acetonitrile in water. Fractions were analyzed by high performance liquid chromatography (HPLC) and the appropriate fractions were combined. After removal of the main volume of acetonitrile on a rotary evaporator, the resulting aqueous suspension is lyophilized to dryness. 3.39 g (38%) of 1 are obtained as a colorless solid.

Example 2: 6 ^A , 6 ^D Diiod-6 ^A , 6 ^D- Dideoxy-β-cyclodextrin, 2 (Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))

In a 40 ml centrifuge tube equipped with a magnetic stirrer, Schlenk nozzle and membrane, 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 are placed N, N-dimethylformamide (DMF) (Aldrich). The resulting suspension is stirred under nitrogen at 80 ° C for 2 hours. After cooling to room temperature, the solid precipitate was filtered off and the supernatant was collected. The solid precipitate was washed with a second portion of anhydrous DMF and the supernatants were combined and evaporated in vacuo. The residue was then dissolved in 14 ml of water, cooled in an ice bath and 0.75 ml (7.3 mmol) of tetrachlorethylene (Aldrich) was added with vigorous stirring. The precipitated product is filtered through a glass filter and washed with a small portion of acetone and dried in vacuum over P ₂ O ₅ for 14 hours. 0.90 g (92%) of 2 are obtained as a white solid.

Example 3: 6 ^A , 6 ^D- Diazido-6 ^A , 6 ^D- dideoxy-β-cyclodextrin, 3 (Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))

In a 100 ml round-bottom flask equipped with a magnetic stirrer, a Schlenk nozzle and a membrane, 1.704 g (1.25 mmol) diiodide of β-cyclodextrin is placed; 0.49 g (7.53 mmol) of sodium azide (EM Science of Gibbstown, NJ) and 10 ml of anhydrous N, N-dimethylformamide. The resulting suspension is stirred at 60 ° C under nitrogen for 14 hours. Then the solvent is distilled off in vacuo. The resulting residue is dissolved in an amount of water sufficient to obtain a 0.2 M solution in salt, and then 11.3 g of Biorad AG501-X8 (D) resin are passed through to remove the remaining salt. The eluate is then lyophilized to dryness to give 1.232 g (83%) 3 as a white amorphous substance, which is introduced into the next step without further purification.

Example 4: 6 ^A , 6 ^D- Diamino-6A, 6 ^D- dideoxy-β-cyclodextrin, 4 (Mungall et al., J. Org. Chem. 1659-1662 (1975)).

In a 250 ml round bottom flask equipped with a magnetic stirrer and membrane, 1.232 g (1.04 mmol) of β-cyclodextrin biazide and 50 ml of anhydrous pyridine (Aldrich) are placed. 0.898 g (3.42 mmol) of triphenylphosphine are added to this suspension with stirring. The resulting suspension is stirred for 1 hour at room temperature and 10 ml of concentrated aqueous ammonia are added. The addition of ammonia is accompanied by vigorous evolution of gas, and the solution becomes homogeneous. After 14 hours, the solvent was removed in vacuo and 50 ml of water was added to the residue. The solid precipitate was filtered off, the filtrate was acidified (pH <4) by adding 10% HCl and transferred onto a Toyopearl SP-650M ion exchange resin column (NH ₄ ⁺ form). Product 4 was eluted in a gradient of 0-0.5 M ammonium bicarbonate. The appropriate fractions were combined and lyophilized to give 0.832 g (71%) of product 4 as a bis (bicarbonate) salt.

Example 5: Copolymer β-cyclodextrin-DSP, 5

A solution of 92.6 mg (7.65 × 10 ^-5 mol) of bis (bicarbonate) of compound 4 in 1 ml of water is placed in a 20 ml scintillation tube. The pH of the solution was adjusted to 10 with 1M NaOH and a solution of 30.9 mg (7.65 × 10 ^-5 mol) of dithiobis (succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 ml of chloroform was added. The resulting two-phase mixture is stirred in a vibrating mixer for 0.5 hour. Then the aqueous layer is decanted and extracted with 3 × 1 ml of fresh chloroform. The aqueous polymer solution is then subjected to gel permeation chromatography (GLP) on Toyopearl HW-40F resin with water as an eluent. Fractions were analyzed by GLP and the corresponding fractions were lyophilized to obtain 85 mg (85%) as a colorless amorphous powder.

Example 6: Copolymer β-cyclodextrin-DSS, 6

The β-cyclodextrin-DSS copolymer, 6, is synthesized similarly to the polymer DSP, 5, except that disuccidinimidyl suberate (DSS, Pierce Chemical Co. of Rockford, IL) is used instead of the DSP reagent. Compound 6 was obtained in 67% yield.

Example 7: Copolymer β-cyclodextrin-DTBP, 7

A solution of 91.2 mg (7.26 × 10 ^-5 mol) of bis (bicarbonate) of compound 4 in 1 ml of water is placed in a test tube for a 20 ml scintillation counter. The pH of the solution was adjusted to 10 with 1M NaOH and a solution of 22.4 mg (7.26 × 10 ^-5 mol) of dimethyl-3,3'-dithiobis (propionimidate) • 2HCl (DTBP, Pierce Chemical Co. of Rockfbrd, IL) was added. ) The resulting homogeneous solution is stirred in a vibrating mixer for 0.5 hour. The aqueous polymer solution is then subjected to gel permeation chromatography (GLP) on Toyopearl HW-40F resin with water as an eluent. Fractions were analyzed using GLP and the appropriate fractions were lyophilized to give 67 mg (67%) as a colorless amorphous powder.

Example 8: Polyethylene glycol (PEG) -600-dicarboxylic acid chloride

5.07 g (about 8.4 mmol) of polyethylene glycol-600-dicarboxylic acid (Fluka Chemical Corp. of Milwaukee, WI) and 10 ml of anhydrous chloroform (Aldrich) are placed in a 50 ml round bottom flask equipped with a magnetic stirrer and reflux condenser. . 3.9 ml (53.4 mmol) of thionyl chloride (Aldrich) are added to this solution with stirring, and the resulting solution is boiled for 1 hour, while gas evolution is observed. The resulting solution was cooled to room temperature and the solvent and excess thionyl chloride were distilled off in vacuo. The resulting solution is stored in a desiccator and used without further purification.

Example 9: Copolymer β-cyclodextrin-PEG 600, 9

A solution of 112.5 mg (8.95 × 10 ^-5 mol) of bis (bicarbonate) 6 ^A , 6 ^D diamino-6 ^A , 6 ^D dideoxy-β-cyclodextrin (4), 50 is placed in a 20 ml scintillation counter tube μl (3.6 × 10 ^-4 mol) of triethylamine (Aldrich) and 15 ml of anhydrous N, N-dimethylacetamide (DMAA, Aldrich). The resulting suspension is treated with 58 mg (9.1 × 10 ^- 5 mol) of acid chloride of polyethylene glycol of 600-dicarboxylic acid, 8. The resulting solution is stirred in a vibrating mixer for 5 minutes, and then left for 1 hour at 25 ° C, during which time the solution becomes homogeneous. The solvent was removed in vacuo and the residue was subjected to gel filtration (gel permeation chromatography) on Toyopearl HW-40F resin with water as an eluent. Fractions were analyzed by GPC and the desired fractions were lyophilized to dryness to give 115 mg (75%) of a colorless amorphous powder.

Example 10: 6 ^A , 6 ^D- Bis- (2-aminoethylthio) -6 ^A , 6 ^D- dideoxy-β-cyclodextrin, 10 (Tabushi, I; Shimokawa, K; Fugita, K. Tetrahedron Lett. 1977, 1527- 1530)

0.91 ml (7.37 mmol) of a 0.81 M solution of sodium 1-aminoethylthiolate in ethanol (Fieser, LF; Fieser, M. Reagents for Organic Synthesis; Wiley) are placed in a 25 ml Schleik flask equipped with a magnetic stirrer and membrane. : New York, 1967; Vol. 3, pp. 265-266). The solution was evaporated to dryness and the solid residue was redissolved in 5 ml of anhydrous DMF (Aldrich). 6 ^A , 6 ^D- diiodo-6 ^A , 6 ^D dideoxy-β-cyclodextrin (2) (100 mg; 7.38 × 10 ^-5 mol) are added and the resulting suspension is stirred at 60 ° C. under nitrogen for 2 hours. After cooling to room temperature, the solution was evaporated and the residue was redissolved in water. After acidification with 0.1 N HCl, the solution was transferred to a Toyopearl SP-650M ion exchange resin column (NH ₄ ⁺ form) and the product was eluted with ammonium bicarbonate in a gradient of 0-0.4 M. The desired fractions were combined and lyophilized to dryness. Obtain 80 mg (79%) of compound 10 as a white powder.

Alternative synthesis of dicysteamine β-CD 10

To a solution of 4.69 g (3.17 mmol) of compound 2 in 100 ml of degassed water was added 0.489 g (6.34 mmol) of freshly distilled cysteamine. The solution was stirred at the boil for 2 hours. After cooling to room temperature and acidifying with 1 N HCl, the solution was transferred onto a Toyopearl SP-650M ion exchange resin column (NH ₄ ⁺ form) and the product was eluted with ammonium bicarbonate in a gradient of 0-0.2 M. The desired fractions were combined and lyophilized to dryness. This yields 1.87 g (39% yield) of a white solid. This solid is characterized by TLC (silica gel, n-PrOH-AcOEt-H ₂ O-NH ₃ aq. 5/3/3/1, detection with ninhydrin), there is a main spot corresponding to 10. Flight time (TOF) obtained MS with a laser desorption / ionization system (MALDI) is recorded on an ELITE 2 meter instrument manufactured by PerSeptive Biosystems, Inc. MALDI-TOF m / z calculated for compound 3: 1252, found: 1253.5 [M + H] ⁺ , 1275, [M + Na] ⁺ , 1291.4 [M + K] ⁺ . ¹³ C NMR (Bruker 500 MHz, D ₂ O) δ ppm: 32.1 (S-CH ₂ ) and 38.8 (CH ₂ -NH ₂ ), 32.9 (C6 adjacent to S), 60.2 (C6 adjacent to OH); 70.8; 71.4; 72.5 (C2, C3, C5), 81.8 (C4), 101.7 (C1).

Example 11: Copolymer β-cyclodextrin (cystamine) -DTBP, 11

A solution of 19.6 mg (1.42 × 10 ^-5 mol) of bis (bicarbonate) of compound 10 in 0.5 ml of 0.1 M NaHCO _{3 is} placed in a 4 ml tube. The solution was cooled in an ice bath and 4.4 mg (1.4 x 10 ^-5 mol) of dimethyl-3,3'-dithiobispropionimidate-2 HCl (DTBP, Pierce Chemical Co. of Rockford, IL) was added. The resulting solution is stirred in a vibrating mixer and left at 0 ° C for 1 hour. The reaction is terminated by adding 1M Tris-HCl and then acidified with 0.1 N HCl to pH 4. The aqueous polymer solution is subjected to gel filtration (gel permeation chromatography) on Toyopearl HW-40F resin. Fractions were analyzed by GPC and the desired fractions were lyophilized to dryness. 21.3 mg (100%) of compound 11 are obtained in the form of a white powder.

Example 12: Copolymer β-cyclodextrin (cystamine) -DMS, 12

In a 10 ml Schlenk flask equipped with a magnetic stirrer and membrane, 200 mg (1.60 × 10 ^-4 mol) of compound 10, 44 μl (3.2 × 10 ^-4 mol) of triethylamine (Aldrich Chemical Co. Milwaukee, WI) are placed ), 43.6 mg (1.60 x 10 ^-4 mol) of dimethylsuberimidate · 2 HCl (DMS, Pierce Chemical Co. of Rockford, IL) and 3 ml of anhydrous DMF (Aldrich Chemical Co. Milwaukee, WI). The resulting suspension is heated at 80 ° C for 18 hours under a constant stream of nitrogen for the entire time until almost all of the solvent has evaporated. The residue was redissolved in 10 ml of water and then the resulting solution was acidified with 10% HCl to pH 4. This solution was passed through an Amicon Centricon filter. Plus-20 5000 NMWL. After washing with water (2 × 10 ml portions), the polymer solution was lyophilized to dryness to give 41.4 mg (18%) of a yellowish-white amorphous solid.

Alternative synthesis: The β-cyclodextrin (cystamine) -DMS copolymer is synthesized as described above (Gonzalez, et al. 1999). In a typical experiment, a solution of bis (bicarbonate) of dicysteamine β-CD 10 (399.6 mg; 0.269 mmol) dissolved in 500 μl of 0.5 M Na ₂ CO _{3 is} placed in a 25 ml test tube. Dimethylsuberimidate · 2 HCl (DMS, Pierce Chemical Co. of Rockford, Illinois, 73.5 mg, 0.269 mmol) was added and the solution was rapidly centrifuged to dissolve the components. The resulting mixture was stirred at 25 ° C for 15 hours. The mixture was then diluted with 10 ml of water and, adding 0.1 N HCl, a pH of less than 4 was obtained. This solution was then dialyzed against a Spectra / Por 7 MWCO3500 dialysis membrane (Spectrum) in dH ₂ O for 24 hours. The dialyzed solution is lyophilized to dryness. ¹³ C NMR (Broker 500 MHz, D ₂ O) δ ppm: 25.8; 26.0; 27.0; 28.7; 29.9; 32.2; 37.5; 38.1; 41.1; 60.0; 71.6; 72.3; 72.6; 80.8; 101.4; 167.9.

Example 13: The formation of a complex with a fixed constant charge of the copolymer with a plasmid.

Typically, equal volumes of a solution of a CD polymer and a fixed-charge DNA plasmid are mixed in the appropriate polymer / plasmid charge ratios. Then, at room temperature, the mixture is balanced and self-assembled. The course of complexation is monitored by transferring an aliquot of the mixture to a 0.6% agarose gel and checking the mobility of the DNA. Free DNA moves when the voltage is applied, while the DNA in the complex is retained in the cell.

A solution of 1 μg of DNA in distilled water with a concentration of 0.1 μg / μl is mixed with 10 μl of copolymer 12 with a polymer amine: DNA phosphate charge ratio of 2, 4, 6, 12, 24, 36, 60 and 120. Add to each solution 1 μg / μl application buffer (40% sucrose; 0.25% bromophenol blue and 200 mM Tris acetate buffer containing 5 mM EDTA (Gao et al., Biochemistry 35: 1027-1036 (1996)). Each DNA sample / the polymer is applied to a 0.6% agarose electrophoretic gel containing 6 μg EtBr / 100 ml in 1 × TAE buffer (40 mm Tris-acetate / 1 mm EDTA), and a voltage of 40 V is applied to the gel for 1 hour. the formation of the DNA / polymer complex is determined by the delay in DNA migration in the gel.The copolymer (12) retains DNA at a charge ratio of 2 or higher, which indicates the formation of the complex under these conditions.

Example 14: Transfection studies using plasmids encoding the luciferase gene (control):

24 hours before transfection, BHK-21 cells were placed in 24-well plates at a cell density of 60,000 cells / well. Plasmids encoding the luciferase gene are mixed with the CD polymer as in Example 13. The medium solution containing the DNA / polymer complexes is added to the cultured cells and replaced with fresh medium after 24 hours incubation at 37 ° C. Cells are lysed 48 hours after transfection. Appropriate substrates for luciferase biotesting are added to the cell lysate. The luciferase activity, measured in units of illumination, is quantified by a luminometer. DNA / polymer complexes successfully transfect BHK-21 cells with a charge ratio of approximately 3 and maximum transfection with a polymer amine / DNA phosphate charge ratio of 40. Cell lysate is also used to determine cell viability using Lowry protein analysis (Lowry et al. Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). Up to a charge ratio of 40, toxicity is not observed.

Example 15: Synthesis of a β-cyclodextrin (cystamine) -DMA copolymer, 13

In a 20 ml scintillation counter tube equipped with a magnetic stirrer, 180 mg (0.131 mmol) of compound 10 and 32 mg of dimethyl adipimide (DMA, Pierce Chemical Co. of Rockford, Illinois) were placed. Add 500 μl of 0.5 M Na ₂ CO ₃ . The resulting solution was covered with foil and stirred overnight. The mixture was acidified with 0.1 N HCl, dialyzed with a Spectrapor MWCO 3500 membrane for 2 hours and lyophilized to give 41 mg of a white, amorphous solid with a molecular weight of 6 kDa as determined by light scattering.

Example 16: Synthesis of a β-cyclodextrin (cystamine) -DMP copolymer, 14

160 mg (0.116 mmol) of compound 10 and 30.1 mg of dimethyl pemilimidate (DMA, Pierce Chemical Co. of Rockford, Illinois) were placed in a 20 ml scintillation counter tube equipped with a magnetic stirrer. Add 500 μl of 0.5 M Na ₂ CO ₃ . The resulting solution was covered with foil and stirred overnight. The mixture was acidified with 0.1 N HCl, dialyzed with a Spectrapor MWCO 3500 membrane for 2 hours and lyophilized to give 22 mg of a white, amorphous solid with a molecular weight of 6 kDa, as determined by light scattering.

Example 17: Copolymer β-cyclodextrin (cystamine) -PEG600, 15

1.564 g (1.25 mmol) of compound 10 and 25 ml of freshly distilled dimethylacetamide (DMAA, Aldrich) are placed in a 100 ml round bottom flask equipped with a magnetic stirrer, Schlenk nozzle and membrane plug. 0.7 ml (4 equiv.) Of triethylamine and a solution of 8 (2.39 g; 3.75 equiv.) In 5 ml of DMAA are added to the suspension. The resulting solution is stirred in a vibrating mixer for 5 minutes, and then left for 1 hour at 25 ° C, during which time the solution becomes homogeneous. Then, the solvent was distilled off in vacuo and the residue was subjected to gel permeation chromatography on Toyopearl HW-40F resin. Fractions were analyzed by GPC and the desired fractions were lyophilized to dryness. A colorless amorphous powder is obtained.

Example 18: Synthesis of β-cyclodextrinosylate, 16 (Melton, L.D., and Slessor, K.N., Carbohydrate Research, 18, p.29 (1971))

A solution of dry β-cyclodextrin (8.530 g; 7.51 mmol) and 200 ml of dry pyridine are placed in a round-bottom flask equipped with a magnetic stirrer and a membrane plug connected to a vacuum pump. The solution was cooled to 0 ° C. and 1.29 g (6.76 mmol) of tosyl chloride were added. The resulting solution was left overnight at room temperature. Pyridine, as far as possible, is removed in vacuo. The residue was recrystallized twice from 40 ml of hot water to obtain 7.54 g (88%) of a white crystalline solid.

Example 19: Synthesis of β-cyclodextriniodide, 17

A compound of 16, 15 equivalents of potassium iodide and DMF was placed in a round-bottom flask with a magnetic stirrer and a Schlenk nozzle. The resulting mixture was heated for 3 hours at 80 ° C, after which the reaction mixture was cooled at room temperature. Then the mixture is filtered, the precipitate is discarded, and the filtrate is evaporated to dryness and again dissolved in water at 0 ° C. Tetrachlorethylene is added and the resulting suspension is vigorously stirred at 0 ° C for 20 minutes. The precipitate was collected on a glass frit (glass filter), washed with acetone and stored over P ₂ O ₅ .

Example 20: Synthesis of β-cyclodextrinthiol-PEG-attached polymer, 18

Stage 1: Synthesis of β-cyclodextrinthiol (K. Fujita, et al., Bioorg. Chem., Vol.11, p. 72 (1982) and K. Fujita, et al., Bioorg. Chem., Vol.11, p .108 (1982))

1.00 g (0.776 mmol) of compound 16, 0.59 g (7.75 mmol) of thiourea (Aldrich) and 7.8 ml of 0.1 N NaOH are placed in a 50 ml round bottom flask with a magnetic stirrer and a Schlenk nozzle. The resulting mixture was heated at 80 ° C for 6 hours in a nitrogen atmosphere. Then 0.62 g (15.5 mmol) of sodium hydroxide was added and the reaction mixture was heated at 80 ° C. under nitrogen for another hour. The reaction mixture was cooled to room temperature, then adjusted to pH 4 with 10% HCl. The total volume of the solution was adjusted to 20 ml, cooled in an ice bath and 0.8 ml of tetrachlorethylene was added. The reaction mixture was vigorously stirred at 0 ° C for 0.5 hour, then the precipitate was collected on a glass filter. The solid was kept in vacuo overnight to give 0.60 g (67%) of a white amorphous solid.

Step 2: 2.33 g (2.11 mmol) of β-cyclodextrinthiol obtained in Step 1, 0.650 g of functionalized PEG (PEG with olefins in the side chain, obtained) are placed in a 100 ml round bottom flask equipped with a magnetic stirrer and reflux condenser. from Yoshiyuki Koyama of Otsuma Women's University, Tokyo, Japan) and 50 ml of dH ₂ O. The resulting mixture is boiled for 12 hours, during which time β-cyclodextrinthiol dissolves. The reaction mixture was cooled to room temperature and the precipitate was separated by centrifugation. The supernatant is dialyzed against water on a Spectra / Por 7 MWCO 1000 membrane. The solution is lyophilized to give a white amorphous solid.

Example 21: Synthesis of a branched polymer PEI-cyclodextrin, 19

Branched PEI (25 kDa, Aldrich) and compound 17 were placed in a 20 ml scintillation counter tube equipped with a magnetic stirrer and then degassed sodium carbonate buffer was added. The resulting solution was stirred at 80 ° C for 4 hours. The mixture was acidified with 0.1 N NaCl, dialyzed using a Spectra / Por 7 MWCO 3500 membrane for 2 days and lyophilized.

Example 21B: Synthesis of Crosslinked Polymer PEI-Cyclodextrin

Branched PEI (M.V. 1200, Aldrich) and difunctionalized cyclodextrin 2 monomer (1 equiv.) Are mixed in dry DMSO. The mixture was stirred at 80 ° C. for 4 days and then dialyzed against water for two days using a Spectra / Por 7 MWCO membrane and lyophilized.

Example 22: Synthesis of Ad-PEG ₃₄₀₀ -Ad

240 mg of 1-aminoadamantane (1.6 mmol, Aldrich) and 288 mg of PEG ₃₄₀₀ (SPA) ₂ (0.085 mmol, Shearwater Polymers) were placed in a glass tube (flask) with a magnetic stirrer. 5 ml of methylene chloride are added and the solution is stirred overnight. The next day, the solution was filtered to remove the by-product n-hydroxysuccinimide, and methylene chloride was distilled off in vacuo. The residue was dissolved in water and centrifuged to remove excess 1-aminoadamantane. The supernatant is then dialyzed overnight in a Pierce Slide-A-Lyzer with MWCO = 3500. The solution was then lyophilized to give 248 mg of Ad-PEG ₃₄₀₀ -Ad as a white fluffy substance.

Example 23: Synthesis of Ad-PEG ₃₄₀₀ -NH ₂

347 mg of FMOC-PEG _3400- NH ₂ (0.110 mmol, Shearwater Polymers) and 155 mg of 1-aminoadamantane (1.0 mmol, Aldrich) were placed in a glass tube (flask) with a magnetic stirrer. 5 ml of methylene chloride are added and the resulting solution is stirred overnight. The next day, the solution was filtered to remove the by-product n-hydroxysuccinimide, and methylene chloride was distilled off in vacuo. The residue was dissolved in water and filtered, removing unreacted 1-aminoadamantane. The solution is then lyophilized to remove water. The FMOC group is removed by dissolving the solid formed in a 20% solution of piperidine in DMF for 20 minutes. The solvent was removed in vacuo and the residue was redissolved in water. The solution was then centrifuged to remove insoluble FMOC and then dialyzed overnight in a Pierce Slide-A-Lyzer with MWCO 3500. The solution was then lyophilized to give 219 mg of Ad-PEG ₃₄₀₀ -NH ₂ as a white fluffy substance.

Example 24: Adamantan-PEG _3400- NH ₂ (Ad-PEG _3400- NH ₂ ).

266 mg of FMOC-PEG _3400- NHS (78.2 μmol, Shearwater Polymers, Huntsville, AL) was placed in a glass tube (flask) with a magnetic stirrer. Then add 10 equiv. 1-adamantylmethylamine (1.5 mmol, Aldrich) dissolved in 3 ml of methylene chloride, and the solution was stirred overnight at room temperature. The solvent was removed in vacuo and water was added to the remaining solution to dissolve the PEG product. The solution is centrifuged at rcf 20K (10 ³ ) for 10 minutes, while the phase of adamantylmethylamine is separated as a liquid with a higher density. The aqueous phase is collected and water is distilled off in vacuo. The remaining viscous liquid was redissolved in a 20% solution of piperidine in DMF to remove the FMOC protection and stirred for 30 minutes at room temperature. The solvent was removed in vacuo, washed with DMF several times, redissolved in water and passed through an ion exchange column to remove unreacted PEG. The first fractions were collected and lyophilized to obtain 222 mg of a white fluffy powder (76% yield) of the desired product, the structure of which was confirmed by MALDI-TOF analysis.

Example 25: Adamantan-PEG ₃₄₀₀ Lactose (Ad-PEG _3400- Lac).

60 mg of Ad-PEG _3400- NH ₂ (16.8 μmol) obtained in Example 24 and 5.0 eq of lactose-monosuccinimidyl (50 mg, Pierce, Rockford, IL) were placed in a glass tube (flask) with a magnetic stirrer. 2 ml of 50 mM NaHCO _{3 was} added and the resulting solution was stirred overnight. The amine reaction is monitored by TNBS analysis (with trinitrobenzenesulfonate), which allows the determination of amine concentrations. At the end of the amine reaction (99% of the amine reacted), the solution was transferred to a dialysis tube (Slide-A-Lyzer, MWCO = 3500, Pierce), dialyzed for 24 hours against water and lyophilized to obtain 65.1 mg of a fluffy white powder ( yield 93%).

Example 26: Synthesis of Ad-PEG ₅₀₀₀

279 mg of PEG ₅₀₀₀ -NHS (0.053 mmol, Shearwater Polymers) was placed in a glass tube (flask) with a magnetic stirrer. Then, 46 μl of 1-adamantylmethylamine (0.42 mmol, Aldrich) dissolved in 3 ml of methylene chloride was added, and the solution was stirred overnight. The next day, the solution was filtered to remove the by-product n-hydroxysuccinimide, and methylene chloride was distilled off in vacuo. The residue is dissolved in water and centrifuged. Excess 1-adamantylmethylamine was separated and the upper aqueous layer was removed and dialyzed overnight in a Pierce Slide-A-Lyzer instrument, MWCO = 3500. The solution was then lyophilized to give 253 mg of a white fluffy solid of Ad-PEG ₅₀₀₀ . According to the analysis (the product was analyzed by HPLC using a Beckman Gold system with a Richards Scientific ELS detector and a C 18 column) the product is pure (retention time of PEG ₅₀₀₀ -NHS: 10.7 min; retention time of the product: 12.0 min; acetonitrile gradient /water).

Alternative synthesis of Adamantan-PEG ₅₀₀₀ (Ad-PEG ₅₀₀₀ )

674 mg of PEG ₅₀₀₀ -NHS (135 μmol, Shearwater Polymers) was placed in a glass tube (flask) with a magnetic stirrer. Then add 5 equiv. 1-adamantylmethylamine (675 μmol, Aldrich) dissolved in 10 ml of methylene chloride, and the solution was stirred overnight at room temperature. The solvent was evaporated in vacuo and water was added to the remaining solution. The solution was centrifuged at rcf 20K for 10 min, during which time the adamantylmethylamine layer separated as a liquid having a higher density. The aqueous phase is collected and dialyzed for 24 hours (Slide-A-Lyzer, MWCO = 3500) against water. The solution was lyophilized and 350 mg of a white fluffy powder was obtained (75% yield, product shown schematically below). According to the analysis (the product was analyzed by HPLC using a Beckman Gold system with a Richards Scientific ELS detector and a C 18 column) the product is pure (retention time of PEG ₅₀₀₀ -NHS: 10.7 min; retention time of the product: 12.0 min; acetonitrile gradient /water). Ad-PEG _{3400 was} prepared in a similar manner (56% yield; product structure was confirmed by MALDI-TOF analysis.

Example 27: Adamantane- (PEG ₅₀₀₀ ) ₂ (Ad- (PEG ₅₀₀₀ ) ₂ ).

315 mg (PEG ₅₀₀₀ ) ₂ -NHS (30 μmol, Shearwater Polymers) was placed in a glass tube (flask) with a magnetic stirrer. Then add 10 equiv. 1-adamantylmethylamine (300 μmol, Aldrich) dissolved in 3 ml of methylene chloride (DCM), and the solution was stirred overnight at room temperature. The solvent was evaporated in vacuo and water was added to the remaining solution to dissolve the PEG product. The solution was centrifuged for 10 min at rfc 20K, during which time the adamantylmethylamine layer separated as a liquid having a higher density. The aqueous phase is collected and dialyzed for 24 hours (Slide-A-Lyzer, MWCO = 3500) against water. The solution was lyophilized and 286 mg of a white fluffy powder was obtained (yield 91%).

Example 28: Adamantan-PEG _3400- fluorescein (Ad-PEG _3400- FITC).

20 mg of Ad-PEG ₃₄₀₀ -NH _{2 was} dissolved in 3 ml of 0.1 M Na ₂ CO ₃ dissolved in 3 ml of 0.1 M Na ₂ CO ₃ in a glass tube with a magnetic stirrer. To this solution 3 eq. fluorescein isocyanate (FITC, Sigma) in DMSO (4 mg / ml, 1.6 ml), the resulting solution was stirred in the dark overnight, transferred to a dialysis tube (MWCO = 3500) and dialyzed in the dark for 48 hours against water. The solution was collected and lyophilized to give 23 mg of a yellow fluffy solid. PEG _3400- FITC was synthesized as a control polymer from PEG _3400- NH ₂ (Shearwater Polymers) in a similar manner to give 23 mg.

Example 29: Synthesis of the peptide GALA

The GALA peptide (sequence: W-E-A-A-L-A-E-A-L-A-E-A-L-A-E-H-L-A-E-A-bL-A-E-A-L-E-A-L-A-A, M.V. 3032) is synthesized using a Biopolymer Synthesis Facility, Institute of Automation, Institute of Biotechnology. Before cleavage of the peptide from the polymer, one third of the polymer is selected for conjugation of adamantane. HPLC analysis of the peptide shows a purity of more than 95%. 1-Adamantylcarboxylic acid (Aldrich) forms a conjugate at the N-terminus of the GALA peptide by DCC condensation. The resulting peptide (GALA-Ad, M.V. 3194) is cleaved from the polymer. HPLC analysis of the peptide shows a purity of more than 90%. The identity of the peptides is confirmed by the MALDI-TOF method (Biopolymer Analysis Facility (installation for analysis of biopolymers), Beckman Institute, California Institute of Technology).

Example 30: Preparation of a composition of the invention using the GALA peptide

Plasmids and oligonucleotides. Plasmid pGL3-CV (Promega, Madison, WI) containing the luciferase gene under the control of the SV40 promoter was amplified using Esherichia Coli and purified using the Qiagen's Endotoxin-free Megaprep kit (Valencia, CA). Fluorescein-labeled oligonucleotides (FITC-oligos, 25mer, 5'-FITC-ACT GCT TAC CAG GGA TTTCAG TGC A-3 ') are synthesized using a biopolymer synthesis device (California Institute of Technology).

Particle formation and characterization. The compositions of the invention are prepared by mixing an equal volume of compound 12 (dissolved in dH ₂ O) with DNA (0.1 mg / ml in dH ₂ O) at an appropriate charge ratio. An equal volume of GALA or GALA-Ad dissolved in 50 mM phosphate buffered saline (PBS, pH 7.2) is then added to the complexes. For example, when studying particle characteristics, a complex of 2 μg plasmid DNA (20 μl) with 12 (20 μl) is obtained with a charge ratio of 5 +/-. Then, 20 μl of a GALA solution, a GALA-Ad solution, or 50 mM PBS (for control samples) are added to the complexes. The solutions are then diluted by adding 1.2 ml of dH ₂ O. Particle size and charge are determined using dynamic light scattering and zeta potential measurements using a ZetaPals dynamic light scattering detector (Brookhaven Instruments Corporation, Holtsville, NY). The results, expressed as mean ± standard deviation for the measurement data, are shown in Figure 2. The hydrodynamic diameter of the compounds 12 / pGL3-CV obtained at a charge ratio of 5 +/-, determined by dynamic light scattering, is 260 nm. 2 μg of plasmid DNA in 20 μl is mixed with an equal volume of compound 12 at a charge ratio of 5 +/-. Then, various GALA or GALA-Ad ratios are added to the particles. The hydrodynamic diameter is determined by measuring dynamic light scattering. Results are expressed as mean ± standard deviation in three dimensions. The GALA peptide undergoes a transition from the water-soluble conformation of the statistical coil at pH 7.5 to a water-insoluble helix at pH 5. The GALA peptide and the GALA peptide modified with adamantane (GALA-Ad) are dissolved in 50 mM PBS (pH 7.2) and added to the therapeutic composition at different peptide / cyclodextrin ratios. The mixture was diluted with dH ₂ O and the particle size was determined by measuring dynamic light scattering (Figure 2). Figure 2 shows the hydrodynamic diameter of GALA (dashed line) and GALA-Ad (solid line) of the modified polyplexes.

Results. Since the particle count rate remains the same at all concentrations of the added peptide, the addition of the peptide does not appear to destroy the composition. The particle size curves for the addition of GALA and GALA-Ad are very similar. The hydrodynamic diameter increases from 250 nm (1% GALA or GALA-Ad) to 400 nm (10% GALA or GALA-Ad). As more peptide is added, the particle size again decreases to the particle size of the unmodified therapeutic composition. The diameter returns to about 200 nm with the addition of 30% or more GALA-Ad and 50% or more GALA. See Figure 2.

Example 31: Inclusion of GALA-modified compositions in BHK-21 cells.

Cell culture. BHK-21 cells were obtained from ATCC (Rockville, MD), and HUH-7 cells were kindly provided by Valigen (Newtown, PA). Both cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 100 Units / ml penicillin, 100 μg / ml streptomycin and 0.25 μg / ml amphotericin in a humidified incubator (atmosphere) at 37 ° C and 5% CO ₂ , and subcultured every 4-5 days. Media and additions are obtained from Gibco BRL (Gaithersburg, MD).

Absorption of the therapeutic composition by culture cells. BHK-21 cells are placed in 6-well plates at a density of 150,000 cells / well and incubated for 24 hours at 37 ° C. Get a complex of 5 μg FITC-oligo with 12 at a charge ratio of 5 +/-. 5 minutes after the start of complexation, 50 μl of GALA or GALA-Ad in 50 mM PBS (pH 7.2) is added to the complexes. The media is removed from the cells and the cells are washed with PBS. For transfection, 900 μl of Optimem is added to the solution of each therapeutic composition, and the entire solution is transferred to cell cultures. The cells are incubated with the transfection mixture for 5 hours, the medium is removed and the PBS cells are washed twice. Cells are harvested by trypsinization and prepared for FACS analysis. Cells are washed twice with wash buffer (Hanks balanced solution containing DNAase and MgCl ₂ ) and resuspended in 500 μl FACS buffer (Hanks equilibrium salt solution, 2.5 mg / ml bovine serum albumin, 10 μg / ml propidium iodide). FACS analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and CellQuest software. The results are shown in Figure 4. As shown in FIG. 4a-d, BHK-21 (4a) cells were transfected using 12 / FITC-Oligo (4b), 12 / FITC-Oligo / 50% GALA (4c), 12 / FITC -Oligo / 50% GALA-Ad (4d). Absorption is determined by flow cytometry. The data are presented in the form of fluorescence curves, with the cell axis being plotted along the y axis, and the fluorescence fluorescence intensity along the x axis.

Example 32: Zeta potential of modified complexes.

2 μg of plasmid DNA in 20 μl is mixed with an equal volume of compound 12 at a charge ratio of 5 +/-. Then, various GALA or GALA-Ad ratios are added to the particles at different peptide / CD ratios and diluted with dH ₂ O. The charge of the particles is determined by measuring the electrophoretic mobility and is represented as the zeta potential of the particle in mV. As found by measuring the zeta potential, the particle charge of the 12 / pGL3-CV compositions with a charge ratio of 5 +/- is +13 mV. Determine the zeta potential of the particles in the presence of peptides, its values are presented in Figure 3 as mean ± standard deviation obtained from three measurements.

Results. Since the GALA peptide is an anionic peptide at pH 7.2 (contains several glutamic acid residues), the association of GALA and GALA-Ad with the compositions lowers their zeta potential. Compositions become negatively charged at 30% GALA (-11 mV) or GALA-Ad (-23 mV). The GALA zeta potential + therapeutic composition at this point is a plateau; adding more GALA only slightly increases the zeta potential (-15 mV at 150% GALA). However, the negative charge on the particles becomes larger at higher concentrations of GALA-Ad. Compositions in which 150% GALA-Ad have a zeta potential value of -42 mV. See FIG. 3.

Example 33: Efficacy of DNA Delivery Compositions

HUH-7 cells: The hepatoma cell line, HUH-7, is also transfected with 12 / FITC-Oligo / 50% at a charge ratio of 5 +/- and 12 / FITC-Oligo / 50% GALA-Ad. DNA uptake is monitored as described for BHK-21 cells. The fluorescence profile for untransfected HUH-7 cells lies in the first decile (Fig. 5a). FITC-Oligo successfully delivered to 95% of HUH-7 cells using compound 12 (Figure 5b). Adding 50% GALA-Ad to the compositions inhibits the absorption of FITC-Oligo by two orders of magnitude, as is observed for BHK-21 cells (Fig. 5c).

Example 34: Efficacy of the compositions of the invention for transfection of the luciferase gene.

The transfection ability of the compositions modified with GALA and GALA-Ad is determined using the delivery of the control luciferase gene to cultured cells. BHK-21 cells were placed in 24-well plates and transfected using 1 μg pGL-CV3 (a plasmid that contains the luciferase gene), complexed with compound 12 at a charge ratio of 5 +/- to form a powdery composite. These powder composites are modified by adding GALA or GALA-Ad at various peptide / cyclodextrin ratios. Cells are lysed within 48 hours after transfection and assayed for luciferase activity. The results shown in Figure 6 are expressed in relative light units (RLU). Data are mean ± SD for three measurements. Background value = 300 RLU.

Cells were successfully transfected using compositions 12 / pGL-CV3 with RLU ~ 1 × 10 ⁵ . The addition of GALA does not significantly affect transfection efficiency. However, modifying the composition with GALA-Ad substantially inhibits transfection. The addition of 1% GALA doubles the transfection to 2 × 10 ⁵ RLU, and 12 / pGL-CV3 / 10% GALA also leads to a slight increase in transfection (1.5 × 10 ⁵ RLU). Addition of 100% GALA reduces transfection by 50% to 5 × 10 ⁴ RLU.

Example 35: Toxicity of the compositions GALA and GALA-Ad.

The toxicity of the compositions modified with GALA and GALA-Ad is determined by measuring the concentration of protein in the cell lysates obtained in transfection experiments. BHK-21 cells were transfected using 1 μg pGL-CV3, complexed with compound 12 at a charge ratio of 5 +/-. Before transfection, various ratios of GALA and GALA-Ad are added to the complexes. Cell viability during transfection in the presence of GALA (shaded columns) and GALA-Ad (unshaded columns) is determined by checking the total protein concentration 48 hours after transfection and normalizing each sample according to the protein level for untransfected cells. Protein concentrations, expressed as mean ± SD in triplicate, are averaged and divided by the average protein concentration of cells transfected using only 12 / pGL-CV3 formulations and expressed as partial cell viability (Figure 7). The addition of GALA and GALA-Ad to the transfection solution does not impart noticeable toxicity to BHK-21 cells.

Example 36: Lactose-β-cyclodextrin-DMS 20 copolymer (Lac-β-cyclodextrin-DMS 20 copolymer).

Compound 12 (20.5 mg, 3 μmol), 10 eq. α-lactose (21 mg, 60 μmol, Sigma) and 18.6 sodium cyanoborohydride (300 μmol) are placed in a glass flask. 1 ml of borate buffer, pH 8.5, was added to the solids, and the resulting solution was shaken quickly and then incubated at 37 ° C in a water bath for 30 hours. The solution was acidified to pH 6.0 by adding 1M HCl and dialyzed against water for 24 hours. Analysis of TNBS for polymer amines shows that the degree of conjugation is 87%. Compound structure 20.

Example 37: Lactose- (CH ₂ ) _6- β-cyclodextrin-DMS 21 copolymer (Lac-C6-β-cyclodextrin-DMS 21 copolymer).

Compound 12 (43.2 mg, 7.4 μmol) and 5.6 equiv. mono (lactosylamido) -mono (succinimidyl) suerate (50 mg, 84 μmol, Pierce) was placed in a glass flask equipped with a magnetic stirrer and dissolved in 2 ml of 50 mM NaHCO ₃ . The resulting solution was stirred overnight. The progress of the reaction is monitored by the disappearance of the terminal groups of the polymer amine by TNBS, which shows a degree of conjugation of 90%. The solution was acidified to pH 5.0 by adding 1M HCl and dialyzed against water for 2 days in a Slide-A-Lyzer, MWCO 3500, Pierce, and then lyophilized. White fluffy powder is obtained with a yield of 70%. The structure of compound 21 is shown in Figure 12.

Example 38: Copolymer of β-cyclodextrin-DMS with terminal PEG ₃₄₀₀ 22; Pegylation before complexing with DNA.

20.3 mg of compound 12 (3 μmol) and 10 eq. FMOC-PEG _3400- NHS (190 mg, 60 μmol) was placed in a glass flask equipped with a magnetic stirrer, and dissolved in 1 ml of 50 mm NaHCO ₃ , pH 8.5. The solution was stirred in the dark at room temperature and then lyophilized. The dry compound is dissolved in 0.5 ml of 20% piperidine in DMF and stirred for 30 minutes to remove FMOC protection. The solvent was removed in vacuo and the remaining viscous liquid was dissolved in water and the pH was adjusted to below 6.0 with 0.1 M HCl. The polymer is separated from unreacted PEG by anion exchange chromatography and lyophilized to give a white fluffy powder. Pattern 22 is shown below.

Pegylation before complexing with DNA. Both 12 and 22 are mixed with plasmid DNA to measure particle size. While βCDP6 12 condenses with the plasmid, giving homogeneous particles with a hydrodynamic diameter of 130 nm, pegylated 22 is not able to condense with DNA. The presence of PEG at the end of the polymer disrupts condensation with DNA.

Example 39 (comparative): Paging by graft copolymerization after complexing with DNA.

A modified technique is used by Ogris et al., Gene Therapy, 595-605 (1999). 5 μg pGL-CV3 in 500 μl dH ₂ O is mixed with an equal volume of PEI (in dH ₂ O) at a charge ratio of 3 +/- or 6 +/-. Powdered 12 / DNA composites are prepared in the same manner as with a charge ratio of 5 +/-. The particle diameter of the powdered composites is determined using dynamic light scattering (DLS). After the formation of the powdery composite, PEG ₅₀₀₀ -SPA (10 mg / ml in DMF) was added to the solution, which was stirred for two hours. In the second stage, after determining the particle size, 500 μl of PBS, pH 7.2, are added to the solution. The solution was incubated for 30 minutes at room temperature before the final particle size was determined by DLS. See the schematic diagram in Figure 8.

In Stage 1, a powdery PEI / DNA or 12 / DNA composite is obtained in 1.2 ml of dH ₂ O. Particle sizes are determined by dynamic light scattering (DLS). To the powder composite solutions obtained in Step 2, PEG ₅₀₀₀ -SPA was added and allowed to react with primary amino groups for 1 hour. The particle size of the pegyated samples is determined using DLS. In Stage 3, 600 μl of PBS, pH 7.2, is added to each sample to test salt resistance of the pegyated particles. Particle size is determined 30 minutes after adding salt to determine the degree of particle aggregation.

Powdered PEI composites are prepared at a charge ratio of 3 +/- and 6 +/-, and Plex powder composites 12 / DNA are prepared at a charge ratio of 5 +/- for Stage 1. PEG ₅₀₀₀ -SPA is added to PEI at a ratio of 10: 1 weight / weight according to a procedure published by Ogris et al .. Gene Therapy, 595-606, 1999. 12 pegylated at 100%, 150% and 200% PEG: amine (mol%). As a control, an unreactive PEG in an amount of 100% is also added to 12. The particle diameter at each stage is shown in the table in Figure 9. The particle size of the powdery PEI composite slightly increases with pegylation (from 58 nm to 65 nm for a charge ratio of 3 +/- and from 55 nm to 60 nm for a charge ratio of 6 +/-) . Pegging protects powdered PEI composites from salt-induced aggregation. While unmodified PEI particles increase in diameter to 80 nm after adding salt, the particle size of the powdery pegylated PEI composite slightly increases to 78 nm (for a charge ratio of 6 +/-) and 115 nm (for a charge ratio of 3 +/-) .

The addition of 150% or 200% PEG ₅₀₀₀ -SPA to the powdered composite based on compound 12 leads to the destruction of the particles; the number of particles drops sharply, and no corresponding correlation of the function is observed. The pegylation of compound 12 appears to prevent polymer / DNA binding. Particle size is maintained at 67 nm after pegylation with 100% PEG ₅₀₀₀ -SPA. However, monitoring of particle size as a function of time shows that the particles are destroyed approximately 30 seconds after the addition of PEG, after which small particles are again observed. Therefore, with the addition of 100% PEG ₅₀₀₀ -SPA, part 12 can be pegylated. Since excess polymer 12 is added relative to DNA (with a charge ratio of 5 +/-), the particles can rearrange so that the unmodified polymer forms polyplexes with a DNA plasmid, then how much of the pegylated polymer remains free in solution. The addition of salt to these particles leads to particle aggregation (300 nm), although the particle size does not reach the particle size of the powdery composite with unmodified 12 (700 nm). In general, pegylation by reaction with the primary amino groups of the polymer after complexing with DNA is apparently effective for polymers with a high molecular weight (mV) with a high charge density. However, reaction with 12, even after complexing with DNA, leads to insufficient stabilization in the presence of salt with the addition of 100% PEG ₅₀₀₀ -SPA and to the destruction of particles at higher concentrations of PEG ₅₀₀₀ -SPA.

Example 40: Pegylation after complexing with DNA due to the formation of an inclusion complex.

Using the following procedure, Adamantan-PEG molecules (Ad-PEG) are added to solutions of pre-prepared compositions of 100% adamantane with cyclodextrin (mol%). PBS is then added to the solutions and the particle size is checked with DLS at intervals of 2 minutes. The results are shown in Figure 10.

Procedure: 2 μg pGL-CV3 in 600 μl dH ₂ O is mixed with an equal volume of 12 (in dH ₂ O) with a charge ratio of 5 +/-. The desired amount of Ad-PEG (10 mg / ml in dH ₂ O) was added and particle size was determined using DLS. To the solution was added 600 μl of PBS, pH 7.2, and particle size was monitored for 8 minutes at 2 minute intervals.

The average particle diameter of un-pegyled 12 increases from 58 to 250 nm within 8 minutes after the addition of salt. The presence of free PEG in the solution does not prevent aggregation (average diameter after adding salt of 240 nm). However, pegylation due to the formation of inclusion complexes with linear Ad-PEG molecules reduces particle aggregation depending on length. 8 minutes after salt addition, the particles pegylated with Ad-PEG ₃₄₀₀ aggregate to 210 nm in diameter, while the size of the aggregated particles with Ad-PEG _3400- Lac is 200 nm. Particles pegylated with Ad-PEG ₅₀₀₀ increase in diameter only to 90 nm after 8 minutes after adding salt and to 160 nm after 2 hours after adding salt. Modification using Ad- (PEG ₅₀₀₀ ) ₂ has a weak effect on aggregation (particle diameter 200 nm after addition of salt).

Stabilization also depends on the density of the PEG (Figure 10A). The average particle diameter, measured 10 minutes after the addition of salt, increases 4.7 times for unmodified polyplexes (from 58 nm to 272 nm), but only 1.2 times for polyplexes modified by adding 150% or 200% adamantane to cyclodextrin.

Example 41: Reduced cell uptake due to pegylation after complexation.

Stage 1. Mixtures for transfection are prepared as follows: An equal volume of cationic 12 is added to 3 μg FITC-Oligos (0.1 μg / ml) with a polymer to DNA charge ratio of 3 +/-. To the complexes add free PEG or Ad-PEG ₅₀₀₀ (obtained in Example 40) in the ratio of PEG to cyclodextrin 1: 1.

Stage 2. Cells HUH-7 are placed in 6-well plates at a density of 3 × 10 ⁵ cells / well and incubated in 4 ml of DMEM + 10% FBS + Antibiotic / Fungicide for 24 hours. After 24 hours, the cells were washed with PBS and 1 ml of Optimem containing transfection mixtures from Stage 1 was added to the cells. After incubation for 15 minutes, the transfection medium was removed, the cells were washed with PBS and 1 ml of Optimem was added to each well. Cells are incubated at 37 ° C for 30 minutes. Cells are then washed with Cell Scrub Buffer, Gene Therapy Systems to remove surface-associated complexes and PBS, and then separated from the wells by trypsin treatment. Cells are then prepared and analyzed by FACS for incorporation of FITC-Oligo. The results are presented below in Table 1. Modification of the complexes with Ad-PEG ₅₀₀₀ reduces the absorption of the FITC-Oligo / polymer complexes.

Table 1. Sample Transfection,% Alone cells 0% Cells + FITC-Oligo 0% Cells + Powder Composite + Free PEG 43% Cells + Ad-PEG ₅₀₀₀ Modified Powder Composite 27%

Example 42: Preparation of composition 12 / Ad-PEG ₅₀₀₀ ⁺ FITC and delivery to cultured cells.

BHK-21 cells were placed in 6-well plates, 200,000 cells / well, and incubated for 24 hours at 37 ° C. Complexation of 3 μg of oligo (0.1 mg / ml in dH ₂ O) with an equal volume of 12 (2 mg / ml in dH ₂ O) is carried out with a charge ratio of 5 +/-. After a 5-minute complexation reaction, 1.5 μl of PEG (PEG) -FITC or Ad-PEG-FITC is added to the complexes. The medium is removed from the cells and the cells are washed with PBS. For transfection, 940 μl of Optimem was added to the solution of each therapeutic composition and the whole solution was transfected into cells, the cells were incubated with the transfection mixture for 4 hours, the medium was removed, the PBS cells were washed and 4 ml of complete medium was added. Cells are incubated for another 24 hours at 37 ° C, the medium is removed and the cells are washed twice with PBS. Cells are harvested by trypsinization and prepared for FACS analysis. Cells are washed twice with wash buffer (Hanks balanced solution containing DNase and MgCl ₂ and resuspended in 500 μl FACS buffer (Hanks balanced solution, 2.5 / ml bovine serum albumin, 10 μg / ml propidium iodide). FACS analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and CellQuest software. The results are shown in Figure 11.

The formation of inclusion complexes with Ad-PEG _3400- FITC causes a markedly increased absorption of fluorescein compared to 12 incubated with Ad-PEG _3400- FITC (43% compared to 14%, FIG. 11). Free Ad-PEG _3400- FITC in media can be absorbed by the cell as part of the pinocytotic or endocytotic pathway. However, Ad-PEG _3400- FITC is also able to be absorbed by the cells and as a complex with 12. It does not seem that modification of the powdered composites 12 with Ad-PEG _3400- FITC at low ratios (10%) inhibits internalization. Most likely, the powdered compositions 12 easily bind to the cell surface and together deliver the Ad-PEG _3400- FITC to the cells because they are internalized. Delivery using powdered formulations 12 leads to higher fluorescence of fluorescein observed in cells transfected with 12 / Ad-PEG _3400- FITC. This method can also be used for co-delivery of low molecular weight therapeutic substances together with the gene of interest.

Example 43: Transfection of HUH-7 cells.

Transfection of luciferase. HUH-7 cells were placed in 24-well plates, 50,000 cells / well, and incubated for 24 hours at 37 ° C. To form a complex, 3 μg of plasmid GL-CV3 (0.1 mg / ml in dH ₂ O) is mixed with an equal volume of 12 (in dH ₂ O) or 21 (see Fig. 13) at different charge ratios. The media is removed from the cell cultures before transfection and the cells are washed with PBS. 600 μl of Optimem was added to each therapeutic composition to form a transfection solution, 230 μl of which was added to each of 3 wells for 4 hours. After four hours, 800 μl of complete medium was added to each well. The medium is changed 24 hours after transfection and the cells are lysed in 50 μl of cell culture lysis buffer (Promega, Madison, WI) 48 hours after transfection. Luciferase activity is determined using a Promega reagent for luciferase assay. The results are shown in Figure 13.

Example 44: Synthesis of Adamantane Derivatized PEI (Ad-PEI)

Polyethyleneimine (PEI) and adamantanecarboxylic acid are mixed in dry CH ₂ Cl ₂ and cooled to 0 ° C. DCC (1 equiv.), 1-hydroxybenzoyl triazole (1 equiv.) And triethylamine (1 equiv.) Were added to the mixture. The solution was slowly brought to room temperature and stirred for 16 hours. The precipitate is filtered off and then the solvent is distilled off in vacuo. Water is added to the remaining yellowish solution. The undissolved solid is separated by centrifugation. The aqueous solution is carefully transferred into a dialysis bag and dialyzed against water for 24 hours. After lyophilization, PEI-CD is obtained.

Example 45: Synthesis of cyclodextrin-PEG (CD-PEG)

PEG-succinimidyl propionic acid (SPA) (Shearwater Polymers) and cyclodextrin-monoamine (1.2 equiv.) Are dissolved in DMSO and stirred for 24 hours at room temperature. The cyclodextrin-PEG product is purified by dialysis.

Example 46: The formation of a powdered composite of Ad-PEI / DNA and subsequent modification using CD-PEG

1 μg of plasmid DNA (0.1 μg / μl in dH ₂ O) was mixed with the Ad-PEI from Example 44 at a charge ratio of 5 +/-. Then, CD-PEG (dissolved in dH ₂ O) was added to the complex in a predetermined ratio of CD: Ad.

Example 47: Stabilization by Pegging: Formulation at High Concentrations

4 μg of plasmid DNA is mixed with an equal volume of polymer mixture (containing polymer cyclodextrin 12 with a charge ratio of 1.5 +/- and, in some cases, Adamantan-PEG ₅₀₀₀ at a ratio of 1 CD: PEG ₅₀₀₀ ) with a final concentration in the range of 0.1 mg / ml-4 mg / ml (see Figure 14). Half of the solution is diluted with 1.2 ml of water and the diameter is determined by dynamic light scattering. The other half of the solution is passed through a Qiagen Quiaquick column to extract the DNA remaining in the solution. DNA concentration is determined by UV adsorption at λ = 260.

Results (Figures 15 and 16): Homogeneous powdery composites consisting of small particles (diameter <100 nm), modified with adamantane-PEG ₅₀₀₀ , can be prepared with concentrations up to and including 4 mg DNA / ml without precipitation. Unmodified polyplexes form large particles (> 300 nm) at concentrations above 0.2 mg / ml, and intense precipitation is observed at all concentrations of the formulation (loss> 50% DNA).

Example 48; Inhibition of non-specific absorption by surface modification of the polyplex

BHK-21 cells are placed in 6-well plates. Cells are transfected with 3 μg FITC-Oligo (final transfection mixture concentration: 0.05 mg DNA / ml), complexed with an equal volume of 12 at a charge ratio of 12 / DNA 2.5 +/-. Powdered composites are then modified using the following linkers:

Anionic Linker: WEAALAEALAEALAEAC Ad-Anionic Linker: Ad-WEAALAEALAEALAEAC Ad peg Ad-peg ₅₀₀₀ Ad-Anionic Linker-PEG Ad-WEAALAEALAEALAEAC-PEG ₅₀₀₀

1 ml of optimem medium was added to the transfection mixture and the whole solution was transferred to the previously washed BHK-21 cells (washed by PBS) for 15 minutes. The medium is then removed and the cells washed with CellScrub, trypsinized and prepared for FACS analysis.

Results: Guest inclusion (adamantane), spacer (anionic linker) and functional group (PEGD ₅₀₀₀ ) act to modify powdered 12 / DNA composites and inhibit non-specific uptake by cultured cells (incorporation into cells). See Figure 17. Optimal inhibition is achieved by a combination of all three components.

Example 49: Absorption of hepatoma cells mediated by galactose

HepG2 cells are placed in 24-well plates at 50,000 cells / well. 1 μg pCMV-Luc is contacted with an equal volume of 12 and modified as follows. Modification using PEG-containing complexing agents is carried out at a ratio of CD: PEG (CD is cyclodextrins in 12) equal to 2: 1.

Powdered composite 12 / pcMV-Luc without modification

Glu-PEG-Pep-Ad Glucose-PEG ₃₄₀₀ -CAEAEAEAE-Ad, 2 CD: 1 PEG Gal-peg-pep-ad Galactose-PEG ₃₄₀₀ -CAEAEAEAE-Ad, 2 CD: 1 PEG Peg-pep-ad PEG ₅₀₀₀ -CAEAEAEAE-Ad, 2 CD: 1 PEG

200 μl of Optimem medium is added to a portion of each transfection mixture and transferred to each cell well. 4 hours after transfection, 800 μl of complete medium is added to each well. 24 hours after transfection, the medium is removed, the cells are washed with PBS and 1 ml of complete medium is added to each well. 48 hours after transfection, cells are washed with PBS, lysed and assayed for luciferase activity. The described transfection procedure is also carried out in the presence of 1 mM glucose or 1 mM galactose as a competitive inhibitor.

Results: Powdered composites modified using Glu-PEG-Pep-Ad or PEG-Pep-Ad have a negative zeta potential and, therefore, are difficult to transfect cells. However, polyplexes modified with Gal-PEG-Pep-Ad show increased transfection, which is inhibited in the presence of free galactose, thereby demonstrating galactose-mediated transfection into hepatoma cells. See Figure 18.

Example 50: Synthesis of a compound of diadamantane.

Reference: Breslow et al., JACS (1996) v118 p8495-8496.

Zhang et al., JACS (1993) v115 p9353-9354.

Anhydrous pyridine (5 ml) was placed in a reactor with a small magnetic stirrer and cooled in an ice bath. Methyl dichlorophosphate (1.0 ml) was added dropwise. The mixture is left in the cold for 15 minutes, during which time a dichlorophosphate precipitate forms. Adamantylethanol dissolved in 5 ml of pyridine was added to the reactor, and after freezing the mixture, the reactor was sealed. The resulting mixture was stirred overnight at room temperature. Then the hermetically sealed reactor is opened and the resulting mixture is poured into a 10% sodium bicarbonate solution (50 ml). Then the resulting solution was evaporated in vacuo. 800 ml of water are added to the solid residue and the product is extracted with 150 ml of ether. The aqueous phase is acidified with 2N HCl to pH = 1.4, and then extracted with 3 × 150 ml of CHCl ₃ : n-BuOH (7: 3). The organic layer was washed with water and the solvent mixture was evaporated in vacuo to give a solid phase. This solid was recrystallized from acetone / hexane to give 27% white solid. Electrospray ionization mass spectrometry indicates the presence of a pure, predetermined product.

Example 51: Synthesis of the compound diadamantane-PEG ₅₀₀₀

Methylene chloride was dried with CaH ₂ at reflux overnight, then distilled immediately before the reaction. To a solution of PEG-epoxide (M.W. 5000) in freshly distilled methylene chloride (0.2 ml), a solution of bis (2- (1-adamantyl) ethyl phosphate (diamantane compound described in Example 51) is slowly added and 0.4 ml methylene chloride. The resulting solution was stirred at 35 ° C. for 4 days. The solvent was evaporated to dryness in vacuo. The resulting solid precipitated by adding 6 ml of water. The resulting mixture was stirred for half an hour at room temperature and then centrifuged to separate the solid phase (unreacted) diadamantane is connected e). The supernatant was dialyzed overnight against 3500MWCO membrane in water and lyophilized to dryness. This gives a white solid with a yield of 99%. MALDI-TOF Analysis shows desired product.

Example 52: Competitive Substitution Experiments between Ad-PEG ₃₄₀₀ and Diadamantane-PEG ₅₀₀₀ :

Competitive absorption experiments are carried out by adding a diAdPEG ₅₀₀₀ solution to a preformed AdPEG ₃₄₀₀ composition, polymer and DNA. A salt solution is then added and particle size is determined as a function of time.

The initial one is obtained by adding solution 12 (16.6 μl of water + 2.61 μl 12 with a concentration of 5 mg / ml + 2.37 μl of AdPEG ₃₄₀₀ with a concentration of 12.5 mg / ml) to a DNA solution (20 μl of DNA with a concentration of 0 1 mg / ml). The following are the characteristics of this composition solution:

[DNA] = 0.05 mg / ml

AdPEG ₃₄₀₀ molar ratio: CD = 1: 1

Charge ratio = 3 +/-

Total volume obtained = 40 μl

This composition was incubated for 10 minutes and a di-AdPEG5K solution (10 mg / ml) was added. The volume of this solution is determined so that the molar ratio between diAdPEG ₅₀₀₀ and AdPEG _{3400 is} 1: 1, 1: 2, 1: 4 or 1: 6. For example, to make the molar ratio 1: 2, 2.38 μl AdPEG _{5000 is} added.

After another 10 minutes of incubation, 1.2 ml of water is added so that it can be read with DLS instruments. The particle size was determined for 10 minutes, and then 600 uL 1 × PBS was quickly mixed with the solution of the composition. Then, the particle size is determined every minute for the next 30 minutes.

For comparison, two other solutions of the composition are prepared. In one case, DiAdPEG ₅₀₀₀ is not added at all. In another case, do not add AdPEG ₃₄₀₀ . You can see that under these conditions, the particle size of the powder composite does not stabilize when using AdPEG ₃₄₀₀ . Salts cause an increase in the average particle diameter from 70 nm to 350 nm in 30 minutes. However, one diAdPEG ₅₀₀₀ does not cause salt tolerance. The particle size remains constant after the addition of the salt solution. This remains true even when di-AdPEG5K is present in an amount of 1/6 of the AdPEG ₃₄₀₀ . The results are shown in Figure 19.

Example 53: pH Sensitive Adamantane-PEG Modifier

The guest-host association constant of the inclusion compound decreases when either the guest or the host is charged. For example, the protonated form (neutral form) of adamantanecarboxylic acid has an association constant of ~ 500000, while the unprotonated (anionic) form of adamantanecarboxylic acid has an association constant of ~ 30000. This can be used to introduce pH-sensitive behavior into a material containing inclusion compounds. For example, it is possible to modify using an adamantane-PEG compound (Ad-PEG) containing secondary amino groups near adamantane. The Ad-PEG compound has high affinity at physiological pHs, but is more readily released at acidic pHs observed in cell endosomes. A milder packing disorder in the endosomes promotes the release of DNA and cellular internalization of polyplexes.

Example 54: Synthesis of pH Sensitive Hydrolyzable Adamantane-PEG Modifiers

PEG5k-NH ₂ (132 mg, 0.0264 mmol) was dissolved in water and cooled to 0 ° C. To the mixture was added a solution of NaOH (5N, 0.053 ml, 0.264 mmol, 10 eq.) And a solution of 1-adamantyl fluoroformate (52 mg, 0.264 mmol, 10 eq.) In THF (3 ml). The mixture was stirred at this temperature for five minutes, and then brought to room temperature and stirred for two hours. THF is removed in vacuo. Insoluble solid is removed by centrifugation. The remaining aqueous solution was transferred onto a Spectra / Por MWCO 35000 membrane and dialyzed against water for two days. After lyophilization, adamantylcarbamate-PEG5k (80 mg) is obtained. The structure of this compound was confirmed by ¹ H NMR, HPLC and MALDI TOF MS.

Synthesis of the Adamantane-Schiff-PEG Hydrolyzable Compound

PEG5K-ALD and 1-adamantylmethylamine (1 eq.) Were mixed in methanol. A few drops of formic acid are added as a Schiff base catalyst. The mixture was stirred at 60 ° C for 12 hours, then the solvent was evaporated in vacuo. The mixture was dialyzed in water to give the desired Adamantane-Schiff-PEG5K compound.

Example 55: Synthesis of the compound Adamantan-PEG-Transferrin (Ad-PEG-Tf), Figure 20

1. Condensation of transferrin by carbohydrate groups.

Stage 1: Synthesis of Ad-PEG-NH ₂ -NH ₂

FMOC-NH-PEG ₅₀₀₀ -NHS (Shearwater Polymers, 0.2 mmol, 1 g) was placed in a round bottom flask with a magnetic stirrer. Tert-butylcarbazate (BOC hydrazide) (Aldrich, 1.6 mmol, 0.2112 mg) dissolved in 7 ml of a mixture of methylene chloride / ethyl acetate (1: 1) was added. The resulting solution was stirred overnight at room temperature. The next day, the solvents are distilled off in vacuo. The FMOC group is removed by dissolving the solid formed in 10 ml of 20% piperidine in dimethylformamide for 5 hours. The solvent was removed in vacuo and the residue was redissolved in water. The resulting solution was centrifuged to remove the insoluble FMOC group, and then dialyzed overnight in a Slide-A-Lyzer from Pierce, 3500MWCO. The solution was then lyophilized to give H ₂ N-PEG ₅₀₀₀ —NH — NH — CO — OtBu.

N-Hydroxysuccinimide (Aldrich, 0.24 mmol, 27.3 mg) and adamantanecarboxylic acid (Aldrich, 0.39 mmol, 71.2 mg) are added to H ₂ N-PEG ₅₀₀₀ -NH-NH-CO-OtBu (2 ) (0.16 mmol, 790 mg) dissolved in 7 ml of methylene chloride. To the resulting solution was added 1,3-dicyulohexylcarbodiimide (Aldrich, 1.6 mmol, 0.326 g) dissolved in 3 ml of methylene chloride. The resulting solution was stirred overnight at room temperature. The next day, the precipitate formed is filtered through a thick glass filter and the filtrate is evaporated on a rotary evaporator in vacuo. The residue was dissolved in 10 ml of water and centrifuged to remove unreacted adamantanecarboxylic acid. The solvent was distilled off in vacuo and the residue was redissolved in 6 ml of 4M HCl in dioxane to remove the deprotected tert.-butoxycarbonyl group. The resulting solution was stirred at room temperature for 4 hours. Then, the solvent was distilled off in vacuo, and the residue was redissolved in water. The resulting solution was dialyzed overnight in a Pierce Slide-A-Lyzer, 3500 MWCO and lyophilized to give 635 mg of Ad-PEG-NH ₂ -NH ₂ .

Stage 2: Synthesis of Conjugate Transferrin-PEG-Ad

A solution of 100 mg (1.28 μmol) of human transferrin (depleted in iron) (Sigma-Aldrich) in 1 ml of 30 mM sodium acetate buffer (pH 5) was subjected to gel filtration on a Sephadex G-25 column (Supelco). The resulting 4 ml of a solution containing transferrin (monitoring: UV absorption at 280 nm) was cooled to 0 ° C and 80 μl of 30 mM sodium acetate buffer (pH 5) containing 4 mg (19 μmol) of sodium periodate was added. The mixture is kept for 2 hours in an ice bath in the dark. An additional gel filtration (Sephadex G-25, 30 mM sodium acetate buffer (pH 5) is used to remove low molecular weight products. A solution containing about 85 mg (1.09 μmol) of oxidized transferrin is obtained. An oxidized transferrin solution is quickly added to a solution containing 54 5 mg (10.9 μmol) of Ad-PEG-NH ₂ -NH ₂ in 1 ml of 100 mM sodium acetate (pH 5). The resulting solution was stirred overnight at room temperature. Then the pH was adjusted to 7.5 by adding 1 M sodium bicarbonate, and at intervals of 1 hour, four portions are added 9.5 mg (150 μmol) cyanoborohydride sodium ida After 18 hours, the pegylated transferrin was purified and concentrated on a Centricon YM-50,000 NMWI instrument (Millipore).

Stage 3: Iron loading of Transferrin-PEG-Ad synthesized by transferrin oxidation

40 mg of the apo-transferrin-based compound (apo-transferrin or apo-transferrin-PEG-Ad) is dissolved in 700 μl of dH ₂ O. 200 μl of 5 mM iron citrate and 100 μl of NaHCO ₃ with a concentration of 84 mg / ml are added to this solution. This solution is left for 2-3 hours and then dialyzed against PBS overnight. The iron load efficiency is calculated by determining the ratio of absorption at 465 nm (oxidized iron) to absorption at 280 nm (tryptophan residues in the protein), and normalized by the ratio A ₄₆₅ / A _{280 of} industrial holo-transferrin. The determined iron loading efficiency for transferrin in oxidative buffer (sodium acetate pH 5) and freshly oxidized transferrin is shown in Figure 21.

Stage 4. The binding affinity of Transferrin-PEG-Ad (synthesized by oxidation of transferrin) with transferrin receptors on PC3 cells.

PC3 cells are incubated with 250 nM fluorescein transferrin (FITC-TF) with varying amounts of unlabeled transferrin and transferrin-PEG-Ad. The association of FITC-hTF cells is evaluated by the FACS method. Unlabeled transferrin competes very effectively with FITC-hTF, while transferrin-PEG-Ad competes very weakly with FITC-hTF, most likely due to reduced receptor affinity. The results are shown in Figure 22.

Example 56: Condensation of transferrin with lysine groups. Figure 23.

Stage 1: Synthesis of VS-PEG _3400- Ad

Vinyl sulfone-PEG _3400- NHS (Shearwater Polymers, 0.147 mmol, 0.5 g) was placed in a round bottom flask with a stirrer and dissolved in 5 ml of DMSO. Adamantylmethylamine (Aldrich, 0.147 mmol, 0.0243 g) was added. The resulting solution was stirred for 1 hour at room temperature. The solvent was removed in vacuo and the residue was redissolved in water. The resulting mixture was dialyzed overnight against a 1000 MWCO membrane (Spectra Horn). The solution is then lyophilized to give Vinylsulfone-PEG _3400- Ad.

Stage 2: Synthesis of conjugate Transferrin-PEG-Ad (Tf-PEG-Ad)

To 109 mg (32.1 μmol) of Vinyl Sulfone-PEG _3400- Ad was added a solution of human transferrin (depleted in iron) (Sigma-Aldrich) in 10 ml of 0.1 M sodium tetraborate buffer (pH 9.4). The resulting solution was stirred at room temperature for 2 hours. The pegylated transferrin is purified from unreacted Vinylsulfone-PEG ₃ -Ad on a Centricon YM-50,000 NMWI instrument (Millipore) and from unreacted transferrin on a hydrophobic column Butyl-650S (Tosoh Biosep) (purity confirmed by HPLC and MALDI-TOF).

Stage 3: Iron loading of Transferrin-PEG-Ad synthesized by condensation through lysine groups

Apo-transferrin and Tf-PEG-Ad are loaded with iron according to the procedure described in Example 55. The excess load of iron is quantified, as described previously. The iron loading efficiency of Tf-PEG-Ad synthesized by condensation by lysine groups is almost 100%.

Example 57: Binding affinity of Transferrin-PEG-Ad (synthesized by condensation by lysine groups) with transferrin receptors on PC3 cells.

PC3 cells are placed in 6-well plates at a density of 125,000 cells / ml. After 24 hours, the cells are exposed with 250 nM FITC-Tf mixed in various concentrations with hTf, hTf-PEG-Ad (synthesized by oxidation of hTf), hTf-PEG-Ad (synthesized by reaction with VS-lysine and purified) and hTf- ( PEG-Ad) ₂ . Absorption after a 20-minute exposure is determined by the FACS method. In contrast to Tf-PEG-Ad synthesized by transferrin oxidation, transferrin compounds synthesized by condensation with lysine effectively compete with FITC-Tf for receptors on the PC3 cell surface. The results are shown in Figure 24.

Example 58: Zeta potential of FITC-Tf-modified polyplexes

An aliquot of 12 equal volumes is added to an aliquot of plasmid DNA (2 μg DNA, 0.1 mg / ml in water) at a charge ratio of 3 +/- to form a powdery composite. Holo-transferrin or holo-Tf-PEG-Ad is added to the powdered composite. The particles are diluted by adding 1.2 ml of water and the zeta potential is determined on a ZetaPals dynamic light scattering instrument (Brookhaven Instruments). The results are shown in Figure 2. Unmodified holo-transferrin is associated with powdered composites via electrostatic interactions. When 2 nmol Tf / μg DNA is added, the powdered composites become neutral (reach a neutral state). Apparently, holo-Transferrin-PEG-Ad (indicated in Figure 25 Tf-PEG-Ad) is associated in the particles of the powdered composite both by electrostatic interaction and by the formation of inclusion compounds. Therefore, there is a stronger association of holo-Tf-PEG-Ad with particles, which clearly shows a continuous decrease in the zeta potential of modified particles at higher concentrations of holo-Tf-PEG-Ad. At 2 nmol Tf / μg, powdered composites modified with holo-Tf-PEG-Ad are negatively charged (zeta potential ~ -7 mV).

Example 59: Synthesis of AD-Phos-PEG ₅₀₀₀ -Galactose

The compound numbers shown below refer to the above diagram.

I. Synthesis of adamantane phosphonic acid 2. Dibenzyl phosphite (0.712 g, 2.71 mmol) is injected with a syringe into a solution of 1-adamantylmethylamine (0.493 g, 2.98 mmol) in dry CCl ₄ under argon. Almost immediately after the addition of dibenzylphosphite, a white precipitate forms. The solution was stirred for 12 hours. CH ₂ Cl ₂ (30 ml) was added to the mixture. The organic layer was washed twice (2 × 40 ml) with dilute acid (acidified water, pH = 4). Then dried the organic layer of MgSO ₄ . The solvent was evaporated in vacuo. The resulting white substance is crystallized from a mixture of solvents CH ₂ Cl ₂ and hexane. Crystals of compound 1 were obtained in the form of needles (0.69 g, 60% yield). The crystalline compound is hydrogenated with hydrogen at a pressure of 15 psi (103.42 kPa) with 10% Pd / C (200 mg) as a catalyst in ethanol (40 ml) for 16 hours. The catalyst is filtered off, the solvent is distilled off from the filtrate in vacuo. Get 2 in quantitative yield. The resulting compound 2 was used without further purification.

II. Synthesis of NH ₂ -PEG ₅₀₀₀ -Halactose 4. FMOC-NH-PEG ₅₀₀₀ -NHS (Shearwater, 760 mg, 0.152 mmol) was dissolved in DMSO (3.7 ml). To this solution is added a solution of galactosamine (385 mg, 1.52 mmol) and diisopropylethylamine (0.264 ml, 1.52 mmol) in DMSO (14 ml). The solution was stirred for 20 minutes and then dialyzed in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.) for 24 hours. The solution is then lyophilized to give 745 mg of FMOC-NH-PEG ₅₀₀₀ -Halactose 3. 3 is dissolved in DMF (12 ml) containing piperidine (3 ml). The solution was stirred for 16 hours. Then DMF is distilled off in high vacuum. 40 ml of water are added to the solid residue. The white solid is separated by centrifugation. The aqueous solution was dialyzed in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.) for 24 hours. The solution was lyophilized to give 625 mg of NH ₂ -PEG ₅₀₀₀ -Galactose 4.

III. Synthesis of Adamantan-Phos-PEG ₅₀₀₀ -Galactose 5. 4 (63 mg, 0.013 mmol) was dissolved in imidazole buffer solution (1 ml, 0.1 N, pH = 6.5). To this solution was added a solution of 2 in CH ₃ CN (4 ml), followed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 100 mg, 40 eq.). The solution was stirred for 16 hours at room temperature. The solution was dialyzed in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.), and then lyophilized to give 5.

Example 60: Synthesis of AD-Glu-Glu-PEG ₅₀₀₀ -Galactose

The compound numbers shown below refer to the above diagram.

I. Synthesis of H-Glu-Glu-Adamantane 7. H-Glu (Bn) -OH (3.55 g, 15 mmol) is dissolved in water (16 ml) containing sodium bicarbonate (1.26 g; 15 mmol). Z-Glu (Bn) -OSu (4.68 g, 10 mmol) in THF (30 ml) was added to the mixture. Then another 30 ml of THF, 20 ml of CH ₃ CN and then 10 ml of 2N NaOH are added to the mixture. The solution was stirred for 16 hours at room temperature. THF and CH ₃ CH were evaporated in high vacuum. 1 N HCl was added to the aqueous mixture, adjusting the pH to 3. Precipitation was observed. The mixture was extracted with chloroform (3 × 30 ml). The organic layer was dried with MgSO ₄ . MgSO _{4 is} filtered off. The organic solvent was evaporated, yielding a white, sticky solid 6. 6 was used in the next step without further purification.

6 (3.51 g, 6.1 mmol) was dissolved in dry THF (40 ml). To this solution, under argon at 0 ° C., 1-adamantylmethylamine (1.007 g, 6.1 mmol), 1-hydroxybenzotriazole (0.93 g, 6.1 mmol), DCC (1.32 g, 6.4 mmol) are added. and diisopropylethylamine (1.06 ml, 6.1 mmol) under argon at 0 ° C. The mixture was then brought to room temperature and stirred overnight. The precipitate is filtered off. Then, THF was removed in vacuo to give a yellow solid. This yellow substance is crystallized from methanol to give crystals 6 in the form of plates (2.1 g, 49%). Then 6 was dissolved in 40 ml of methanol and shaken in a hydrogenation apparatus in the presence of 200 mg of 10% Pd / C at a hydrogen pressure of 25-30 psi (172.37-206.84 kPa). The catalyst is filtered off after 24 hours. H-Glu-Glu-AD 7 was obtained in quantitative yield after distillation of methanol in vacuo. 7 use without further purification.

II. Synthesis of AD-Glu-Glu-PEG ₅₀₀₀ -Galactose 9. Vinylsulfone (VS) -PEG ₅₀₀₀ -NHS (Shearwater, 423 mg, 0.085 mmol) and galactosamine (216 mg, 0.85 mmol) are added to the PBS solution (2.25 ml, 1x, pH 7.2). The solution was stirred for 1 hour and then dialyzed in water (4 × 4 L) for 24 hours using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). Then the solution is lyophilized. Product 8 was analyzed using MALDI-TOF MS and HPLC. 8 is dissolved in a borate buffer solution (6 ml, 0.1 N, pH 9.4). Compound 7 (121 mg) was dissolved in DMSO (2 ml) and then added to the polymer solution. The mixture was stirred at 35 ° C for 16 hours, and then at 50 ° C for 17 hours. The reaction is monitored by HPLC. The polymer was dialyzed using a 3500 MWCO membrane and lyophilized to obtain 419 mg of AD-Glu-Glu-PEG ₅₀₀₀ -Galactose 9 in 90% yield.

Example 61: Synthesis of AD-Glu-Glu-PEG ₅₀₀₀

Synthesis of AD-Glu-Glu-mPEG ₅₀₀₀ 10. mPEG ₅₀₀₀ -SPA (Shearwater, 300 mg, 0.06 mmol) and 7, Example 60, were dissolved in DMSO (2 ml) and CH ₃ CN (1 ml). The mixture was stirred at room temperature for 24 hours. The solution is then dialyzed in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.) for 24 hours. The solution was lyophilized to give 276 mg of Ad-Glu-Glu-mPEG ₅₀₀₀ 10. Structure 10 was confirmed by MALDI-TOF MS, HPLC and ¹ H NMR.

Example 62: The preparation of transferrin and PEG-modified polyplexes.

Polyplexes (ratio of polymer charge to DNA charge 3 +/-) modified using Tf-PEG-AD (or Tf- (PEG-AD) ₂ ) and PEG-AD (or PEG-Glu-Glu-AD) can be prepared in the following way. Take equal volumes of all components. Tf-PEG-AD (or Tf- (PEG-AD) ₂ ) is added to a solution of 12 in water. An aliquot of PEG-AD (or PEG-Glu-Glu-AD) is added to this mixed solution. This ternary polymer mixture is then added to the DNA solution. The solution was carefully mixed by pipetting, and the particle size, zeta potential, and salt resistance were determined as described above. The zeta potential of particles can be changed ("tuned") by changing the relative ratios of Tf-PEG-AD (or Tf- (PEG-AD) ₂ ) and PEG-AD (or PEG-Glu-Glu-AD). Some examples of changes in the zeta potential and particle size depending on the modification of the particles are shown in Figures 26, 27 and 28.

Example 63: Adamantane Anionic Peptide-PEG ₃₄₀₀ -galactose / Glucose (AD-nen-PEG-gal-glu). Anionic peptide (sequence: E-A-E-A-E-A-E-A-C) is synthesized using a device for the synthesis of biopolymers (Biopolymer Synthesis Facility, Beckman Institute, California Institute of Technology) using an automatic synthesizer. Before cleavage of the peptide from the polymer, adamantanecarboxylic acid (ASA, Aldrich) conjugates to the N-terminus of the peptide in the presence of DCC. The resulting peptide (ASA-E-A-E-A-E-A-E-A-C, M.V. 1084) was cleaved from the polymer and analyzed by the MALDI-TOF method.

Galactose- and glucose-PEG _3400- vinylsulfone (gal / glu-PEG ₃₄₀₀ -VS) are obtained in about 95% yield by reaction of NHS-PEG ₃₄₀₀ -VS (Shearwater Polymers) with 20 equivalents of glucosamine or galactosamine (Sigma) in physiological phosphate buffered solution, pH 7.2, for two hours at room temperature. The solution is subjected to intensive dialysis against water, and then lyophilized. Anionic peptide thiols (two equivalents) are reacted with galactose-PEG _3400- VS or glucose-PEG _3400- VS in 50 mM sodium borate buffer (pH 9.5) containing 10 mM TCEP. The solution was acidified and the precipitate (insoluble below pH 9.0) was removed by centrifugation. The supernatant is collected, intensively dialyzed and lyophilized. The structure of the desired products is confirmed by the MALDI-TOF method.

Example 64: Synthesis of naphthalene-PEG ₅₀₀₀

500 mg of PEG ₅₀₀₀ —NHS (0.1 mmol, Shearwater Polymers) was added to a glass jar with stirrer. 146 μl of 1-naphthalenemethylamine (1 mmol, 10 equiv., Aldrich) dissolved in 8 ml of methylene chloride are added and the solution is stirred for 16 hours. Then the solvent is removed in vacuo. 20 ml of water are added to the mixture. The insoluble precipitate is removed by centrifugation. The aqueous solution is dialyzed with a Spectra / Por 3500 MWCO membrane for 24 hours. The solution was then lyophilized to give a white fluffy solid naphthalene-PEG ₅₀₀₀ . The product was analyzed by ¹ H NMR, MALDI-TOF MS and reverse phase HPLC. Naphthalene-PEG _{5000 is} synthesized by a similar procedure (yield 56%; product structure is confirmed by the MALDI-TOF method).

Example 65: Synthesis of Naphthalene-PEG ₅₀₀₀ -Galactose

Vinyl sulfone (VS) -PEG ₅₀₀₀ -NHS (Shearwater, 423 mg, 0.085 mmol) and galactosamine (216 mg, 0.85 mmol) are added to the PBS solution (2.25 ml, 1x, pH 7.2). The solution was stirred for 1 hour and then dialyzed in water (4 × 4 L) for 24 hours using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution was then lyophilized to give Vinylsulfone-PEG ₅₀₀₀ -Galactose. Product analyzed by MALDI-TOF MS and HPLC. 300 mg (0.06 mmol) of Vinyl Sulfone-PEG ₅₀₀₀ -Halactose are dissolved in borate buffer solution (3 ml, 0.1 N, pH 9.4). 1-Naphthalenemethylamine (8.8 μl, 0.06 mmol) was dissolved in DMSO (3 ml) and then added to the polymer solution. The mixture was stirred at 55 ° C for 36 hours. The polymer was dialyzed using a 3500 MWCO membrane and lyophilized to give Naphthalene-PEG ₅₀₀₀ -Galactose.

It should be understood that the above discussion and examples are a detailed description of only certain preferred embodiments of the invention. Those skilled in the art will recognize that various modifications and equivalents can be made without departing from the spirit and scope of the invention.

Claims (26)

1. The compound of the formula

where J is -NH-, -C (= O) NH- (CH ₂ ) _d -, -NH-C (= O) - (CH ₂ ) _d -, -CH ₂ SS-, -C (= O) O- (CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Y) O-,

peptide or polypeptide residue, or -NH- (C = O) -CH (R ¹ ) -NH- (C = O) -CH (R ¹ ) -NH-; Y denotes additional host / guest functionality; R ¹ is - (CH ₂ ) _a —CO ₂ H, an ester or salt thereof; or - (CH ₂ ) _a —CONH ₂ ; PEG (PEG) means —O (CH ₂ CH ₂ O) _z -, where z varies from 2 to 500; L is H, -NH ₂ , -NH- (C = O) - (CH ₂ ) _e - (C = O) -CH ₂ -, -S (= O) ₂ -CH = CH ₂ -, - SS- , C (= O) O- or carbohydrate residue; the host is selected from adamantyl, naphthyl, cholesterol, cyclodextrin, or any combination thereof; a is 0 or 1; b is 0 or 1; d has a value in the range from 0 to 6; e matters in the range from 1 to 6; n has a value in the range from 0 to 6; q has a value in the range of 1 to 5; w has a value in the range from 1 to 5; y is 1; x is 1; and z has a value in the range of 1 to 5. 2. The compound according to claim 1, where q is 1; w is 1 and z is 1. 3. The compound according to claim 2, where

and Y is adamantyl. 4. A composition intended for the delivery of a therapeutic agent containing a powdery composite of a polymer and a therapeutic agent and a complex for including said polymer and a complexing agent, the complexing agent containing a functional group and the polymer containing one or more cyclodextrin fragments. 5. The composition according to claim 4, characterized in that the polymer has host functionality, and the complexing agent has guest functionality. 6. The composition according to claim 4, characterized in that the polymer has guest functionality, and the complexing agent has host functionality. 7. The composition according to claim 4, characterized in that the polymer has the functionality of a host and a guest and containing a mixture of complexing agents has the functionality of a guest and a host. 8. The composition according to one of claims 5, 6 or 7, characterized in that the host functionality is selected from cyclodextrin, carcerand, cavitand, crown ether, cryptand, cucurbituril, calixarene, spherand, or any combination thereof. 9. The composition according to one of paragraphs.5, 6 or 7, characterized in that the complexing agent further comprises a spacer group. 10. The composition according to one of paragraphs.5, 6 or 7, characterized in that the guest functionality is selected from adamantane, diadamantane, naphthalene and cholesterol. 11. The composition of claim 10, wherein the host functionality is cyclodextrin, and the guest functionality is adamantane or diadamantane. 12. The composition according to one of claims 4 to 7, characterized in that the functional group is selected from a ligand, a nuclear localization signal, an endosomal release peptide, an endosomal release polymer, a second therapeutic agent, a stabilizing polymer / hydrophilic polymer for stabilization, or any combination thereof; the spacer group is selected from a single bond, a phosphate group, a polyethylene glycol and a short anionic peptide sequence. 13. The composition according to one of claims 4 to 7, characterized in that the therapeutic agent is selected from an antibiotic, steroid, polynucleotide, low molecular weight pharmaceutical substance, virus, plasmid, peptide, peptide fragment, chelating agent, biologically active macromolecules and any combination thereof. 14. The composition according to item 13, wherein the therapeutic agent is a polynucleotide. 15. The composition according to claim 4, characterized in that the polymer is a cyclodextrin-containing polymer, and the complexing agent further comprises a guest guest selected from adamantane and diadamantane. 16. The composition according to clause 15, wherein the therapeutic agent is selected from an antibiotic, steroid, polynucleotide, low molecular weight pharmaceutical substance, virus, plasmid, peptide, peptide fragment, chelating agent, biologically active macromolecules and any combination thereof. 17. The composition according to clause 16, wherein the therapeutic agent is a polynucleotide. 18. The composition according to p. 15, characterized in that the complexing agent is a compound of the formula:

Where J is -NH-, -C (= O) NH- (CH ₂ ) _d -, -NH-C (= O) - (CH ₂ ) _d -, -CH ₂ SS-, -C (= O) O - (CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,

or —NH— (C = O) —CH (R ¹ ) —NH— (C = O) —CH (R ¹ ) —NH—; Ad is adamantyl; R ¹ is - (CH ₂ ) _a —CO ₂ H, an ester or salt thereof; or - (CH ₂ ) _a —CONH ₂ ; PEG (PEG) means —O (CH ₂ CH ₂ O) _z -, where z varies from 2 to 300; L is H, -NH ₂ , -NH- (C = O) - (CH ₂ ) _e - (C = O) -CH ₂ -, -S (= O) ₂ -HC = CH ₂ -, -SS- , —C (═O) O— or a carbohydrate residue; a is 0 or 1; b is 0 or 1; d has a value in the range from 0 to 6; e matters in the range from 1 to 6; n has a value in the range from 0 to 6; y is 1; and x is 1. 19. The composition according to claim 4, characterized in that the polymer is a cyclodextrin-containing polymer, and the complexing agent contains an inclusion guest, which is a compound according to claim 4 or 5. 20. The composition according to claim 19, wherein the therapeutic agent is selected from an antibiotic, steroid, polynucleotide, low molecular weight pharmaceutical substance, virus, plasmid, peptide, peptide fragment, chelating agent, biologically active macromolecules, or any combination thereof. 21. The composition according to claim 20, characterized in that the therapeutic agent is a polynucleotide. 22. The method of preparing a composition according to claim 4, comprising the step of combining a therapeutic agent, a polymer and a complexing agent to form a composition, wherein the polymer and therapeutic agent form a powdery composite, and the polymer and complexing agent form an inclusion complex. 23. The method according to item 22, wherein the therapeutic agent is first combined with the polymer to form a powdery composite, and then the powdery composite is combined with a complexing agent so that the polymer and complexing agent form an inclusion complex. 24. The method according to item 22, wherein the polymer is first combined with a complexing agent to form an inclusion complex and the inclusion complex is combined with a therapeutic agent so that the polymer and therapeutic agent form a powdery composite. 25. A method for delivering a therapeutic substance, comprising the step of administering to a person in need of a therapeutic agent, a therapeutically effective amount of a composition according to claims 4, 5 or 7. 26. A method for delivering a therapeutic substance, comprising the step of administering to a person in need of a therapeutic agent, a therapeutically effective amount of a composition according to claims 15-18.

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