Compositions containing inclusion complexes

专利摘要:
The present invention provides a composition containing a polymer, a therapeutic drug, and a complexing agent. The polymer interacts with the complexing agent in host-guest or guest-host interactions to form inclusion complexes. The compositions of the present invention can be used to deliver therapeutic drugs in the treatment of various diseases. Polymers and complexing agents of the particulate mixture may be used to introduce functional groups into the therapeutic composition. The invention also relates to a method of preparing the composition. The method consists of combining the therapeutic drug, a polymer bearing host / guest functional groups, and a complexing agent bearing guest / host functional groups to form a therapeutic composition. The complexing agent and the polymer form an inclusion complex. The present invention also relates to a method of delivering a therapeutic drug. According to this method, a therapeutically effective amount of a therapeutic composition according to the present invention is administered to a mammal (eg, a human or animal) in need of the therapeutic drug.
公开号:KR20030081354A
申请号:KR10-2003-7008188
申请日:2001-12-19
公开日:2003-10-17
发明作者:수지에 황푼；헥토르 곤잘레즈；마크이. 데이비스；나탈리에 벨로로크；지안준 쳉
申请人:캘리포니아 인스티튜트 오브 테크놀로지；인설트 테라페틱스, 인코퍼레이티드；
IPC主号:

专利说明:

COMPOSITIONS CONTAINING INCLUSION COMPLEXES
[2] Cyclodextrins are cyclic polysaccharides bearing naturally occurring D (+)-glucopyranose units with α- (1,4) bonds. The most common cyclodextrins are alpha (α) -cyclodextrin, beta (β) -cyclodextrin and gamma (γ) -cyclodextrin, which each have six, seven or eight glucopyranose units. Structurally, the cyclodextrin's cyclic nature takes a toric or donut-like form, an apolar or hydrophobic cavity inside, a secondary hydroxyl group located on one side of the cyclodextrin toric, and one on the other side. Possesses primary hydroxyl groups. Thus, cyclodextrins are schematically represented as follows, taking (β) -cyclodextrin as an example:
[3]
[4] The portion where the secondary hydroxyl group is located has a wider diameter than the portion where the primary hydroxyl group is located. Hydrophobicity of the cyclodextrin internal cavity allows for the inclusion of various 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); FR 665 169).
[5] Cyclodextrins form inclusion complexes with various drugs that may be suitable for the hydrophobic cavities of the cyclodextrins, or non-covalent complexes with other biologically active molecules, such as oligonucleotides and derivatives thereof, for delivery of various therapeutic drugs. Is being used. For example, US Pat. No. 4,727,064 describes a pharmaceutical formulation consisting of a drug with substantially low water solubility and an amorphous water soluble cyclodextrin-based mixture. The drug forms an inclusion complex with the cyclodextrin of the mixture. US Pat. No. 5,691,316 describes a cyclodextrin cell delivery system for oligonucleotides. In such a system, the oligonucleotides may form a non-covalent complex with the cyclodextrin, or alternatively the oligonucleotide may covalently bind to adamantane which non-covalently binds with the cyclodextrin.
[6] Various cyclodextrin-bearing polymers and methods for their preparation are also known in the art (Comprehensive Supramolecular Chemistry, Volume 3, JL Atwood et al., Eds., Pergamon Press (1996)). Processes for producing polymers are described in US Pat. No. 5,608,015. According to this process, cyclodextrin derivatives include α, β-unsaturated acids or acid halide monomers of derivatives thereof, or α, including terminal isocyanate functional groups or derivatives thereof. The cyclodextrin derivatives are obtained by reacting cyclodextrins with compounds such as carbonyl glides and acid anhydrides, and the resulting polymers are cyclodextrins with side chains separated from the acid polymer main chain. Hold the unit.
[7] U.S. Patent 5,276,088 describes a process for synthesizing cyclodextrin polymers by reacting polyvinyl alcohol or derivatives thereof with cyclodextrin derivatives or by copolymerizing the cyclodextrin derivatives with vinyl acetate or methyl methacrylate. The resulting cyclodextrin polymer has a cyclodextrin component as a pendant component separated from the main chain of the polymer.
[8] Biodegradable drug polymer assemblies with supramolecular structures are described in WO 96/09073 A1 and US Pat. No. 5,855,900. The assembly comprises a plurality of drug-containing cyclic compounds made by binding the drug to α, β or γ-cyclodextrin, and then arranging the drug / cyclodextrin compound along the linear polymer with biodegradable components bound to both ends of the polymer. It contains. This assembly is reported to be capable of releasing drugs in response to specific biodegradation resulting from the disease. In general, these assemblies are referred to as "necklace" cyclodextrin polymers.
[9] However, there is still a need in the art for more effective non-viral delivery systems that exhibit properties such as, for example, increased stability (eg, under physiological conditions) and effective targeting capabilities. The present invention fulfills these needs.
[10] Summary of the Invention
[11] The present invention provides a composition containing a polymer, a therapeutic drug, and a complexing agent. The polymer interacts with the complexing agent in host-guest or guest-host interactions to form inclusion complexes. The compositions of the present invention can be used to deliver therapeutic drugs in the treatment of various diseases. Polymers and complexing agents can be used to introduce functionality into the composition.
[12] The present invention provides a composition comprising a particulate mixture of a polymer and a therapeutic drug and an encapsulation composite of the polymer and a complexing agent. The polymer of the particulate mixture can retain host functional groups and form an encapsulation complex with the guest complexing agent. Alternatively, at least one polymer of the particulate mixture retains guest functional groups and forms inclusion complexes with the host complexing agent. In other embodiments, the polymer or complexing agent may have both host and guest functional groups forming an inclusion complex. This allows multiple complexing agents to form inclusion complexes that can be combined with the therapeutic composition. This also allows multiple functional groups to be incorporated into the therapeutic compositions of the invention.
[13] The invention also relates to a method of preparing the therapeutic composition. The method consists of combining the therapeutic drug, a polymer bearing host / guest functional groups, and a complexing agent bearing guest / host functional groups to produce a therapeutic composition. The complexing agent forms an inclusion complex with the polymer.
[14] The present invention also relates to a method of delivering a therapeutic drug. According to this method, a therapeutically effective amount of a composition according to the invention is administered to a mammal (eg, a human or an animal) in need of a therapeutic drug. Accordingly, the present invention provides for the treatment of diseases using the compositions of the present invention to deliver appropriate therapeutic agents.
[1] The present invention relates to compositions and methods for use in delivering a therapeutic drug. More specifically, the present invention relates to compositions containing polymers, therapeutic agents, and complexing agents, wherein the polymers interact with the complexing agent in host-guest or guest-host interactions. Act to form the encapsulation complex. The compositions of the present invention can be used to deliver therapeutic drugs in the treatment of various diseases.
[15] In the drawings illustrating various embodiments of the invention, compound (12) is βCDP6. Mixtures containing nucleic acids and cationic polymers in the particulate mixture are called polyplexes. Brief description of the drawings is as follows.
[16] 1 depicts the structure of various adamantane-PEG molecules.
[17] 2 shows the hydrodynamic diameter (Example 30) of GALA and GALA-Ad modified compositions.
[18] 3 shows the zeta potential (Example 32) of GALA and GALA-Ad modified compositions.
[19] 4 shows the uptake of GALA and GALA-Ad modified compositions by Example BHK-21 cells (Example 31).
[20] 5 depicts uptake of GALA and GALA-Ad modified compositions by HUH-7 cells (Example 33).
[21] FIG. 6 depicts luciferase transfection (Example 34) of BHK-21 cells with 12-based compositions modified with GALA and GALA-Ad modified β-cyclodextrin-DMS copolymer.
[22] 7 shows the toxicity of GALA and GALA-Ad modified polyplexes against BHK-21 cells (Example 35).
[23] 8 shows a pegylation scheme (Example 39) after DNA-complexation by grafting.
[24] 9 shows particle size (Example 39) of PEI and 12 (βCDP6) polyplexes after DNA-complexing.
[25] FIG. 10 shows stabilization of polyplex compositions by PEGylation (Example 40).
[26] 11 shows co-delivery (Example 42) of 12 polyplexes with PEG ₃₄₀₀ -FITC.
[27] 12 shows the structure of Lactose-12 (Example 37).
[28] FIG. 13 shows transfection of 12 and LAC-CDP6 polyplexes with HUH-7 cells (Example 43).
[29] 14 shows an overview (Example 47) of an experimental protocol.
[30] 15 shows particle diameters (Example 47).
[31] 16 depicts DNA loss by complex precipitation (Example 47).
[32] 17 shows the inclusion complex (Example 48) changing the 12 / DNA mixture.
[33] 18 depicts transfection of modified polyplexes into HepG2 cells (Example 49).
[34] 19 shows a competitive substitution experiment (Example 52).
[35] 20 shows the synthesis of Adamantane-PEG-transferrin (Ad-PEG-Tf) (Example 55).
[36] FIG. 21 shows ion loading for transferrin (Example 55).
[37] FIG. 22 depicts binding affinity transferrin-PEG-Ad (Example 55).
[38] 23 depicts transferrin binding via lysine functional groups (Example 56).
[39] 24 depicts the binding affinity of transferrin-PEG-AD to transferrin receptors in PC3 cells (Example 57).
[40] FIG. 25 shows zeta potential change and particle size (Example 58) as a function of particle modification in transferrin and PEG-modified polyplexes.
[41] FIG. 26 shows zeta potential measurement, Ad-anionic-PEG (Example 62).
[42] 27 shows stability measurement (Example 62).
[43] 28 shows the addition of increasing transferrin complexing agent (Example 62).
[44] 29 shows the synthesis of histylated 12.
[45] 30 shows pH-sensitive polymers (synthesis of secondary amine-containing polymers) for endosome escape.
[46] The present invention relates to compositions that use inclusion complexes to deliver therapeutic agents. Inclusion complexes are molecular compounds with the characteristic structure of adducts, where one of the compounds (host molecule) spatially surrounds at least a portion of the other compound. Enclosed compounds (guest molecules) are located in the cavity of the host molecule without affecting the skeletal structure of the host. It is a feature of the encapsulation composite that the size and shape of the available cavities does not substantially change regardless of some modification. A "host" can be any host compound or molecule known in the art. Examples of suitable hosts include cyclodextrins, carcerands, cavitands, crown ethers, cryptands, cucurbituril, calixarenes, spe Lands, etc., but is not limited to these. Suitable encapsulation guests for complexing agents are known in the art and include, but are not limited to, adamantane, diadamantane, naphthalene, cholesterol, and the like.
[47] Cyclodextrins are suitable hosts that can interact with various ions and molecular types, and the resulting inclusion compounds belong to the group of "host-guest" complexes. Several requirements must be met to realize host-guest correlation; One of these is that the binding sites of the host and guest molecules must be complementary in terms of stereoelectronics. Cyclodextrins may form inclusion complexes with compounds having a size appropriate to the dimensions of the cavity. However, the degree of complex formation depends on the polarity of the guest molecule. In addition, complex formation with molecules significantly larger than the cavities is possible in such a way that specific functional groups or side chains penetrate carbohydrate channels (J. Szejtli, Akademial Kiado, Cyclodextrins and their inclusion complexes, Budaoest, 1982).
[48] The composition of the present invention contains at least one polymer and at least one therapeutic drug in the form of a particulate mixture of the polymer and the therapeutic drug. In addition, the therapeutic composition contains one or more complexing agents. At least one polymer of the particulate mixture interacts with the complexing agent in a host-guest or guest-host interaction to form an inclusion complex between the polymer and the complexing agent. Polymers and complexing agents can be used to introduce functional groups into the compositions of the present invention. In one embodiment, at least one polymer of the particulate mixture forms an inclusion complex with a complexing agent that retains host functional groups and retains guest functional groups. In another embodiment, at least one polymer of the particulate mixture forms an inclusion complex with a complexing agent that retains guest functional groups and retains host functional groups. In another embodiment, the polymer of the particulate mixture retains both host and guest functional groups and forms encapsulation complexes with the guest complexing agent and host complexing agent.
[49] 1. Particulate Mixture
[50] The particulate mixture of the therapeutic drug and the polymer is a combination or integration of the therapeutic drug and the polymer. The particulate mixture is a linking structure containing one or more therapeutic agents in a multi-dimensional polymer network. Homopolymers or polymer mixtures may be used. At least one polymer in the mixture not only can form a multi-dimensional polymer network of the particulate mixture, but also has host and / or guest functional groups capable of forming inclusion complexes with one or more complexing agents, as described below.
[51] A. Polymer
[52] Any type of polymer capable of forming a particulate mixture with the therapeutic drug and having host and / or guest functional groups can be used in the compositions of the present invention. The polymer is a linear or branched polymer. The polymer may be a homopolymer or a copolymer. The copolymer used may be any copolymer or branched copolymer. The polymer is preferably dispersible in water, more preferably water soluble. For example, suitable polymers include polysaccharides, polyesters, polyamides, polyethers, polycarbonates, polyacrylates, and the like. Therapeutic polymers have a low toxicity profile, preferably no toxicity or cytotoxicity. As described below, suitable polymers for use in the present invention are cyclodextrin-based polymers. The water soluble linear cyclodextrin copolymer described below preferably has a molecular weight of 3,000 to 100,000, more preferably 3,000 to 50,000.
[53] According to the invention, the polymer in the particulate mixture may be a single polymer or a mixture of two or more polymers, the same or different. Each polymer of the particulate mixture further retains or is further modified to retain such crosslinking groups, through which the bonding of the polymers forming the particulate mixture can be achieved.
[54] At least one polymer of the particulate mixture is a polymer capable of forming an inclusion complex. "Polymers capable of forming inclusion complexes" include non-covalent bonds (eg, van der Waals forces, hydrogen bonds, dipole-dipole interactions, ion-bonds, hydrophobic interactions, etc.). ) May be any polymer capable of achieving one or more host-guest bonds with other compounds (complexing agents) or substituents on the compound. In other words, at least one polymer bears host / guest functional groups and forms inclusion complexes with the complexing agent or substituents on the complexing agent. Host / guest functional groups may be part of the polymer backbone, or may be present in substituents or in suspended or branched chains. An example of a polymer having a host functional group in the polymer backbone is the linear cyclodextrin polymer described below. An example of a polymer having a guest functional group that is not part of the polymer backbone is a polymer having pendant adamantane functional groups. Other examples of "hosts" suitable for use as polymers include carceronds, cavitands, crown ethers, cryptands, cucurbiturils, calixarenes, spetses Spherand and the like. Suitable encapsulation guests for such hosts are known to those skilled in the art, and include, but are not limited to, adamantane, diadamantane, naphthalene, cholesterol, and the like.
[55] In suitable embodiments, the polymer may have different types of host / guest functional groups, or the polymer may have both host and guest functional groups. This allows for more flexibility for the discrete encapsulation composites formed in certain polymers. Retaining both host and guest functional groups in multiple hosts, multiple guests, or the same polymer increases the variety of functional groups introduced into the therapeutic composition of the present invention through inclusion complexes.
[56] As a result of the host-guest bond, the polymer interacts with the complexing agent to form an inclusion complex. Suitably, the inclusion complexes resulting from non-covalent interactions or bindings exhibit binding constants of approximately> 10 ² , preferably> 10 ³ , more preferably> 10 ⁴ . Typically, the binding constant is 10 ² -10 ⁶ .
[57] The polymer of the particulate mixture can be modified with one or more ligands. The ligand can be introduced after formation of the particulate mixture via ligand modification of the polymer of the therapeutic drug and / or the particulate mixture. The ligand can be any ligand that enables targeting and / or binding to the desired cell. As will be appreciated by those skilled in the art, targeting and binding to cells include cellular receptor attachment, which can lead to receptor mediated endocytosis. If two or more ligands are attached, these ligands may be the same or different. Examples of suitable ligands include, but are not limited to, vitamins (eg folic acid), proteins (eg transferrin and monoclonal antibodies), monosaccharides (eg galactose), peptides, polysaccharides. As will be appreciated by those skilled in the art, the choice of ligand depends on the type of delivery desired. Alternatively, the ligand may be a membrane penetrable agent such as the TAT protein of HIV-1. TAT protein is a viral transcriptional activator that actively enters the cell nucleus (Torchilin, V. P. et al, PNAS. 98, 8786-8791, (2001)).
[58] In a suitable embodiment of the present invention, at least one of the polymers of the particulate mixture is a substantially linear polymer having host and / or guest functional groups capable of forming inclusion complexes. Substantially linear polymers can be made by any method known in the art. The polymer may be made from suitable monomers capable of forming inclusion complexes, or monomer mixtures in which at least one of the monomers has a host / guest functional group. The host / guest functional group may be present in the polymer chain, in a suspended (or branched) form, or as an end group in the polymer chain. Alternatively, after the polymer is formed, it can be made to be a substantially linear polymer capable of forming an inclusion complex by modifying it to retain host and / or guest functional groups as described above. The substantially linear polymer may be a block copolymer, where the block introduces properties such as host functional groups, water-dispersible and / or water soluble. Examples of such blocks include linear polyethyleneimine (PEI), linear cyclodextrin-bearing polymers, bis (2-aminoethyl) -1,3-propanediamine (AEPD), N ₂ , N ₂ , N ₃ , N _3- ( 3′-PEG ₅₀₀₀ aminopropane) -bis (2-aminoethyl) -1,3-propanediammonium-di-trifluoroacetate (AEPD-PEG).
[59] In another suitable embodiment, the polymer used to form the particulate mixture is a cyclodextrin-bearing polymer, more preferably a substantially linear cyclodextrin polymer described below. The polymer may be a polymer having polyethyleneimine (PEI) or pendant cyclodextrin. Linear cyclodextrin copolymers are polymers that contain a cyclodextrin component as an integral part of the polymer backbone. Polymers having a cyclodextrin component that is not part of the main polymer chain and is attached in a form separated from the polymer backbone may also be used in the compositions of the present invention. A linear cyclodextrin-retained polymer is part of the polymer backbone and at least one cyclodextrin component. May be any linear polymer having Suitably, the cyclodextrin-bearing polymer is water soluble. More suitably, the linear cyclodextrin-bearing polymer is a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer described below. Cyclodextrin functionality in the polymer provides a host functional group that allows the polymer to form inclusion complexes. Substantially linear polymers capable of forming inclusion complexes may further retain other functional groups (eg, thiol groups), or may be further modified to retain them.
[60] Linear Cyclodextrin-Holded Polymers
[61] Linear cyclodextrin copolymers that can be used to form particulate mixtures are divalent linking cyclodextrins of linear cyclodextrin polymers through positions 2, 3 or 6 of one or more glucopyranose rings of cyclodextrins in the linear copolymer backbone. retains a substituted or unsubstituted cyclodextrin component that binds a difunctional component to a bifunctional group. As found in WO 00/01734, such linear cyclodextrin copolymers have repeat units of formula la, lb or combinations 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., Bellocq, N. and Davis, M. (2001) Effects of structure of Beta-Cyclodextrin-Containing Polymers on Gene Delivery. Bioconjugate Chem, 12 (2), 280-290.
[62]
[63]
[64] In formulas (Ia) and (Ib), C is a substituted or unsubstituted cyclodextrin monomer, and A is a comonomer covalently bonded to cyclodextrin C. Polymerization of the cyclodextrin monomer C precursor and comonomer A precursor results in a linear cyclodextrin copolymer. Within a single linear cyclodextrin copolymer, the cyclodextrin monomer C units may be the same or different and the comonomer A may be the same or different.
[65] The cyclodextrin monomer precursor can be any cyclodextrin or derivative thereof known in the art. As mentioned above, cyclodextrins are cyclic polysaccharides having 6 to 8 naturally occurring D (+)-glucopyranose units with α- (1,4) bonds. Suitably, the cyclodextrin monomer precursors are cyclodextrins each having six, seven or eight glucose units, ie alpha (α) -cyclodextrin, beta (β) -cyclodextrin, gamma (γ)- Cyclodextrin. The cyclodextrin derivative may be any substituted cyclodextrin known in the art, wherein the substituent does not interfere with the copolymerization with the comonomer A precursor as described below. Cyclodextrin derivatives may be neutral, cationic or anionic. Examples of suitable substituents include hydroxyalkyl groups such as hydroxypropyl, hydroxyethyl; Ether groups such as dihydroxypropyl ether, methyl-hydroxyethyl ether, ethyl-hydroxyethyl ether, ethyl-hydroxypropyl ether; Alkyl groups such as methyl; Saccharides such as glucosyl and maltosyl; Acid functional groups such as carboxylic acid, phosphorous acid, phosphinous acid, phosphonic acid, phosphoric acid, thiophosphonic acid, sulfonic acid; Imidazole group; Sulfate group; Protected thiol groups are included.
[66] The cyclodextrin monomer precursor can be further modified chemically to promote copolymerization of the cyclodextrin monomer precursor and the comonomer A precursor as described below. Chemical modification of the cyclodextrin monomer precursor allows for polymerization, i.e., formation of a bifunctional cyclodextrin component, only at two positions of each cyclodextrin component. The numbering for the C1-C6 positions of each glucopyranose is as follows:
[67]
[68] In a suitable embodiment, the polymerization proceeds at C2, C3, C6 or a combination thereof of the cyclodextrin components. For example, one cyclodextrin monomer precursor may be polymerized at two C6 positions, and another cyclodextrin monomer precursor may be polymerized at C2 and C6 positions of the cyclodextrin component. The letter designation for the relative position of each glucopyranose ring in the cyclodextrin is (for β-cyclodextrin):
[69]
[70] In a suitable embodiment of the linear cyclodextrin copolymer, the cyclodextrin monomer C has formula II:
[71]
[72] In formula (II), n and m are integers that define the total amount of glucopyranose units in the cyclodextrin monomer along with the other two glucopyranose rings. Formula II represents a cyclodextrin monomer that can be polymerized at two C6 positions of a cyclodextrin unit. Examples of cyclodextrin monomers of formula (II) include 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 ^A , 6 ^c -dideoxy-γ-cyclodextrin (n = 1, m = 5), 6 ^A , 6 ^D -dide Oxy-γ-cyclodextrin (n = 2, m = 4), 6 ^A , 6 ^E -dideoxy-γ-cyclodextrin (n = 3, m = 3), but are not limited to these.
[73] In another suitable embodiment, the linear cyclodextrin copolymer can have a glucose-ring-opened cyclodextrin monomer C unit, wherein at least one glucopyranose ring of the cyclodextrin is open while maintaining the cyclodextrin ring system. Formula III represents glucopyranose-ring-opened cyclodextrins having ring openings at positions C2, C3:
[74]
[75] P is 5 to 7 in formula (III). In formula (III), at least one D (+)-glucopyranose unit of the cyclodextrin monomer undergoes ring opening to allow polymerization at the C2 and C3 positions of the cyclodextrin unit. Cyclodextrin monomers of formula III, such as 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 commercially available from Carbomer (Westborough, Mass.). Examples of cyclodextrin monomers of formula III include 2 ^A , 3 ^A -dideoxy-2 ^A , 3 ^A -dihydro-α-cyclodextrin, 2 ^A , 3 ^A -dideoxy-2 ^A , 3 ^A -dihydro- β-cyclodextrin, 2 ^A , 3 ^A -dideoxy-2 ^A , 3 ^A -dihydro-γ-cyclodextrin (2,3-dideoxy-α-cyclodextrin, 2,3-dideoxy-β, respectively -Cyclodextrin, 2,3-dideoxy-γ-cyclodextrin), but is not limited to these.
[76] Comonomer A precursors are any straight-chain or branched symmetrical or asymmetric compound that connects two cyclodextrin monomers to each other immediately after reaction with the cyclodextrin monomer precursor as described above. Suitably, the comonomer A precursor is a compound having at least two crosslinking groups that achieve the reaction and thus the binding of the cyclodextrin monomers. Examples of possible crosslinking groups which are the same or different and which are terminally or internally of each comonomer A precursor include, but are not limited to, amino, acid, ester, imidazole and acyl halide functional groups and derivatives thereof. In suitable embodiments, the two crosslinkers are identical and located at the ends. Immediately after copolymerization of the comonomer A precursor with the cyclodextrin monomer precursor, the two cyclodextrin monomers combine the primary hydroxyl portion of one cyclodextrin monomer with the primary hydroxyl portion of the other cyclodextrin monomer, or one cyclodextrin The second hydroxyl portion of the monomers and the secondary hydroxyl portion of the other cyclodextrin monomers can be joined, or the primary hydroxyl portion of one cyclodextrin monomer and the secondary hydroxyl portion of the other cyclodextrin monomer can be linked to each other. Thus, combinations of such bonds may be present in the final copolymer.
[77] Both comonomer A precursor and comonomer A of the final copolymer are neutral, cationic (e.g. have a proton-receiving functional group such as a quaternary ammonium group) or anionic (e.g. protons such as sulfate, phosphate or carbohydrate anionic functional groups) Retain the lost functional group). The counterion of the charged comonomer A precursor or comonomer A may be any suitable counterion or counter cation (e.g., the counterion of the cationic comonomer A precursor or comonomer A is a halide (e.g., , Chloride) anions). The charge of comonomer A in the copolymer can be adjusted by adjusting the pH conditions.
[78] Examples of suitable comonomer A precursors include cystamine, 1,6-diaminohexane, diimidazole, dithioimidazole, spermine, dithiospermine, dihistidine, dithiohistidine, succinimide (eg , Dithiobis (succinimidyl propionate) (DSP) and disuccinimidyl suverate (DSS), imidate (e.g., dimethyl 3,3'-dithiobispropion-imideate (DTBP)) It is not limited to these.
[79] Copolymerization of the comonomer A precursor with the cyclodextrin monomer precursor leads to the formation of a linear cyclodextrin copolymer having a comonomer A linkage of the formula:
[80]
[81]
[82]
[83]
[84] In the above formula, x = 1-50, y + z = x. Preferably, x = 1-30. More preferably x = 1-20. In suitable embodiments, comonomer A has a biodegradable bond, such as a disulfide bond. In addition, comonomer A includes acid-labile functional groups (eg esters) and other acid-labile functional groups known to those skilled in the art.
[85] In other suitable embodiments, comonomer A precursor and comonomer A may be selectively selected to achieve the desired purpose. For example, charged polymers are not necessary to deliver small molecule therapeutic drugs, and comonomer A may be or have a hydrophilic functional group, such as polyethylene glycol, to further enhance water solubility. In the case of a polypeptide therapeutic drug, such as DNA or protein, comonomer A carries a cationic charge that enhances the ability of the linear cyclodextrin copolymer to form a particulate mixture with the polypeptide therapeutic drug. In addition, the linear cyclodextrin copolymer may have a mixture of comonomer A functional groups.
[86] Linear cyclodextrin copolymers can be made by copolymerizing a cyclodextrin monomer precursor with a suitable leaving group distributed to a comonomer A precursor that can substitute for the leaving group. The same or different leaving group may be any leaving group known in the art, which may be substituted immediately after copolymerization with the comonomer A precursor.
[87] The linear cyclodextrin copolymers iodineize the cyclodextrin monomer precursors to form a diiodinated cyclodextrin monomer precursor and copolymerize the biiodated cyclodextrin monomer precursor with a comonomer A precursor to formula Ia, Ib, or a combination thereof as described above. To form a linear cyclodextrin copolymer having a repeating unit of.
[88] Another method of preparing linear cyclodextrins is to iodide the cyclodextrin monomer precursors, as described above, to form biiodated cyclodextrin monomer precursors of Formula IVa, IVb, IVc, or mixtures thereof:
[89]
[90]
[91]
[92] Biiodinated cyclodextrins can be made by methods known in the art (Tabusfi et al. J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al. J. Am. Chem. 106, 4580-). 4584 (1984)). For example, β-cyclodextrin is reacted with biphenyl-4,4'-disulfonyl chloride in the presence of anhydrous pyrimidine to form a biphenyl-4,4'-disulfonyl chloride added β-cyclodextrin, which is iodide It can be reacted with potassium to produce biiodine-β-cyclodextrin. Cyclodextrin monomer precursors are iodinated only at two sites. As described above, the diiodinated cyclodextrin monomer precursor can be copolymerized with a comonomer A precursor to produce a linear cyclodextrin polymer having repeat units of Formulas Ia, Ib, or a combination thereof. Where appropriate, the iodine or iodine group may be substituted with other known leaving groups.
[93] An iodine group or other suitable leaving group may be substituted with a functional group that allows reaction with the comonomer A precursor as described above. For example, the diiomidated cyclodextrin monomer precursor of Formulas IVa, IVb, IVc, or mixtures thereof can be aminated to form a biaminoated cyclodextrin of Va, Vb, Vc, or mixtures thereof:
[94]
[95]
[96]
[97] Biaminolated cyclodextrin monomer precursors can be made by any means known in the art (Tabusfi et al. J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al. J. Am. Chem 106, 4580-4584 (1984). For example, biiodine-β-cyclodextrin can be reacted with sodium azide and then reduced to form a doubleamino-β-cyclodextrin. Cyclodextrin monomer precursors are only aminoated at two positions. As described above, the diaminolated cyclodextrin monomer precursor can be copolymerized with a comonomer A precursor to produce a linear cyclodextrin polymer having repeat units of Formulas Ia, Ib, or a combination thereof. However, the amino functional group of the biaminolated cyclodextrin monomer precursor does not need to be attached directly to the cyclodextrin component.
[98] Alternatively, ^- SCH ₂ CH ₂ with an amino group of pictures of the same component with NH ₂ cyclodextrin monomer precursor of iodine or by introducing an amino functionality substituted groups other suitable leaving formula Ⅴd, Ⅴe, Ⅴf, Ⅴg, Ⅴh, Ⅴi or Mixtures of these can form biaminoated cyclodextrin monomer precursors:
[99]
[100]
[101]
[102]
[103]
[104]
[105] The linear cyclodextrin copolymer can also be made by reducing the linear oxidized cyclodextrin copolymer as described below. This method can be carried out when the comonomer A does not have a reducing component or a functional group such as disulfide bond.
[106] The linear cyclodextrin copolymer can be oxidized by introducing at least one oxidizing cyclodextrin monomer into the copolymer such that the oxidizing cyclodextrin copolymer is an integral part of the polymer backbone. Linear cyclodextrin copolymers having at least one oxidized cyclodextrin monomer are defined as linear oxidized cyclodextrin copolymers. The linear oxidized cyclodextrin is then a bifunctional component (mainly component A) that connects the cyclodextrin of the linear cyclodextrin polymer via positions 2, 3 or 6 of one or more glucopyranose rings of the cyclodextrin in the linear copolymer backbone. To a substituted or unsubstituted cyclodextrin component that is bound to a bifunctional group, wherein the glucopyranose ring of the cyclodextrin component is oxidized. Cyclodextrin monomers can be oxidized at the secondary or primary hydroxyl sites of the cyclodextrin component. If one or more oxidizing cyclodextrin monomers are present in the linear oxidizing cyclodextrin copolymer, the same or different oxidizing cyclodextrin monomers may be present in the primary hydroxyl moiety, the secondary hydroxyl moiety, or both. For example, a linear oxidized cyclodextrin copolymer bearing oxidized secondary hydroxyl groups has at least one unit of formula VIa or VIb:
[107]
[108]
[109] In formulas VIa and VIb, C is a substituted or unsubstituted oxidized cyclodextrin monomer and A is a comonomer covalently bonded to oxidized cyclodextrin C. In the formulas VIa and VIb, oxidation of the secondary hydroxyl group leads to ring opening of the cyclodextrin component and generation of aldehyde groups.
[110] The linear oxidation cyclodextrin copolymer can be made by oxidation of the linear cyclodextrin copolymer as described above. Oxidation of linear cyclodextrin copolymers can be accomplished by oxidation techniques known in the art (Hisamatsu et al., Starch 44: 188-191 (1992)). Suitably, an oxidant such as sodium peroxide is used. As will be appreciated by those skilled in the art, the degree of oxidation under standard oxidation conditions varies from copolymer to copolymer. Thus, in one embodiment the linear oxidation copolymer can have one oxidized cyclodextrin monomer. In other embodiments, substantially all cyclodextrin monomers of the copolymer can be oxidized.
[111] Other methods of preparing linear oxidized cyclodextrin copolymers include the oxidation and comonomer A of a biiodated or diaminoated cyclodextrin monomer precursor to form an oxidized diiodinated or biaminoated cyclodextrin monomer precursor as described above. A copolymerization step of a iodinated or biaminoated cyclodextrin monomer precursor oxidized to a precursor. In suitable embodiments, the oxidized diiodinated cyclodextrin monomer precursor of formula (VIIa), (VIIb), (VIIc) or mixtures thereof:
[112]
[113]
[114]
[115] Oxidized cyclodextrin monomers can be made by oxidation of the biiodated cyclodextrin monomer precursor of Formulas IVa, IVb, IVc, or mixtures thereof, as described above. In another embodiment, the oxidized biaminoated cyclodextrin monomer precursor of formula (VIIa), (b), (c) or mixtures thereof is prepared by amination of the oxidized biiodated cyclodextrin monomer precursor of formula (VIIa), (b), (c) or mixtures thereof. Can make:
[116]
[117]
[118]
[119] In another embodiment, the oxidized biaminoated cyclodextrin monomer precursor of formula (VIIa), (b), (c), (d), (d), (f), or mixtures thereof is selected from the group consisting of iodine, other suitable leaving group ^- can be made by substituting an amino group which have a SCH ₂ CH ₂ NH ₂ components.
[120]
[121]
[122]
[123]
[124]
[125]
[126] Alternatively, the oxidized diiodic dicarboxylic acid or biaminoated cyclodextrin monomer precursor as described above oxidizes the cyclodextrin monomer precursor to form an oxidized cyclodextrin monomer precursor, which then doubles the oxidized cyclodextrin monomer. It can be made by iodide and / or diaminoation. The amine groups of any biaminoated oxidized cyclodextrin monomer may be present in protected form to avoid unwanted side effects. As mentioned above, the cyclodextrin component may be modified with other leaving groups and other amino group-containing functional groups except for the iodine group. The oxidized diiodinated or diaminoated cyclodextrin monomer precursor can then be copolymerized with a comonomer A precursor to form a linear oxidized cyclodextrin copolymer.
[127] The linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer terminates with at least one comonomer A precursor or a hydrolyzate of comonomer A precursor. As a result of terminating the cyclodextrin copolymer with at least one comonomer A precursor, there is one free derivatizing group per linear cyclodextrin copolymer or linear oxidized cyclodextrin copolymer as described above. For example, the derivative group can be an acid group or a derivative group that can be hydrolyzed to an acid group. In accordance with the present invention, derivatives can be further modified chemically to enhance the properties of the cyclodextrin copolymers, such as colloidal safety and transfection efficiency. For example, derivatives can be modified by reaction with PEG to form cyclodextrin copolymers terminated with PEG and increase colloidal stability, or reacted with histidine or imidazole acetic acid to form cyclodextrin copolymers terminated with imidazolyl and Intracellular (eg endosome) release and transfection efficiency can be increased (FIGS. 29 and 30).
[128] Additional chemical properties can be carried out on the cyclodextrin copolymer via modified derivatives. For example, modified leaving groups can be used to extend polymer chains by linking linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers to the same or different cyclodextrin or non-cyclodextrin polymers. The polymers added are the same or different linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers, which can also be terminated with comonomer A precursors for further modification.
[129] Alternatively, at least two identical or different linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers having a terminal derivative or a terminal modified derivative as described above may react and bind to each other via a functional group or modified derivative. have. Suitably, degradable components, such as disulfide bonds, are formed immediately after the reaction of the functional group or modified derivative. For example, modification of the end derivatives to cysteines can be used to produce linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers having free thiol groups. Reaction with the same or different cyclodextrin copolymers bearing free thiol groups also results in disulfide bonds between the two copolymers. Functional groups or modified derivatives can be selected to provide binding that exhibits different rates of degradation (eg, via enzymatic degradation), and thus provide a time control system for the therapeutic drug, if desired. The resulting polymer may be crosslinked as found herein. As noted herein, the therapeutic agent may be added before or after crosslinking of the polymer. Ligands can also be linked to the cyclodextrin copolymer via modified derivatives. For example, a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer can be modified with a ligand attached to a cyclodextrin copolymer. The ligand may be attached to the cyclodextrin copolymer via cyclodextrin monomer C or comonomer A. Suitably, the ligand is attached to the cyclodextrin component of the cyclodextrin copolymer (WO 00/01734).
[130] Branched cyclodextrin-bearing polymer
[131] In addition, the polymer of the particulate mixture bearing host and / or guest functional groups may be a substantially branched polymer such as branched polyethyleneimine (PEI) or branched cyclodextrin-bearing polymer, preferably branched cyclodextrin-bearing It may be a polymer. The branched cyclodextrin-bearing polymer may be any water soluble branched polymer having at least one cyclodextrin component, which component may be part of the polymer backbone and / or suspended from the polymer backbone. Branched cyclodextrin-bearing polymers are branched cyclodextrin copolymers or branched oxidized cyclodextrin copolymers. Branched cyclodextrin copolymers or branched oxidized cyclodextrin copolymers are linear cyclodextrin copolymers or linear oxidized cyclodextrin copolymers in which dependent chains are branched as described above. Branching dependent chains may further carry various derivatives or substituents, for example hydroxyl, amino, acid, ester, amino, keto, formyl, nitrogen groups. Branching dependent chains may also carry cyclodextrins or other host or guest functional components. Molecular dependent chains can also be modified with ligands. Such ligand modifications include, but are not limited to, attachment of ligands to cyclodextrin components in branching dependent chains.
[132] Suitably, the branched cyclodextrin-bearing polymer is a branched cyclodextrin copolymer or branched oxidized cyclodextrin copolymer, wherein the branched heterochain has a cyclodextrin component. If the branching dependent chain has a cyclodextrin component, the cyclodextrin component can promote inclusion complex formation and encapsulation of the therapeutic drug. Suitably, the cyclodextrin component of the branching dependent chain, together with the cyclodextrin component in the polymer backbone, may promote inclusion complex formation and encapsulation of the therapeutic drug. Branched cyclodextrin-bearing polymers can be made by any method known in the art, including derivatization (eg, substitution) of polymers (eg, linear or branched PEI) with cyclodextrin monomer precursors. Examples of polymers having pendant cyclodextrins 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., Maromel. Rapid Commun. 17, 731 (1996), and Bachmann, et al., J. Carbohydrate Chemistry 17, 1359 (1998) (Weickenmeier paper describes cyclodextrin side chain polyesters, their synthesis and inclusion of adamantane derivatives). ). Branched cyclodextrin-bearing polymers may be crosslinked as described above.
[133] The poly (ethyleneimine) (PEI) used in the present invention has an average molecular weight of approximately 800 to 800,000 daltons, preferably 2,000 to 100,000 daltons, more preferably 2,000 to 25,000 daltons. PEI can be linear or branched. Suitable PEI compounds are commercially available from a variety of sources, including polyethyleneimine from Aldrich Chemical Company, polyethyleneimine from Polysciences, POLYMIN poly (ethyleneimine) and LUPASOL ^™ poly (ethyleneimine) from BASF Corporation.
[134] Other host-functional polymers
[135] As mentioned above, at least one polymer of the particulate mixture is a polymer capable of forming an inclusion complex. Along with various methods of preparation, polymers having suitable cyclodextrin host functional groups have been described above. In the same way any linear or branched polymer bearing host functional groups can be used in the practice of the present invention. Other examples of "hosts" suitable for use with polymers include, but are not limited to, carvitand, crown ethers, cryptand, cucurbituril, calixarenes, and Sperland ( spherands), but not limited to these. Polymers of these other hosts can be made in the same manner as described above for cyclodextrin-bearing polymers. The host of interest can be reacted with a suitable comonomer A which derives a functional group such as a hydroxyl group to attach a leaving group such as iodine, tosylate and the like and substitutes the leaving group to form a host copolymer. Alternatively, the host may possess or be derived to possess a functional group such as an amine or carboxyl group, such that the host undergoes a condensation reaction with comonomer A and forms a host copolymer. The host copolymer can then be made to have a mixture of host functional groups in the branch when the polymer backbone and copolymer are branched.
[136] Guest functional polymer
[137] The guest functional polymer can be any polymer capable of forming an inclusion complex with a host-functional complexing agent. Typically, guest functional groups are present in the side chain or end groups. An example of a polymer having a guest functional group that is not part of the polymer backbone is a polymer having a pendant adamantane group. Examples of inclusion functional groups that can be incorporated into the polymer include, but are not limited to, adamantane, diamantane, naphthalene, cholesterol.
[138] B. Treatment Drug
[139] In accordance with the present invention, at least one therapeutic agent may be encapsulated in a polymer as described above to form a particulate mixture. A "therapeutic drug" includes any active agent used pharmacologically or therapeutically and an active compound or agent used for sterilization purposes as described below. Examples of such therapeutic agents (or active agents) are described below. Encapsulation is defined as any means by which a therapeutic agent binds to a polymer (eg, electrostatic interaction, hydrophobic interaction, actual encapsulation). The degree of binding can be measured by techniques known in the art, including fluorescence irradiation, DNA mobility irradiation, light injection, electron microscopy, depending on the therapeutic drug. For example, a therapeutic composition containing a multi-dimensional polymer network made from the polymer and DNA of the aforementioned particulate mixture in a delivery mode can be used to assist transfection, ie, the influx of DNA into animal (eg, human) cells ( 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 Polyethylenmine ", College of Pharmacy, The Ohio State University m published on web, revised manuscript July 5, 2001). If the therapeutic drug is nucleic acid-based (eg, DNA), the polymer of the therapeutic drug that forms the mixture may be in the form of a "polyplex". Polyplexes are mixtures between nucleic acids and accounting polymers (Felgner, et al. "Nomenclature for Synthetic Gene Delivery Systems". Hum. Gene Ther. 8, 511-512 (1997)).
[140] Mixtures of therapeutic agents can be used in the compositions of the present invention. Immediately after forming the particulate mixture, the therapeutic drug may or may not retain biological or therapeutic activity. Immediately after release from the polymer of the therapeutic composition, in particular the particulate mixture, the activity of the therapeutic drug is restored or in the case of a prodrug the potential for activity is restored. Thus, the particulate mixture protects the therapeutic drug from loss of activity due to degradation, for example, and provides enhanced bioavailability. As such, the compositions of the present invention can be used to provide stability, in particular storage or degradation stability, to a therapeutic drug or any active chemical compound. Encapsulation of a lipid-compatible therapeutic drug is not complete but enhances the solubility of the lipid-compatible therapeutic drug. The therapeutic agent may be further modified with a ligand prior to or after formation of the particulate composition or therapeutic composition.
[141] The therapeutic drug may be a synthetic or naturally occurring biologically active therapeutic drug of lipid affinity or hydrophilicity, including therapeutic drugs known in the art (Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, 13th Edition, 2001, Merck and Co) , Inc., Whitehouse Station, NJ). These drugs include small molecule therapeutics, antibiotics, steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA, antisense oligonucleotides, viruses, chimeric polynucleotides), plasmids, peptides, peptide fragments, small molecules (e.g. doxorubicin), Chelating agents (eg DESFERAL, ethylenediaminetetraacetic acid (EDTA)), natural products (eg taxol, amphotericin), and other biologically active macromolecules (eg proteins and enzymes). US Pat. No. 6,048,736 describes an active drug (therapeutic drug) used as a guest to form an inclusion compound with a cyclodextrin polymer. Small molecule therapeutic agents are not only therapeutic agents in the mixture particles but can also be covalently bonded to the polymer in the mixtures of other embodiments. Suitably, the covalent bonds are reversible (eg, in prodrug form, or via biodegradable bonds such as disulfide bonds) and provide other methods of delivering therapeutic agents.
[142] 2. Complexing agent
[143] According to the invention, the complexing agent is a compound having a host / guest functional group capable of forming an inclusion complex with the polymer in a particulate mixture bearing the corresponding guest / host functional group. As mentioned above, guest complexing agents can be used to modify the polymer of the particulate mixture bearing host functional groups, or the monomers of the polymer bearing host functional groups to form inclusion complexes. In addition, as described above, the host complexing agent may act as a host to the polymer guest functional group to form an encapsulation composite with at least one polymer of the particulate mixture. The complexing agent may have two or more encapsulation functional groups. For example, complexing agents having two encapsulation functional groups can be guest, guest; Host, host; Or host, guest complexing agent. The complexing agent may have a mixture of multiple host and / or guest functional groups. This functional group may be, for example, a ligand, a hydrophilic or hydrophobic functional group, another therapeutic drug, or the like. The complexing agent may also comprise a spacer functional group between the encapsulating guest / host and the functional group.
[144] Suitably, the complexing agent exhibits a binding constant of approximately> 10 ² , preferably approximately> 10 ³ , more preferably approximately> 10 ⁴ . Typically, the binding constant is 10 ² -10 ⁶ . Suitable encapsulating guests for complexing agents are known in the art and include, but are not limited to, adamantane, diadamantane, naphthalene, cholesterol, and the like (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); Amiel et al., Advances in Colloid and Interface Science, 79, 105-122 (1999); Sandier et al., Langmuir, 16, 1634-1642 (2000).
[145] Complexing agents possess functional groups that provide advantages to the compositions of the present invention. Simply adding functional groups, such as hydroxyl or amine functional groups, is one way to introduce functional groups. In suitable embodiments, the complexing agent may not only form inclusion complexes with the polymer of the particulate mixture, but may also alter the mixture to promote cell contact, trafficking and / or cellular uptake and release, for example. Any complexing agent known in the art can be used. Examples of suitable "functional" functional groups include ligands, nuclear batch signals (Zanta et al., Proc. Natl. Acad. Sci. USA, 96, pp. 91-96 (1999)), endosomal release peptides, endosomal release polymers , Membrane permeants, or mixtures thereof, but are not limited to these. The nuclear batch signal (NLS) can be any nuclear batch signal known in the art. The endosomal releasing peptide or polymer may be any endosome releasing peptide or polymer known in the art (eg, HA-2 and GALA) (“Gene delivery by negatively charged peptides” Simoes S, Slepushkin V, Gaspar R, de Lima MCP, Duzgunes N, GENE THERAPY 5: (7) 955-964JUL 1998). An example of a cell membrane penetrant (or cell membrane penetrant) is a TAT protein derived from HIV-1. TAT protein is a viral transcriptional activator that actively enters the cell nucleus (Torchilin, V. P. et al, PNAS. 98, 8786-8791, (2001)).
[146] In addition, complexing agents may be functionalized with polymers which increase solubility or provide stabilization, especially under biological conditions. Stabilization of the composition can be accomplished by the use of a complexing agent having hydrophilic or lipocompatible functional groups. Suitable types of hydrophilic functional groups are polyethylene glycols or polyethylene glycol-containing copolymers (PEG). Suitable polyethylene ethylene glycols are of the formula HO (CH ₂ CH ₂ O) _z H, wherein z is from 2 to 500, preferably 10-300. PEG ₆₀₀ , PEG ₃₄₀₀ , PEG ₅₀₀₀ represent polyethylene glycols that can be used in the present invention. In general, the higher the molecular weight of PEG in the complexing agent, the higher stabilization of the composition is achieved. Higher molecular weight PEGs are preferred. Suitable complexing agents are pegylated adamantane or pegylated diamantane. The structure of some adamantane-PEG molecules useful as complexing agents is shown in FIG. 1. In order to increase the lipid affinity (hydrophobicity), the complexing agent may comprise a lipid affinity functional group such as long chain alkyl, fatty acid or the like. The choice of a lipid affinity group depends on the degree of lipid affinity desired. In the present application, the complexing agent can be modified with any type of functional group to introduce the desired properties into the composition. Complexing agents can be made with standard organic engineering. Using a mixture of different complexing agents allows for more variation and specificity in achieving the desired composition properties.
[147] Spacer functional groups can be used to link the functional groups to the complexing agent. The spacer functional group can be any spacer functional group known in the art that does not negatively affect the properties of the guest complexing agent or functional group. For example, the spacer functional group may be a direct bond to which the functional group directly binds the complexing agent. Alternatively, the spacer functional group may be water soluble, anionic at physiological pH, or a component with fusogenic activity under acidic conditions. Suitably, the spacer functional group enhances the binding affinity of the polymer and the complexing agent in the inclusion complex (eg, anionic spacer functional groups bearing glutamic acid residues, carboxylic acid functional groups, etc.). Spacer functional groups also retain reducible bonds (eg disulfide bonds), the reduction of which releases functional groups from the complexing agent. Examples of suitable spacer functional groups include, but are not limited to, direct bonds, polyglutamic acid, GALA, polyethylene glycol (PEG).
[148] The functional group may be an additional therapeutic agent. The therapeutic agent may be reversibly bound to the complexing agent (eg, via prodrug form or biochemical binding). This provides a method of delivering additional therapeutic drugs through complexing agents.
[149] As noted above, the complexing agents used in the compositions herein are of the formula:
[150]
[151] here,
[152] J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
[153] ,
[154] Peptide or peptide residues, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
[155] Ad is adamantyl;
[156] R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
[157] PEG is -O (CH ₂ CH ₂ O) _z -wherein z is from 2 to 500;
[158] L is H, -NH ₂ , -NH- (C = O)-(CH ₂ ) _e- (C = O) -CH _2- , -S (= O) ₂ -HC = CH _2- , -SS- , -C (= 0) O- or a carbohydrate moiety;
[159] a is 0 or 1;
[160] b is 0 or 1;
[161] d is 0 to 6;
[162] e is 1 to 6;
[163] n is 0 to 6;
[164] y is 0 or 1;
[165] x is 0 or 1.
[166] The complexing agent may be a compound of the formula:
[167]
[168] Wherein the variables are the same as above, z is 1-5, q is 1-5, w is 1-5.
[169] As noted above, examples of guest functional groups include, but are not limited to, adamantyl, naphthyl, cholesterol, and preferred host functional groups are cyclodextrins. As indicated in the formula, a mixture of host and guest functional groups is present in the complexing agent.
[170] A preferred group of complexing agents possessing adamantane guest functional groups are of the formula:
[171]
[172] here,
[173] J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
[174] ,
[175] Peptide or peptide residue, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
[176] Ad is adamantyl;
[177] R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
[178] PEG is -O (CH ₂ CH ₂ O) _z -wherein z is from 2 to 500;
[179] L is H, -NH ₂ , -NH- (C = O)-(CH ₂ ) _e- (C = O) -CH _2- , -S (= O) ₂ -HC = CH _2- , -SS- , -C (= 0) O- or a carbohydrate moiety;
[180] a is 0 or 1;
[181] b is 0 or 1;
[182] d is 0 to 6;
[183] e is 1 to 6;
[184] n is 0 to 6;
[185] y is 0 or 1;
[186] x is 0 or 1.
[187] With the use of functionalized complexing agents, the compositions of the present invention can be modified or functionalized to promote cell contact and / or cell entry. In order to achieve multiple functions and / or advantages, the composition may form two or more types of inclusion complexes using complexing agents having different functional groups. As mentioned above, ligands can be used to modify the polymer or complexing agent of the particulate mixture. Thus, a composition according to the present invention may have one or more ligands through the inclusion complex and thus may have one or more sites for cell targeting and / or delivery. Particulate mixtures having multiple ligand- or other-functionalized complexing agents can be stabilized by addition of complexing agents having stabilizing or soluble functional groups such as PEGylated complexing agents.
[188] Since the polymer may form multiple inclusion complexes with mixtures of several functionalized complexing agents, the therapeutic compositions of the present invention may contain, for example, multiple therapeutic agents, different ligands, and / or various stabilizing polymers. When the complexing agent is functionalized as a therapeutic drug or prodrug, forming multiple inclusion complexes allows for the delivery of multiple therapeutic agents in the same therapeutic composition. If a ligand is present, the entire combination (or cocktail) of the therapeutic agent may be directed to a particular cell type, disease, or other therapeutic use.
[189] Functionalized guest complexing agents can be made by any means known in the art (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).
[190] 3. Preparation of the composition according to the present invention
[191] The present invention also relates to a method for producing the composition. The method consists of combining the therapeutic agent, a polymer bearing host / guest functional groups and a complexing agent to produce a therapeutic composition. The complexing agent that acts as a guest / host forms an inclusion complex with the polymer. In another embodiment, the therapeutic agent and polymer are first combined to form a particulate mixture, and then the particulate mixture is combined with a complexing agent to produce an encapsulation complex of the therapeutic composition. In addition, the composition may first combine the polymer and the complexing agent to form an encapsulation complex, which may then be combined with the therapeutic drug to produce a particulate mixture and ultimately a composition of the present invention.
[192] A. Formation of Polymer-Drug Particulate Mixture
[193] The particulate mixture of the therapeutic drug and the polymer may be made by any suitable means known in the art. For example, the particulate mixture may be formed by simply contacting, mixing, or dispersing the therapeutic drug with the polymer. For example, the polymer and the therapeutic agent may be mixed in a solvent in which both are dissolved, in which the polymer is dissolved but the therapeutic drug is dispersed, or in a solvent in which the polymer and the therapeutic agent are dispersed but which stabilizes the particulate mixture. In pharmaceutical use, the solvent may be any physiologically acceptable aqueous solution. The particulate mixture may be formed by combining the polymer with the therapeutic agent, self-bonding the polymer, or by chemical means. Prior to the formation of the particulate mixture, the polymer of the particulate mixture is not present in a substantially bonded structure such as a polymer gel. However, depending on the nature of the polymer and the therapeutic drug, the polymer as part of the particulate mixture may form a substantially bonded structure such as a gel. The particulate mixture may also be made by polymerizing the same or different monomers which form a linear or branched polymer in the presence of the therapeutic drug. The particulate mixture can also be made by polymerizing the same or different monomers that can form linear or branched polymers in the presence of the therapeutic agent, where the therapeutic agent serves as a template for polymerization (Trubetskoy et al., Nucleic). Acids Research, Vol. 26, No. 18, pp. 4178-4185 (1998)).
[194] The content of polymer and therapeutic drug used may be any amount that allows the particulate mixture to aggregate. Typically, polymers are used in excess of therapeutic drugs. The polymer used to form the particulate mixture has a cation or anionic charge as in positively charged comonomer A or polyalkylene imine (eg PEI) and the therapeutic agent has a charge such as anionic polynucleotide The ratio of polymer to therapeutic drug can be expressed in terms of charge ratio. The charge ratio is the ratio of polymer charge to therapeutic drug charge. As shown in the examples, the particulate mixture of the cationic cyclodextrin polymer and the anionic DNA is formulated with a 5 +/− charge, which is the 5 cationic charge of the cyclodextrin polymer to the monoanionic charge of the DNA. The charge ratio is any ratio that allows the particulate mixture to achieve the required minimum charge ratio and be present in excess of the minimum charge ratio. If the polymer and / or drug is not charged, the ratio of polymer to drug product can be expressed in weight, molar or concentration as is known in the art.
[195] In accordance with the present invention, the polymer of the particulate mixture may be treated under conditions sufficient to form a particulate mixture comprising a therapeutic drug and a multi-dimensional polymer network. Such multi-dimensional polymer networks are described in WO 00/33885. In WO 00/33885, the treatment of the polymer of the particulate composite under conditions sufficient to form a multi-dimensional polymer network is achieved with any suitable reaction conditions, including the addition of additional agents or reactants that promote the binding of the polymer of the particulate mixture to the therapeutic drug. can do. Polymers can be linked through covalent, non-covalent bonds (eg, ionic bonds), or non-covalent interactions (eg, van der Waals interactions) between polymers. Intrapolymer covalent, non-covalent, or non-covalent interactions of the polymers are also possible. As a result of this bonding, the polymer of the particulate mixture interacts to form a multi-dimensional polymer network.
[196] In one embodiment of the invention, a crosslinking reaction is included in forming a particulate mixture comprising a therapeutic agent and a multi-dimensional polymer network. For example, if the polymer of the particulate mixture is a single polymer molecule, the polymer promotes crosslinking, or molecules, oligomers, or discrete molecules that form crosslinks such that crosslinking within the polymer of the particulate mixture or actual crosslinking with a single polymer molecule is achieved. React with the polymer. Similarly, if the polymer of the particulate mixture is a mixture of two or more polymers, the polymer may be reacted with molecules, oligomers, or discrete polymers that promote crosslinking or form crosslinks.
[197] The resulting crosslinking is a crosslinking within the polymer and / or between polymers, preferably between polymers, of the particulate mixture.
[198] The crosslinker can be any crosslinker known in the art. The crosslinker may be any oligomer or polymer (eg, polyethylene glycol (PEG) polymer, polyethylene polymer) capable of promoting crosslinking in the polymer of the particulate mixture or actually crosslinking with these polymers. Crosslinking oligomers or polymers may be the same or different as the polymer of the particulate mixture. Similarly, the crosslinker can be any suitable molecule capable of crosslinking with the polymer of the particulate mixture. The crosslinker itself may carry a ligand.
[199] As found in WO 00/33885, the degree of bonding of the polymers of the particulate mixture forming the multi-dimensional polymer network varies from partial to full bond. By varying the degree of bonding of the polymers, short chain polymers can be made to exhibit the properties of long chain polymers while retaining the properties of the desired short chain polymers. For example, long chain polymer properties promote overall stability, ie resistance to degradation, until they reach the target cell, while short chain polymer properties promote DNA release within the target cell. This duality allows therapeutic compositions containing therapeutic agents and multi-dimensional polymer networks to exhibit improved stability and higher storage stability under non-physiological / physiological conditions. It is also possible to control the release of the therapeutic agent by varying the degree of binding of the polymer of the therapeutic composition.
[200] The particle size of the particulate composition depends on the polymer and the therapeutic agent used to form the composition of the present invention. As revealed in the examples below, the particle size is 50-1000 nm, preferably 50-500 nm. Typically, the formation of inclusion complexes does not significantly increase particle size. The composition is kept as individual particles. As described below, compositions containing pegylated complexing agents show excellent stability in salt solutions. Suitably the composition is stable under physiological conditions and can be used as a means of delivery of a therapeutic drug in the treatment of various diseases.
[201] B. Formation of inclusion complex
[202] Encapsulation composites can be made by any suitable means known in the art. For example, the encapsulation composite can be formed by simply contacting, mixing, or dispersing the particulate mixture and the complexing agent. For example, the particulate mixture and the complexing agent may be mixed in a solvent in which both are dissolved, a particulate mixture or a complexing agent is dissolved but the other is dispersed, or a solvent in which the particulate composite and the complexing agent are dispersed but which stabilizes the encapsulated composite. Suitably, the inclusion complex is formed by adding a complexing agent to the particulate mixture in the same container used to mix the polymer and the therapeutic drug. In pharmaceutical use, the solvent may be any physiologically acceptable aqueous solution.
[203] Complexing agents are added to the mixture particles in any molar ratio relative to the moles of host and / or guest functional groups present in the polymer of the mixture forming the inclusion complex. In general, the complexing agent is added in a 1: 1 molar ratio relative to the moles of host and / or guest functional groups. Lower molar ratios (excess guest and / or guest functional groups in the polymer) can be used if the composition has at least one complexing agent and at least one host / guest functional group in the polymer and forms an encapsulated composite. Excess complexing agents may also be used. Typically, the molar ratio of complexing agent to mole of polymeric host and / or guest functional groups is 0.01: 1 to 1: 0.01, preferably 0.5: 1 to 1: 0.5. If multiple complexing agents are used, the molar ratio of the individual complexing agents is selected by the functional groups desired to be introduced into the composition. For example, in certain compositions the PEGylated stabilizing complexing agent may be present in a ratio of 0.9: 1 and the ligand containing complexing agent may be present in small amounts, eg 1-2% of the total complexing agent. Typically, the total content of complexing agents in such compositions is inherent in the aforementioned ranges.
[204] 4. Compositions and Methods of Treatment
[205] Therapeutic compositions of the invention can be made in the form of solids, liquids, suspensions or emulsions. Suitably, the therapeutic composition of the present invention is in a form that can be injected intravenously. Other modes of administration of the therapeutic compositions according to the present invention include oral administration, inhalation, topical administration, extra-intestinal, intravenous, intranasal, intraocular, Methods known in the art, such as intracranial or intraperitoneal injection and pulmonary administration, are included, but are not limited to these. The method of administration often depends on the formulation of the therapeutic composition. Prior to administration, the therapeutic composition may be separated and purified by any means known in the art, such as centrifugation, dialysis and / or lyophilization.
[206] The present invention relates to a pharmaceutical composition consisting of an effective amount of a therapeutic composition according to the invention and a physiologically acceptable carrier. Suitable solid or liquid galenic formulations are, for example, granules, powders, coated tablets, microcapsules, suppositories, syrups, elixirs, suspensions, emulsions, eye drops or injectable solutions. Additives commonly used in pharmaceutical compositions include, but are not limited to, excipients, disintegrants, binders, coatings, swelling agents, lubricants, flavors, sweeteners, or solubilizers. More specifically, the additives frequently used are, for example, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactalbumin, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols, solvents. Solvents include sterile water and monohydric or polyhydric alcohols (eg glycerol).
[207] Depending on the type of therapeutic agent used, the therapeutic compositions of the invention may be congenital or acquired diseases, such as cystic fibrosis, Gaucher's disease, muscular dystrophy, AIDS, cancer (eg, multiple myeloma, leukemia, melanoma, ovarian carcinoma), cardiovascular Diseases (eg, progressive heart failure, stenosis, hemophilia), neurological diseases (eg, brain trauma). The method of treatment according to the invention consists in administering to a human or mammal a therapeutically effective amount of a therapeutic composition according to the invention. As will be appreciated by those skilled in the art, the therapeutically effective amount is determined on a case by case basis. Factors to consider include, but are not limited to, therapeutic diseases and the body characteristics of patients suffering from such diseases.
[208] 6. Other uses
[209] Encapsulation composites of the invention can also be used to deliver chemicals used in agriculture. In another embodiment of the invention, the "therapeutic drug" is a biologically active compound used for sterilization and agricultural use. These biologically active compounds include compounds known in the art. For example, agriculturally suitable biologically active compounds include, but are not limited to, fertilizers, fungicides, herbicides, insecticides, fungi. Fungicides are also used for water-treatment in treating tap water and industrial waters (eg cooling water, white water systems in papermaking). Aqueous systems susceptible to microbial attack or degradation are found in the leather, textile, coating and paint industries. Examples and uses of such fungicides are described in US Pat. Nos. 5,693,631, 6,034,081, 6,060,466. Compositions containing the active agents identified in these can be used in the same manner known for the active ingredient itself. In particular, since such uses are not pharmaceutical uses, the polymers of the mixture need not meet the toxicity profile required for pharmaceutical use.
[210] The following examples are intended to illustrate the present invention and do not limit the present invention.
[211] material. β-cyclodextrin (Cerestar USA, Inc., Hammond, IN) is dried for 12 hours under vacuum (<0.1 mTorr) at 120 ° C. prior to use. Biphenyl-4,4'-disulfonyl chloride (Aldrich Chemical Company, Inc., Milwauke, WI) is recrystallized from chloroform / hexanes. Potassium iodine is ground with a mortar and pestle and dried in an oven at 200 ° C. All other reagents are obtained from commercial suppliers and used without further purification. Polymer samples are analyzed at 1.0 mlmin ⁻¹ flow rate on a Hitachi HPLC system equipped with Anspec RI detector, Precision DLS detector, Progel-TSK G3000 _PWXL column, using 0.3 M NaCl or water as eluent.
[212] Example 1 Biphenyl-4,4′-Disulfonyl-A, D-Capped-β-cyclodextrin, Compound 1 (Tabushi et al. J. Am. Chem. Soc. 106, 5267-5270 (1984))
[213] A 500 ml round bottom flask equipped with a magnet rod, Schlenk adapter, septum is filled with 7.92 g (6.98 mmol) dry β-cyclodextrin and 250 ml anhydrous pyridine (Aldrich Chemical Company, Inc.). The resulting solution is stirred at 50 ° C. under nitrogen and 2.204 g (6.28 mmol) biphenyl-4,4′-disulfonyl chloride are added in equal portions four times at 15 minute intervals. After stirring for an additional 3 hours at 50 ° C., the solvent is removed in vacuo and the residue is subjected to reverse phase column chromatography using a concentration gradient of 0-40% acetonitrile in water. Aliquots are analyzed by high performance liquid chromatography (HPLC), and suitable aliquots are collected. After removal of acetonitrile in a rotary evaporator, the resulting aqueous suspension is freeze dried. This gives 3.39 g (38%) Compound 1 as a colorless solid.
[214] Example 2 6 ^A , 6 ^D -diode-6 ^A , 6 ^D -dideoxy-β-cyclodextrin, compound 2 (Tabusi et al. J. Am. Chem. 106, 4580-4584 (1984))
[215] 40 ml centrifuge tube with magnetic rod, Schlenk adapter, septum was 1.02 g (7.2 mmol) compound 1, 3.54 g (21.3 mmol) dry ground potassium iodine (Aldrich), 15 ml anhydrous N, N Fill with dimethylformamide (DMF) (Aldrich). The resulting suspension is stirred at 80 ° C. for 2 hours under nitrogen. After cooling to room temperature, the solids are separated by filtration and the supernatant is collected. The solid precipitate is washed with a second portion of anhydrous DMF and the supernatant is collected and concentrated in vacuo. The residue is then dissolved in 14 mL water and cooled in an ice solution, after which 0.75 mL (7.3 mmol) tetrachloroethylene (Aldrich) is added with rapid stirring. The precipitated product is filtered through a medium glass frit, washed with a small amount of acetone and then dried in P ₂ O ₅ under vacuum for 14 hours. 0.90 g (92%) Compound 2 is obtained as a white solid.
[216] Example 3 6 ^A , 6 ^D -diazaid-6 ^A , 6 ^D -dideoxy-β-cyclodextrin, Compound 3 (Tabusi et al. Tetrahedron Lett. 18, 1527-1530 (1997))
[217] A 100 ml round bottom flask equipped with a magnet rod, Schlenk adapter, septum was used as 1.704 g (1.25 mmol) β-cyclodextrin diiodine, 0.49 g (7.53 mmol) sodium azide (EM Science, Gibbstown, NJ), Fill with 10 ml anhydrous N, N-dimethylformamide (DMF). The resulting suspension is stirred at 60 ° C. for 14 hours under nitrogen. The solvent is then removed under vacuum. The resulting residue is dissolved in sufficient water to form a 0.2 M salt solution and passed through 11.3 g Biorad AG501-X8 (D) resin to remove residual salts. The eluate is then freeze dried to yield 1.232 g (83%) Compound 3 as a white amorphous solid, which is used in the next step without further purification.
[218] Example 4 6 ^A , 6 ^D -Diamino-6 ^A , 6 ^D -dideoxy-β-cyclodextrin, Compound 4 (Mungall et al., J. Org. Chem. 1659-1662 (1975))
[219] A 250 ml round bottom flask equipped with a magnet rod and septum is filled with 1.232 g (1.04 mmol) β-cyclodextrin bisazide and 50 ml anhydrous pyridine. 0.898 g (3.42 mmol) triphenylphosphine is added to this stirred suspension. The resulting suspension is stirred at room temperature for 1 hour, after which 10 ml concentrated aqueous ammonia is added. The addition of ammonia is achieved by rapid gas release and the solution becomes homogeneous. After 14 hours, the solvent is removed in vacuo and the residue is triturated with 50 ml water. The solid was filtered off and the filtrate is an ion exchange column containing the acidified (pH <4), since Toyopearl SP-650M (MH ₄ ⁺ form) resin with 10% HCl. Product 4 elutes with a concentration gradient of 0-0.5 M ammonium bicarbonate. Suitable aliquots are collected and lyophilized to yield 0.832 g (71%) product (4) as bis (hydrocarbon carbonate) salt.
[220] Example 5 β-cyclodextrin-DSP Copolymer, Compound 5
[221] A 20 ml scintillation vial is filled with a solution of 92.6 mg (7.65 × 10 ⁻⁵ mol) bis (hydrogen carbonate) salt of compound 4 dissolved in 1 ml water. The pH of the solution was adjusted to 10 with 1M NaOH and then a solution of 30.9 mg (7.65x10 ^-5 mol) dithiobis (succiminidyl propionate) (DSP, Pierce Chemical Co., Rockford, IL) dissolved in 1 ml chloroform. Add. The resulting biphasic mixture is stirred for 0.5 h with a Vortex mixer. The aqueous layer is then decanted and extracted with 3 x 1 ml of fresh chloroform. The aqueous polymer solution is then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin with water as eluent. Aliquots are analyzed by GPC and suitable aliquots are lyophilized to yield 85 mg (85%) as colorless amorphous powder.
[222] Example 6 : β-cyclodextrin-DSS copolymer, compound 6
[223] β-cyclodextrin-DSS copolymer (Compound 6) is similar to DSP polymer (Compound 5), except that disuccinimidyl suverate (DSS, Pierce chemical Co., Rockford, IL) was used instead of DSP reagent. Synthesized in a manner. Compound (6) is obtained in 67% yield.
[224] Example 7 : β-cyclodextrin-DTBP copolymer, compound 7
[225] A 20 ml scintillation vial is filled with a 91.2 mg (7.26 × 10 ⁻⁵ mol) bis (hydrogen carbonate) salt solution of compound 4 dissolved in 1 ml water. The pH of the solution was adjusted to 10 with 1M NaOH, then 22.4 mg (7.26x10 ^-5 mol) dimethyl 3,3'-dithiobis (propionimate) .2HCl (DTBP, Pierce Chemical Co., Rockford, IL) solution Add. The resulting homogeneous solution is stirred for 0.5 h with a Vortex mixer. The aqueous polymer solution is then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin. Aliquots are analyzed by GPC and suitable aliquots are lyophilized to yield 67 mg (67%) as colorless amorphous powder.
[226] Example 8 Polyethylene Glycol (PEG) 600 Diacid Chloride, Compound 8
[227]
[228] A 50 ml round bottom flask equipped with a magnetic rod and septum is 5.07 g (ca. 8.4 mmol) polyethylene glycol 600 diacid (Fluka Chemical Corp., Milwauke, WI) and 10 ml anhydrous chloroform (Aldrich). Fill it with 3.9 mL (53.4 mmol) thionyl chloride (Aldrich) is added to this stirred solution, and the resulting solution is heated to reflux for 1 hour, during which time gas evolution is evident. The resulting solution is cooled to room temperature, after which the solvent and excess thionyl chloride are removed under vacuum. The resulting oil is stored in a dry box and used without further purification.
[229] Example 9 β-cyclodextrin-PEG 600 Copolymer, Compound 9
[230]
[231] 20 ml scintillation vial was used to prepare 50 ml of 112.5 mg (8.95x10 ^-5 mol) bis (hydrocarbon carbonate) salt of 6 ^A , 6 ^D -diamino-6 ^A , 6 ^D -dideoxy-β-cyclodextrin (Compound 4). Fill with a solution of (3.6x10 ^-4 mol) triethylamine (Aldrich), 5 ml anhydrous N, N-dimethylacetamide (DMAc, Aldrich). The resulting suspension is then treated with 58 mg (9.1 × 10 ⁻⁵ mol) polyethylene glycol 600 diacid chloride (Compound 8). The resulting solution was stirred for 5 minutes with a Vortex mixer and then left at 25 ° C. for 1 hour, during which time it became homogeneous. The solvent is removed in vacuo and the residue is subjected to gel permeation chromatography on Toyopearl HW-40F resin with water as eluent. Aliquots are analyzed by GPC and suitable aliquots are lyophilized to yield 115 mg (75%) as colorless amorphous powder.
[232] Example 10 6 ^A , 6 ^D -bis- (2-aminoethylthio) -6 ^A , 6 ^D -dideoxy-β-cyclodextrin (Compound 10) (Tabushi, I: Shimokawa, K; Fugia, K. Tetrahedron Lett. 1977,1527-1530)
[233]
[234] A 25 ml round bottom flask equipped with a magnetic rod and septum is filled with 0.91 ml (7.37 mmol) of 0.81 M sodium 2-aminoethylthiolate solution in ethanol (Fieser, LF; Fiester, M. Reagents for Organic) Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266). The solution is evaporated to dryness and the solid is dissolved again in 5 ml anhydrous DMF (Aldrich). 6 ^A , 6 ^D -diode-6 ^A , 6 ^D -dideoxy-β-cyclodextrin (Compound 2) (100 mg, 7.38 × 10 ⁻⁵ mol) was added and the resulting suspension was added at 60 ° C. under nitrogen. Stir for time. After cooling to room temperature, the solution is concentrated in vacuo and the residue is dissolved again in water. After the reaction mixture was acidified with 0.1N HCl, the solution is Toyopearl SP-650M ion-exchange column was added to the (MH ₄ ⁺ form), the product is eluted with a 0-0.4 M ammonium bicarbonate gradient. Suitable aliquots are collected and lyophilized. This gives 80 mg (79%) Compound 10 as a white powder.
[235] Alternative Synthesis of Dicysteamine β-CD (Compound 10)
[236] To a 4.69 (3.17 mmol) solution of Compound 2 dissolved in degassed 100 mL water is added 0.489 g (6.34 mmol) of freshly purified cysteamine. The solution is stirred at reflux for 2 hours. After cooling to room temperature and acidified with 1N HCl, the solution is Toyopearl SP-650M ion-exchange column was added to the (MH ₄ ⁺ form), the product is eluted with a 0-0.2 M ammonium bicarbonate gradient. Suitable aliquots are collected and lyophilized. This gives 1.87 g (39% yield) white solid. The solid is characterized by TLC (silica gel, n-PrOH-AcOEt-H ₂ O-NH ₃ aq5 / 3/3/1, detection by ninhydrin), showing a major spot corresponding to compound 10. Matrix-Assisted Laser High Resolution Matrix Assisted Laser Desorption / Ionization (MALDI) Flight Time (TOF) mass spectra are recorded on a 2 meter ELITE device provided by PerSeptive Biosystems, Inc. MALDI-TOF m / z calculated for compound 3: 1252, found: 1253.5 [M + H} ⁺ , 1275.5 {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).
[237] Example 11 β-cyclodextrin (citamine) -DTBP copolymer, compound 11
[238]
[239] A 4 ml vial is filled with a 19.6 mg (1.42 × 10 ⁻⁵ mol) bis (hydrocarbon carbonate) salt solution of compound 10 dissolved in 0.5 ml 0.1M NaHCO ₃ . The solution is cooled in an ice solution and then 4.4 mg (1.4 × 10 ⁻⁵ mol) dimethyl 3,3′-dithiobispropionimate-2HCl (DTBP, Pierce Chemical Co., Rockford, IL) is added. The resulting solution is stirred with a Voltex mixer and left at 0 ° C. for 1 hour. The reaction is stopped with 1M Tris-HCl and then acidified to pH 4 with 0.1N HCl. The aqueous polymer solution is then subjected to gel permeation chromatography on Toyopearl HW-40 resin. Aliquots are analyzed by GPC and suitable aliquots are lyophilized. This gives 21.3 mg (100%) Compound 11 as white powder.
[240] Example 12 β-cyclodextrin (citamine) -DMS copolymer, compound 12
[241]
[242] A 10 ml Shlenk flask equipped with a magnetic rod and septum was used for preparing 10 mg of 200 mg (1.60 × 10 ⁻⁴ mol) compound, 44 μl (3.2 × 10 ⁻⁴ mol) triethylamine (Aldrich Chemical Co., Milwauke, WI), Filled with 43.6 mg (1.60 × 10 ⁻⁴ mol) dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co., Rockford, IL), 3 ml anhydrous DMF (Aldrich Chemical Co, Milwauke, WI). The resulting slurry is heated to 80 ° C. for 18 hours under constant nitrogen flow, during which time most of the solvent is evaporated. The remaining residue is dissolved again in 10 ml water and the resulting solution is acidified to pH 4 with 10% HCl. The solution is then passed through an Amicon Centricon Plus-20 5,000 NMWL centrifugal filter. After washing with 2 × 10 mL water, the polymer solution is lyophilized to yield 41.4 mg (18%) greyish white amorphous solid.
[243] Alternative Synthesis :
[244] β-cyclodextrin (citamine) -DMS copolymers are synthesized as reported in Gonzalez, et al. 1999. In a typical experiment, a 25 ml vial is filled with a bis (hydrogen carbonate) salt solution of dicysteamine β-CD (Compound 10) (399.6 mg, 0.269 mmol) dissolved in 500 μl of 0.5 M Na ₂ CO ₃ . Dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co., Rockford, IL; 73.5 mg, 0.269 mmol) is added and the solution is briefly centrifuged to dissolve the components. The resulting mixture is stirred at 25 ° C. for 15 hours. The mixture is then diluted with 10 ml water and the pH is lowered below 4 by addition of 1N HCl. The solution is then dialyzed for 24 hours on a Spectra / Por 7 MWCO 3500 Spectrum in dH ₂ O. The dialysed solution is freeze dried. ¹³ C NMR (Bruker 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.
[245] Example 13 : Formation of permanently charged copolymer complexes with plasmids
[246] In general, a fixed equal volume of charged CD-polymer and DNA plasmid solution dissolved in water are mixed at an appropriate polymer / plasmid charge ratio. The mixture is then equilibrated at room temperature and self-combined. Complexation success is monitored by transferring a small amount of the mixture to a 0.6% agarose gel and examining the DNA mobility. Free DNA migrates under applied voltage, while immobilized DNA is retarded in the wells.
[247] 1 μg DNA in distilled water is mixed with 10 μl copolymer (Compound 12) in polymer amine at a concentration of 0.1 μg / μl: DNA phosphate charge ratio of 2.4, 6, 12, 24, 36, 60, 120. 1 μg / μL dropping buffer (40% sucrose, 0.25% bromophenol blue, 5 mM EDTA-containing 200 mM Tris-acetate buffer (Gao et al., Biochemistry 35: 1027-1036 (1996)) was added to each solution. Each DNA / polymer sample was added to a 0.6% agarose electrophoresis gel containing 6 μg EtBr / 100ml in 1 × TAE buffer (40 mM Tris-acetate / 1 mM EDTA) and 40V was added to the gel for 1 hour. The degree of DNA / polymer complexation is manifested as DNA retardation in the gel migration pattern Copolymer 12 retards DNA at a charge ratio of at least two, suggesting complexation under these conditions.
[248] Example 14 Transfection Studies with Plasmids Encoding Luciferase Reporter Gene
[249] BHK-21 cells are plated in 24 well plates at a cell density of 60,000 cells / well 24 hours prior to transfection. The plasmid encoding the luciferase gene is mixed with the CD-polymer as in Example 13. The medium solution containing the DNA / polymer complex is added to the cultured cells and incubated at 37 ° C. for 24 hours and then replaced with fresh medium. Cells are lysed 48 hours after transfection. A substrate suitable for luciferase light analysis is added to the cell lysate. Luciferase activity measured in terms of generated light units is quantified by a luminometer. The DNA / polymer complex successfully transfects BHK-21 cells at a charge ratio of at least 3 and shows a maximum transfection at a polymer amine: DNA phosphate charge ratio of 40. Cell lysates are also used to measure cell viability by Lowry protein analysis (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275 (1951)). No toxicity was observed up to a charge rate of up to 40.
[250] Example 15 Synthesis of β-cyclodextrin (citamine) -DMA Copolymer, Compound 13
[251]
[252] A 20 ml scintillation vial with a magnetic rod is filled with 180 mg (0.131 mmol) compound 10 and 32 mg dimethyl adipidamide (DMA, Pierce Chemical Co., Rockford, IL). 500 μl of 0.5 M Na ₂ CO ₃ is added thereto. The resulting solution is wrapped in a thin film and stirred overnight. The mixture is then acidified with 0.1N HCl and dialyzed with Spectrapor MWCO 3,500 membrane for 2 days and lyophilized to yield 41 mg of white amorphous solid (Mw = 6 kDa, measured by light dispersion).
[253] Example 16 Synthesis of β-cyclodextrin (citamine) -DMP Copolymer, Compound 14
[254]
[255] A 20 ml scintillation vial with a magnetic rod is filled with 160 mg (0.116 mmol) compound 10 and 30.1 mg dimethyl pimelimidate (DMA, Pierce Chemical Co., Rockford, IL). 500 μl of 0.5 M Na ₂ CO ₃ is added thereto. The resulting solution is wrapped in a thin film and stirred overnight. The mixture is then acidified with 0.1N HCl and dialyzed with Spectrapor MWCO 3,500 membrane for 2 days and lyophilized to yield 22 mg of white amorphous solid (Mw = 6 kDa, measured by light dispersion).
[256] Example 17 β-cyclodextrin (citamine) -PEG600 copolymer, compound 15
[257]
[258] A 100 ml round bottom flask equipped with a magnet rod, Schlenk adapter, septum is filled with 1.564 g (1.25 mmol) compound 10 and 25 ml of freshly distilled dimethylacetamide (DMAc, Aldrich). To the slurry is added a solution of compound 8 (2.39, 3.75 eq) dissolved in 0.7 ml (4 eq) triethylamine and 5 ml DMAc. The resulting solution was stirred for 5 minutes with a Voltex mixer and then left at 25 ° C. for 1 hour, during which time it became homogeneous. The solvent is removed in vacuo and the residue is subjected to gel permeation chromatography on Toyopearl HW-40F resin with water as eluent. Aliquots are analyzed by GPC and suitable aliquots are lyophilized to yield a colorless amorphous powder.
[259] Example 18 β-Cyclodextrin-Tosylate, Synthesis of Compound 16 (Melton, LD, and Slessor, KN, Carbohydrate Research, 18, p. 29 (1971))
[260]
[261] A 500 ml round bottom flask equipped with a magnet rod, vacuum adapter, septum is filled with a solution of dry β-cyclodextrin (8.530 g, 7.51 mmol) and 200 ml dry pyridine. The solution is cooled to 0 ° C. and then 1.29 g (6.76 mmol) tosyl chloride is added. The resulting solution is warmed to room temperature overnight. Pyridine is removed in vacuo where possible. The resulting residue is then recrystallized twice from 40 ml hot water to yield 7.54 (88%) of white crystalline solid.
[262] Example 19 Synthesis of β-cyclodextrin-iodine, Compound 17
[263]
[264] Round bottom flasks with magnetic rods and Schlenk adapters are filled with compound 16, 15 equivalents of potassium iodine, DMF. The resulting mixture is heated at 80 ° C. for 3 hours and then cooled to the reaction room temperature. The mixture is then filtered to remove the precipitate, the filtrate is evaporated to dryness and dissolved again in water at 0 ° C. Tetrachloroethylene is added and the resulting slurry is vigorously stirred at 0 ° C. for 20 minutes. The solids are collected in secondary glass frit, softened with acetone and stored in P ₂ O ₅ .
[265] Example 20 Synthesis of β-cyclodextrin-thiol-PEG Added Polymer, Compound 18
[266] Step 1: Synthesis of β-cyclodextrin-thiol (K. Fujita, et al., Bioorg. Chem., Vol. 11, p. 72 (1982) and K. Fujita, et al., Bioorg. Chem., Vol 11, p. 108 (1982))
[267] A 50 ml round bottom flask equipped with a magnet rod and a Schlenk adapter was filled with 1.00 g (0.776 mmol) compound 16, 0.59 g (7.75 mmol) thiourea (Aldrich), 7.8 mL 0.1 N NaOH solution. The resulting mixture is heated at 80 ° C. for 6 hours under nitrogen. Then 0.62 g (15.5 mmol) sodium hydroxide are added and the reaction mixture is heated at 80 ° C. for 1 additional hour under nitrogen. The reaction is cooled to room temperature and then acidified to pH 4.0 with 10% HCl. The total solution volume is brought to 20 ml and cooled in an ice solution, followed by the addition of 0.8 ml tetrachloroethylene. The reaction mixture is vigorously stirred at 0 ° C. for 0.5 h, after which the precipitated solid is collected in a thin glass frit. The solid is blown overnight to yield 0.60 g (67%) white amorphous solid.
[268] Stage 2: A 100 ml round bottom flask equipped with a magnetic rod and reflux condenser was prepared using a 2.433 g (2.11 mmol) β-cyclodextrin-thiol, 0.650 g functionalized PEG (PEG with pendant olefins, Yoshiyuki Koyama of Otsuma Women's University, Tokyo, Japan), 50 ml dH ₂ O. The resulting mixture is heated at reflux for 12 hours during which time β-cyclodextrin-thiol is dissolved. The reaction mixture is cooled to room temperature and the precipitated solids are removed by centrifugation. Supernatant is dialyzed on a Spectra / Por 7 MWCO 1,000 membrane. The solution is lyophilized to yield an amorphous white solid.
[269]
[270]
[271] Example 21 Synthesis of Branched PEI-Cyclodextrin Polymers, Compound 19
[272] A 20 ml scintillation vial with a magnetic rod is filled with branched PEI (25 kD, Aldrich) and compound 17. To this is added degassed sodium carbonate buffer. The resulting solution is stirred at 80 ° C. for 4 hours. The mixture is acidified with 0.1 N HCl and dialyzed on Spectra / Por MWCO 3,500 membrane for 2 days and lyophilized.
[273] Example 21B Synthesis of PEI-cyclodextrin crosslinked polymer
[274] Branched PEI (Mw 1200, Aldrich) and bifunctional cyclodextrin monomer 2 (1 eq) are mixed in dry DMSO. The mixture is stirred at 80 ° C. for 4 days and then lyophilized with dialysis on Spectra / Por MWCO 10,000 membrane for 2 days.
[275] Example 22 Synthesis of Ad-PEG ₃₄₀₀ -Ad
[276] 240 mg 1-aminoadamantane (1.60 mmol, Aldrich) and 288 mg PEG ₃₄₀₀ (SPA) ₂ (0.085 mmol, Shearwater Polymers) are added to a glass vial with a magnetic rod. To this is added 5 ml dichloromethane and the solution is stirred overnight. The next day, the solution is filtered to remove n-hydroxysuccinimide by-product and dichloromethane is removed in vacuo. The residue is dissolved in water and centrifuged to remove excess 1-aminoadamantane. The supernatant is then dialyzed overnight at Pierce's Slide-A-Lyzer (MWCO = 3500). The solution was lyophilized to give 248 mg Ad-PEG ₃₄₀₀ -Ad as a white downy solid.
[277] Example 23 Synthesis of Ad-PEG ₃₄₀₀ -NH ₂
[278] 347 mg FMOC-PEG ₃₄₀₀ -NH ₂ (0.110 mmol, Shearwater Polymers) and 155 mg 1-aminoadamantane (1.0 mmol, Aldrich) are added to a glass vial equipped with a magnetic rod. 5 ml dichloromethane is added thereto and the resulting solution is stirred overnight. The next day, the solution is filtered to remove n-hydroxysuccinimide by-product and dichloromethane is removed in vacuo. The residue is dissolved in water and filtered to remove unreacted 1-aminoadamantane. The solution is then freeze dried to remove water. The FMOC functional group is removed by dissolving the resulting solid for 20 minutes in 20% piperidine dissolved in DMF. The solvent is removed in vacuo and the residue is dissolved again in water. The solution is centrifuged to remove undissolved FMOC and then dialyzed overnight in Pierce's Slide-A-Lyzer (MWCO = 3500). The solution is then lyophilized to yield 219 mg Ad-PEG ₃₄₀₀ -NH ₂ as a white downy solid.
[279] Example 24: adamantane _{-PEG 3400 -NH 2 (Ad-PEG} 3400 -NH 2)
[280] 266 mg FMOC-PEG _3400- NHS (78.2 μmol, Shearwater Polymers, Huntville AL) is added to a glass vial equipped with a magnetic rod. Then, 10 eq. Dissolved in 3 ml offdichloromethane. 1-adamantane-methylamine (1.5 mmol, Aldrich) is added and the solution is stirred overnight at room temperature. The solvent is removed under vacuum and water is added to the residual solution to dissolve the PEG product. The solution is centrifuged for 10 minutes at 20K rcf, where adamantane-methylamine is phase-separated into a more dense liquid. The aqueous portion is collected and the water is removed under vacuum. The remaining viscous liquid is redissolved in 20% piperidine dissolved in DMF for FMOC deprotection and stirred for 30 minutes at room temperature. The solvent is removed in vacuo, washed several times with DMF, dissolved in water again and operated on an anion exchange column to remove unreacted PEG. The first aliquot was collected and lyophilized to yield 222 mg of the desired product as a white downy powder (76% yield) (confirmed by MALDI-TOF analysis).
[281] Example 25 adamantane-PEG ₃₄₀₀ -lactose (Ad-PEG _3400- Lac)
[282] 60 mg Ad-PEG ₃₄₀₀ -NH ₂ (16.8 μmol) and 5.0 eq lactose-monosuccinimidyl (50 mg, Pierce, Rockford, IL) made in Example 24 are added to a glass vial equipped with a magnetic rod. Then 2 ml of 50 mM NaHCO ₃ are added and the resulting solution is stirred overnight. The reaction of the amine is monitored by TNBS analysis, which measures the amine concentration. Immediately after completion of the amine reaction (99% amine reaction), the solution was transferred to a dialysis tube (Slide-A-Lyzer, MWCO = 3500, Pierce) and dialyzed in water for 24 hours, lyophilized to give 65.1 mg white downy powder ( 93% yield).
[283] Example 26 Synthesis of Ad-PEG ₅₀₀₀
[284] 279 mg PEG ₅₀₀₀ -NHS (0.053 mmol, Shearwater Polymers) is added to a glass vial equipped with a magnetic rod. To this was added 46 μl 1-adamantane methylamine (0.42 mmol, Aldrich) dissolved in 3 mL dichloromethane and the solution was stirred overnight. The next day, the solution is filtered to remove n-hydroxysuccinimide by-product and dichloromethane is removed in vacuo. The residue is dissolved in water and centrifuged. Excess 1-adamantane methylamine is phase-separated, the upper aqueous phase removed and dialyzed overnight in Pierce's Slide-A-Lyzer (MWCO = 3500). The solution is then lyophilized to give 253 mg Ad-PEG ₅₀₀₀ as a white downy solid. The product was found to be pure for analysis on a Beckman Gold HPLC system equipped with a Richard Scientific ELS detector and a C18 column (retention time of PEG ₅₀₀₀ -NHS: 10.7 min; retention time of product: 12.0 min; acetonitrile / water gradient ).
[285] Alternative Synthesis: Adamantane-PEG ₅₀₀₀ (AD-PEG ₅₀₀₀ )
[286] 674 mg PEG ₅₀₀₀ -NHS (135 μmol, Shearwater Polymers) is added to glass vials equipped with a magnetic rod. 5 eq. Dissolved in 10 ml dichloromethane. 1-adamantane methylamine (675 μmol, Aldrich) is added and the solution is stirred overnight at room temperature. The solvent is removed in vacuo and water is added to the residual solution. The solution is centrifuged for 10 minutes at 20K rcf, where adamantane-methylamine is phase-separated into a more dense liquid. The aqueous portion is collected and dialyzed in water for 24 hours (Slide-A-Lyzer, MWCO = 3500). The solution was lyophilized to give 530 mg white downy powder (75% yield, product shown below). The product was found to be pure for analysis on a Beckman Gold HPLC system equipped with a Richard Scientific ELS detector and a C18 column (retention time of PEG ₅₀₀₀ -NHS: 10.7 min; retention time of product: 12.0 min; acetonitrile / water gradient ). AD-PEG ₃₄₀₀ is synthesized in a similar protocol (56% yield; confirmed by Maldi-TOF analysis).
[287]
[288] Example 27 Adamantane- (PEG ₅₀₀₀ ) ₂ (Ad- (PEG ₅₀₀₀ ) ₂ )
[289] 315 mg (PEG ₅₀₀₀ ) ₂ -NHS (30 μmol, Shearwater Polymers) is added to a glass vial equipped with a magnetic rod. 10 eq dissolved in 3 mL DCM. 1-adamantane methylamine (300 μmol, Aldrich) is added and the solution is stirred overnight at room temperature. The solvent is removed under vacuum and water is added to the residual solution to dissolve the PEG product. The solution is centrifuged for 10 minutes at 20K rcf, where adamantane-methylamine is phase-separated into a more dense liquid. The aqueous portion is collected and dialyzed in water for 24 hours (Slide-A-Lyzer, MWCO = 3500). The solution was lyophilized to give 286 mg white fluffy powder (91% yield).
[290] Example 28: adamantane -PEG ₃₄₀₀ - fluorescein (Ad-PEG ₃₄₀₀ -FITC)
[291] 20 mg Ad-PEG ₃₄₀₀ -NH ₂ is dissolved in 3 ml 0.01 M Na ₂ CO ₃ in a glass vial equipped with a magnetic rod. To the solution was added 3 eq fluresin isothiocyanate (FITC, Sigma) dissolved in DMSO (4 mg / ml, 1.6 ml), and the resulting solution was stirred overnight in the dark, followed by dialysis tube (MWCO = 3500). ) And dialysate for 48 hours against water in the dark. The solution is collected and lyophilized to yield 23 mg yellow downy solid. PEG _3400- FITC is synthesized with 23 mg of control polymer from PEG ₃₄₀₀ -NH ₂ (Shearwater Polymers) using the same protocol.
[292] Example 29 Synthesis of GALA Peptides
[293] GALA peptides (SEQ ID NO: W-E-A-A-L-A-E-A-L-A-E-A-L-A-E-H-L-A-E-A-L-A-E-A-L-E- A-L-A-A, MW 3032) are synthesized by Biopolymer Synthesis Facility (Beckman Institute, California Institute of technology) using an automatic sequencing device. Prior to cleaving peptides from the resin, one third of the resin is left for adamantane conjugate. Analysis of peptides by HPLC suggests greater than 95% purity. L-adamantane-carboxylic acid (Aldrich) conjugates to the N-terminus of the GALA-peptide by DCC binding chemistry. The resulting peptide (GALA-Ad, MW 3194) is cleaved from the resin. Analysis of the peptides by HPLC suggests greater than 90% purity. The identity of the peptide is confirmed by MALDI-TOF analysis (Biopolymer Analysis Facility, Beckman Institute, California Institute of Technology).
[294] Example 30 Preparation of a Composition According to the Invention Using a GALA Peptide
[295] Plasmids and Oligonucleotides . The plasmid pGL3-CV (Promega, Madison, Wis.) Carrying the luciferase gene under the control of the SV40 promoter was amplified with Esherichia Coli and purified by Qiagen's Endotoxin-Free Megaprep Kit (Valencia, CA). Fluresin-labeled oligonucleotides (FITC-oligo, 25-mer, 5'-FITC-ACT GCT TAC CAG GGA TTT CAG TGC A-3 ') are synthesized by the Biopolymer Synthesis Facility (California Institute of Technology).
[296] Particle Formation and Characterization . The compositions of the present invention are prepared by mixing equal volumes of compound 12 (dissolved in dH ₂ O) and DNA (0.1 mg / ml in dH ₂ ) at a suitable charge ratio. Equal volumes of GALA or GALA-Ad dissolved in 50 mM phosphate buffered saline (PBS, pH 7.2) are then added to the complex. For example, in a particle characterization study, 2 μg plasmid DNA (20 μl) is combined with compound 12 (20 mL) at a 5 +/− charge ratio. Then 20 μl GALA solution, GALA-Ad solution or 50 mM PBS (control sample) are added to the complex. The solution is then diluted with 1.2 ml dH ₂ O addition. Particle size and charge are determined by dynamic light dispersion and zeta potential measurements, respectively, using a ZetaPals dynamic light dispersion detector (Brookhaven Instruments Corporation, Holtsville, NY). These results, expressed as mean ± standard deviation of these values, are shown in FIG. 2. The hydrodynamic diameter of the compound 12 / pGL3-CV composition made at the 5 +/- charge ratio was determined by dynamic light scattering and was found to be 260 nm. 2 μg plasmid DNA at 20 μl is mixed with an equal volume of compound 12 at a 5 +/− charge ratio. Thereafter, various proportions of GALA or GALA-Ad are added to the particles. Hydrodynamic diameter is determined by light scatter measurement. Results are expressed as mean ± standard deviation of three measurements. GALA peptides transition from water soluble random coil conformation at pH 7.5 to water soluble helix at pH 5. GALA and Adamantane-modified GALA (GALA-Ad) peptides are dissolved in 50 mM PBS pH 7.2 and added to the therapeutic composition at various peptide / cyclodextrin ratios. The mixture is diluted with dH ₂ O and the particle size is determined by dynamic light dispersion (FIG. 2). 2 shows the hydrodynamic diameter (dashed line) of GALA and the hydrodynamic diameter (solid line) of the GALA-Ad modified polyplex.
[297] Result . Since the particle count rate is the same at all concentrations of the added peptide, the addition of peptide does not appear to destroy the composition. The particle size profile is very similar as a function of GALA and GALA-Ad addition. 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 is reduced back to the size of the unmodified therapeutic composition. The diameter returns to approximately 250 nm with addition of at least 30% GALA-Ad and at least 50% GALA (FIG. 2).
[298] Example 31 Uptake of GALA-Modified Compositions into BHK-21 Cells
[299] Cell culture . BHK-21 cells were purchased from ATCC (Rockville, MD) and HUH-7 cells were donated from Valigen (Rockville, MD). Both cell lines were incubated in DMEM supplemented with 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin in a humidified incubator operating at 37 ° C. 5% CO ₂ and 4 -Passage every five days. Media and supplements were purchased from Gibco BTL (Gaithersburg, MD).
[300] Uptake of Therapeutic Compositions by Cultured Cells . BHK-21 cells are plated in 6-well plates at 150,000 cells / well and incubated at 37 ° C. for 24 hours. 5 μg FITC-oligo is combined with compound 12 at a 5 +/− charge ratio. After 5 minutes of complexation time, 50 μl GALA or GALA-Ad dissolved in 50 mM PBS pH 7.2 is added to the complex. The medium is removed from the cells and the cells are washed with PBS. For transfection, 900 μl Optimen is added to each treatment composition solution and the entire solution is transferred to the cells. Cells are incubated with the transfection mixture for 5 hours, then the medium is removed and the cells washed twice with PBS. Cells are harvested by trypsinization and prepared for FAC analysis. Cells are washed twice in wash buffer (Hank Balance Salt Solution containing DNase and MgCl ₂ ) and in 500 μl FACS buffer (Hank Balance Salt Solution, 2.5 mg / ml Bovine Serum Albumin, 10 μg / ml Propidium Iodide). Resuspend. FACS assays are performed with the FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) And CellQuest software. The results are shown in FIG. As shown in FIG. 4 ad, BHK-21 cells (4a) were compound 12 / FITC-oligo (4b), compound 12 / FITC-oligo / 50% GALA (4c), compound 12 / FITC-oligo / 50% Transfect with GALA-Ad (4d). Uptake is confirmed by flow cytometry. Data is presented in fluorescence profiles, with cell populations plotted along the y-axis and fluresin fluorescence intensity plotted along the x-axis.
[301] Example 32 Zeta Potential of Modified Composites
[302] 2 μg plasmid DNA at 20 μl is mixed with an equal volume of compound 12 at a 5 +/− charge ratio. Various ratios of GALA or GALA-Ad are added to the particles at various peptide / CD ratios and diluted with dH ₂ O. Particle charge is determined by electrophoretic measurements and expressed as particle zeta potential (mV). Particle charge of the compound 12 / pGL3-CV composition at 5 +/- charge ratio was determined by zeta potential measurement, which was found to be +13 mV. The zeta potential of the particles in the presence of the peptide is measured and the results are expressed as mean ± standard deviation of three measurements (FIG. 3).
[303] Result . Since GALA peptides are anionic peptides at pH 7.2 (having multiple glutamic acid residues), the binding of the composition with GALA and GALA-Ad reduces zeta potential. The composition is negatively charged by 30% GALA (-11 mV) or GALA-Ad (-23 mV). Zeta potential of the GALA + therapeutic composition solution is stagnant at this point; Only by adding more GALA does the zeta potential increase slightly (-15 mV at 150% GALA). However, the particles are more negatively charged at higher GALA-Ad concentrations. The composition to which 150% GALA-Ad was added has a zeta potential of -42 mV (FIG. 3).
[304] Example 33 DNA Delivery Efficiency of Compositions
[305] HUH-7 cells : liver cancer cell line, HUH-7 is also transfected with Compound 12 / FITC-oligo and Compound 12 / FITC-oligo / 50% GALA-Ad compositions at a 5 +/− charge ratio. DNA uptake is monitored as revealed in BHK-21 cells. Fluorescence profiles for untransfected HUH-7 cells are in the first decile (FIG. 5A). FITC-oligo is successfully delivered by compound 12 to 95% of HUH-7 cells (FIG. 5B). Addition of 50% GALA-Ad to the composition inhibits FITC-oligo uptake in two size digits, as observed in BHK-21 cells.
[306] Example 34 Luciferase Transfection Efficiency of Compositions According to the Invention
[307] Transfection ability of GALA and GALA-Ad modified compositions is measured by delivery of the luciferase reporter gene to cultured cells. BHK-21 cells are plated on 24-well plates and transfected with 1 μg pGL-CV3 (plasmid containing luciferase gene) complexed with Compound 12 to form a particulate mixture at 5 +/− charge ratio. . These particulate mixtures are modified by the addition of GALA or GALA-Ad at various peptide / cyclodextrin ratios. Cells are lysed 48 hours after transfection and assayed for luciferase activity, and the results shown in FIG. 6 are reported in relative luminosity (RLU). Data is expressed as mean ± SD of 3 experiments. Background = 300RLV.
[308] Cells are successfully transfected with a Compound 12 / pGL-CV3 composition at RLU ˜1 × 10 ⁵ . The addition of GALA does not significantly affect transfection efficiency. However, modification of the composition with GALA-Ad significantly inhibits transfection. 1% GALA doubled transfection with 2 × 10 ⁵ RLU and Compound 12 / pGL-CV 3/10% GALA also resulted in slightly higher transfection (1.5 × 10 ⁵ RLU). The addition of 100% GALA reduced transfection by 50% with 5x10 ⁴ RLU.
[309] Example 35 Toxicity of GALA and ALA-Ad Compositions
[310] Toxicity of GALA and ALA-Ad modified compositions is confirmed by measuring protein concentrations of cell lysates obtained in transfection experiments. BHK-21 cells are transfected with 1 μg pGL-CV3 complexed with compound 12 at a 5 +/− charge ratio. Prior to transfection, various lq rates of GALA and ALA-Ad are added to the complex. Cell viability for transfection in the presence of GALA (filled bar) and ALA-Ad (white bar) was analyzed 48 hours after transfection and analyzed for total protein concentration and protein levels for untransfected cells. Verify by standardizing the sample. Protein concentrations are expressed as mean ± SD of 3 replicates, which are averaged and divided by the average protein concentration of cells transfected with Compound 12 / pGL-CV3 composition alone (Figure 7). The addition of GALA and GALA-Ad to the transfection solution results in observable toxicity to BHK-21 cells.
[311] Example 36 lactose-β-cyclodextrin-DMS copolymer, compound 20 (Lac-β-cyclodextrin-DMS copolymer)
[312] Compound 12 (20.5 mg, 3 μmol), 10 eq α-lactose (21 mg, 60 μmol, Sigma), 18.6 mg sodium cyanoborohydride (300 μmol) is added to the glass vial. 1 ml borate buffer, pH 8.5, is added to the solid and the resulting solution is briefly stirred and incubated for 30 hours in a 37 ° C. water bath. The solution is acidified to pH 6.0 with the addition of 1M HCl and dialyzed in water for 24 hours. TNBS analysis for polymer amines confirmed 87% conjugation. Structure of Compound 20.
[313] Example 37 Lactose- (CH ₂ ) ₆ -β-cyclodextrin-DMS Copolymer, Compound 21 (Lac-C6-β-cyclodextrin-DMS Copolymer)
[314] Compound 12 (43.2 mg, 7.4 μmol) and 5.6 eq mono (lactosylamido) mono (succinimidyl) suberate (50 mg, 84 μmol, Pierce) were added to a glass vial equipped with a magnetic rod and 2 ml Dissolve in 50 mM NaHCO ₃ . The resulting solution is stirred overnight. The reaction was followed by monitoring the disappearance of polymer amine end groups by TNBS analysis, where 90% conjugation was identified. The solution is acidified to pH 5.0 with 1M HCl addition and the resulting solution is dialyzed in water for 2 hours in Pierce MWCO 3500 Slide-A-Lyzer prior to freeze drying. A white downy powder is obtained in 70% yield. The structure of compound 21 is shown in FIG. 12.
[315] Example 38 PEG ₃₄₀₀ -terminated β-cyclodextrin-DMS copolymer, compound 22; Pre-DNA Complexing PEGylation
[316] 20.3 mg Compound 12 (3 μmol) and 10 eq FMOC-PEG _3400- NHS (190 mg, 60 μmol) are added to a glass vial equipped with a magnetic rod and dissolved in 1 ml of 50 mM NaHCO ₃ , pH 8.5. The solution is stirred for 20 hours in the dark at room temperature and lyophilized. The solid is dissolved in 0.5 ml 20% piperidine dissolved in DMF and stirred for 30 minutes for FMOC deprotection. The solvent is removed in vacuo, the remaining viscous liquid is dissolved in water and the pH is acidified to 6.0 or below with 0.1 M HCl. The polymer is separated from unreacted PEG by anion exchange chromatography and lyophilized to yield a white downy powder. The structure of compound 22 is shown below.
[317]
[318] Pre-DNA Complexing PEGylation . Compounds 12 and 22 are mixed with plasmid DNA for particle size determination. βCDP6 Compound 12 may be used to convert plasmid DNA into hydrodynamic diameter; Pegylated Compound 22 cannot condense DNA, while condensing to 130 nm uniform particles. The presence of PEG at the polymer end disrupts DNA condensation.
[319] Example 39 (Comparative) Post-DNA Complexing PEGylation by Grafting
[320] The procedure used is modified in Ogris et al., Gene Therapy, 6, 595-605 (1999). 5 μg pGL3-CV dissolved in 500 μl dH ₂ O is mixed with equal volume of PEI (in dH ₂ O) at a charge ratio of 3 +/- or 6 +/-. Compound 12 / DNA particulate mixtures are prepared in the same manner at a 5 +/− charge ratio. The particle diameter of the particulate mixture is measured by dynamic light dispersion (DLS). After formation of the particulate mixture, PEG ₅₀₀₀ -SPA (10 mg / ml / DMF) is added and mixed for 2 hours at room temperature. In the second step after particle size measurement, 500 μl PBS, pH 7.2 is added to the solution. The solution is incubated for 30 minutes at room temperature and then the final particle size is measured by DLS (FIG. 8).
[321] In step 1, the PEI / DNA or Compound 12 / DNA particulate mixture is formed in 1.2 ml dH ₂ O. Particle size is measured by dynamic light dispersion. In step 2, PEG ₅₀₀₀ -SPA is added to the particulate mixture solution and reacted with the polymer primary amine group for 1 hour. The size of the "pegylated" sample is measured by DLS. In step 3, 600 μl PBS, pH 7.2 is added to each sample to check the salt stability of PEGylated particles. Particle size is measured 30 minutes after salt addition to confirm the degree of particle agglomeration.
[322] In step 1, the PEI particulate mixture is organized at 3 +/- and 6 +/- charge ratios and the Compound 12 / DNA particulate mixture is organized at 5 +/- charge ratios. PEG ₅₀₀₀ -SPA is described in Ogris et al. Add to PEI at 10: 1 w / w following the procedure described in Gene Therapy 6, 595-606, 1999. Compound 12 is PEGylated with 100%, 150%, 200% PEG / amino (mol%). As a control, unreacted PEG is also added to compound 12 in 100%. The particle diameter at each step is shown in the table of FIG. 9. The PEI particulate mixture increases slightly in size immediately after PEGylation (58 nm to 65 nm at 3 +/− charge ratio; 55 nm to 60 nm at 6 +/− charge ratio). PEGylation protects the PEI-particulate mixture from salt-induced aggregation. Unmodified PEI particles increase in diameter up to 800 nm after salt addition, while PEGylated PEI particulate mixtures are sized at 78 nm (at 6 +/- charge ratio) and 115 nm (at 3 +/- charge ratio). Slightly increased.
[323] Addition of 150% and 200% PEG ₅₀₀₀ -SPA to the compound 12-based particulate mixture results in particle destruction; The particle count decreases drastically and no consistent correlation function is observed. PEGylation of compound 12 is likely to prevent polymer / DNA binding. Particle size is maintained at 67 nm after PEGylation with 100% PEG ₅₀₀₀ -SPA. However, in monitoring particle size as a function of time, it was found that after about 30 seconds from PEG addition, the particles were destroyed and then small molecules were observed again. Thus, addition of 100% PEG ₅₀₀₀ -SPA can PEGylate a portion of compound 12. Since polymer 12 is added in excess compared to DNA (at a 5 +/- charge ratio), the unmodified polymer forms a polyplex with plasmid DNA and the particles are kept so that most PEGylated polymer remains free in solution. Can be rearranged. Salt addition to these particles causes particle agglomeration (300 nm), but not about the level of an unmodified 12 particulate mixture (700 nm). In summary, post-DNA complexation PEGylation in the reaction with polymer primary amino groups is believed to be effective for high MW polymers with high charge density. However, reaction with Compound 12, even post-DNA complexation, leads to a lack of salt stabilization at 100% PEG ₅₀₀₀ -SPA addition and particle breakdown at higher PEG ₅₀₀₀ -SPA concentrations.
[324] Example 40 Post-DNA Complexing PEGylation by Inclusion Complex Formation
[325] Using the following procedure, adamantane-PEG (Ad-PEG) molecules are added to cyclodextrin (mol%) in a solution of the composition previously formed with 100% adamantane. PBS is then added to the solution and particle size is monitored by DLS every two minutes. The results are shown in FIG.
[326] Procedure: 2 μg pGL3-CV dissolved in 600 μl dH ₂ O is mixed with an equal volume of compound 12 (in dH ₂ O) at a 5 +/− charge ratio. Desired content of Ad-PEG (10 mg / ml, in dH ₂ O) is added and particle size is measured by DLS. 600 μl PBS, pH 7.2 is added to the solution and particle size is monitored for 8 minutes at 2 minute intervals.
[327] The average diameter of unpegylated Compound 12 particles increases from 58 nm to 250 nm within 8 minutes after salt addition. The presence of free PEG in solution does not prevent aggregation (average diameter of 240 nm after salt addition). However, PEGylation through inclusion complexes with linear Ad-PEG molecules reduces particle aggregation in a length dependent manner. The particles pegylated with salt added after 8 minutes, Ad-PEG ₃₄₀₀ are agglomerated to 210 nm in diameter, and pegylated with Ad-PEG ₃₄₀₀ -Lac particles are aggregated up to 200 nm in diameter. Pegylated particles with Ad-PEG ₅₀₀₀ increase in diameter to 90 nm after 8 minutes of salt addition and to 160 nm after 2 hours. Straining with Ad- (PEG ₅₀₀₀ ) ₂ slightly affects aggregation (particle diameter 200 nm after salt addition).
[328] Stabilization also proceeds in a PEG-density-dependent manner (FIG. 10A). The average particle diameter measured 10 minutes after salt addition increases 4.7 times (58 nm to 272 nm) in the unmodified polyplex, but only 1.2 in the polyplex modified with the addition of 150% or 200% adamantane cyclodextrin. Fold increases.
[329] Example 41 Reduced Cell Uptake by Post-Complexed PEGylation
[330] Step 1: The transfection mixture is prepared as follows: an equal volume of cationic compound 12 is added to 3 μg FITC-oligo (0.1 μg / μL in water) with 3 +/− charge ratio polymer: DNA. . To the complex is added free PEG or Ad-PEG ₅₀₀₀ (Example 40) in a 1: 1 PEG: cyclodextrin ratio.
[331] Step 2: HUH-7 cells are plated in 6-well plates at 3 × 10 ⁵ cells / well and maintained in 4 ml DMEM + 10% FBS + antibiotic / antifungal for 24 hours. After 24 hours, cells are washed with PBS and 1 ml Optimen containing one step of transfection mixture is added to the cells. After 15 minutes of incubation, the transfection medium is removed, the cells are washed with PBS and 1 ml Optimen is added to the cells in each well. The cells are incubated for another 30 minutes at 37 ° C. Cells are then washed with Cell Scrub Buffer (Gene Therapy Systems) to remove surface-bound complexes and PBS and separated from wells by trypsin treatment. The cells are then analyzed for FITC-oligo uptake by FACS assay. The results are shown in Table 1 below. Modification of the complex with Ad-PEG ₅₀₀₀ reduces the uptake of the FITC-oligo / polymer complex.
[332] Table 1.
[333] sampleTransfection Rate Cell alone0% Cell + FITC-Ups0% Cell + Particulate Mix + Free PEG43% Cell + Modified Ad-PEG ₅₀₀₀ Particulate Mixture27%
[334] Example 42 Compound 12 / Ad-PEG _3400- FITC Composition Formulation and Delivery to Cultured Cells
[335] BHK-21 cells are plated in 6-well plates at 200,000 cells / well and incubated at 37 ° C. for 24 hours. 3 ㎍ oligo (from _{0.1 ㎎ / ㎖, dH 2 O} ) is combined with (at _{2 ㎎ / ㎖, dH 2 O} ) 5 +/- 12 equivalent to the compound of the volume of the charge ratio. After 5 minutes of complexation time, 1.5 μl PEG-FITC or Ad-PEG-FITC (10 μg / ml at dH ₂ O) is added to the complex. The medium is removed from the cells and the cells are washed with PBS. For transfection, 940 μL Optimen is added to each treatment composition solution and the total solution is transferred to the cells. Cells are incubated with the transfection mixture for 4 hours, then the medium is removed and cells are washed with PBS and added to 4 ml complete medium. The cells are incubated for an additional 24 hours at 37 ml, then the medium is removed and the cells are washed twice with PBS. Cells are harvested by trypsinization and prepared for FAC analysis. Cells are washed twice in wash buffer (Hank Balance Salt Solution containing DNase and MgCl ₂ ) and in 500 μl FACS buffer (Hank Balance Salt Solution, 2.5 mg / ml Bovine Serum Albumin, 10 μg / ml Propidium Iodide). Resuspend. FACS assays are performed with the FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) And CellQuest software. 11 shows the results.
[336] Ad-PEG ₃₄₀₀ -FITC and sealed closure body formed increases the fluorescein absorbing substantially in the compound 12 incubated with _{Ad-PEG 3400 -FITC (43%} vs. 14%, Fig. 11). Free Ad-PEG ₃₄₀₀ -FITC in the medium can enter cells as part of the phagocytosis or endocytosis pathway. However, Ad-PEG ₃₄₀₀ -FITC can enter cells even when combined with compound 12. At low rates (10%) Ad-PEG ₃₄₀₀ -FITC modification of the 12 particulate mixture does not appear to inhibit internalization. Rather, the 12 particulate mixture readily binds to the cell surface and delivers Ad-PEG ₃₄₀₀ -FITC to the cell to internalize it. 12 particulate mixture-assisted delivery results in the higher fluresin fluorescence observed in Compound 12 / Ad-PEG ₃₄₀₀ -FITC transfected cells. The method can also be applied to co-delivery of small molecule therapeutic agents with genes of interest.
[337] Example 43 Transfection of HU47 Cells
[338] Luciferase Transfection . HUH-7 cells are plated in 24-well plates at 50,000 cells / well and incubated for 24 hours at 37 ° C. A 3 μg pGL3-CV plasmid (0.1 mg / ml at dH ₂ O) is combined with equal volumes of compound 12 or 21 (FIG. 13) at various charge ratios. Prior to transfection, the medium is removed from the cells and the cells are washed with PBS. 60 μl Optimen is added to each treatment composition to make a transfection solution, and 230 μl of the solution is added to each of the three wells for 4 hours. Medium is replaced after 24 hours of transfection and cells are lysed in 50 μl cell culture lysis buffer (Promega, Madison, WI) after 48 hours of transfection. Luciferase activity is analyzed with Promega's luciferase assay reagent. The results are shown in FIG.
[339] Example 44 Synthesis of Adamantane-Derived PEI (Ad-PEI)
[340] Polyethylenimine (PEI) and adamantane carboxylic acid are mixed in dry CH ₂ Cl ₂ and cooled to 0 ° C. DCC (1 eq.), 1-hydroxybenzoyltriazole (1 eq.), Traethylamine (1 eq.) Are added to the mixture. The solution is slowly warmed to room temperature and stirred for 16 hours. The precipitate is removed by filtration and then the solvent is removed in vacuo. Water is added to the remaining yellowish solid. Insoluble solids are removed by centrifugation. The aqueous solution is carefully transferred to the dialysis bag and dialyzed in water for 24 hours. After freeze drying, PEI-CD is obtained.
[341] Example 45 Synthesis of Cyclodextrin-PEG (CD-PEG)
[342] PEG-succinimidyl propionic acid (SPA) (Shearwater Polymers) and cyclodextrin-monoamine (1.2 eq.) Are dissolved in DMSO and stirred at room temperature for 24 hours. Cyclodextrin-PEG products are purified by dialysis.
[343] Example 46 Formation of Ad-PEI / DNA Particle Mixture and Subsequent Modification with CD-PEG
[344] 1 μg plasmid DNA (0.1 μg / μl, in dH ₂ O) is mixed with the Ad-PEI of Example 42 at a 5 +/− charge ratio. The CD-PEG (dissolved in dH ₂ O) of Example 46 is then added to the complex in the desired CD: Ad ratio.
[345] Example 47 Stabilization by PEGylation: Formulation at High Concentrations
[346] 4 μg plasmid DNA was substantiated with equal volume of polymer mixture (cyclodextrin polymer 12 at 2.5 +/− charge ratio 12, in some cases 1 CD: 1 PEG ₅₀₀₀ at various final DNA concentrations ranging from 0.1 mg / ml to 4 mg / ml). Butane-containing PEG ₅₀₀₀ or PEG ₅₀₀₀ ) (Fig. 14). Half of the solution is diluted with 1.2 ml water and the diameter is measured by dynamic light dispersion. The other half of the solution is passed through a Qiagen Qiaquick column to extract the DNA remaining in the solution. DNA concentration is measured by UV absorbance at λ = 260.
[347] Results (FIGS. 15 and 16): Small, uniform particulate mixtures (diameter <100 nm) modified with adamantane-PEG ₅₀₀₀ can be organized up to 4 mg DNA / ml concentration without precipitation. Unmodified polyplexes form large particles (> 300 nm) at concentrations greater than 0.2 mg / ml and extensive precipitation is observed at all formulation concentrations (> 50% DNA loss).
[348] Example 48 Inhibition of Non-Specific Absorption by Polyplex Surface Modification
[349] BHK-21 cells are plated in 6-well plates. Cells are transfected with 3 μg FITC-oligo (final concentration of transfection mixture: 0.05 mg DNA / ml) complexed with equal volume of compound 12 at 2.5 +/− charge ratio. The particulate mixture is then transformed into the following linker:
[350] Anionic Linker: WEAALAEALAEALAEAC
[351] Ad-anionic linker: Ad-WEAALAEALAEALAEAC
[352] Ad-PEG: Ad-PEG ₅₀₀₀
[353] Ad-anionic linker-PEG: Ad-WEAALAEALAEALAEAC-PEG ₅₀₀₀
[354] 1 ml Optimem is added to the transfection mixture and the whole solution is transferred to prewashed BHK-21 cells (washed with PBS) for 15 minutes. The medium is then removed, cells are washed with CellScrub, trypsinized and prepared for FAC analysis.
[355] Results: Compound 12 / DNA particulate mixture was modified with inclusion guest (adamantane), spacer (anionic linker), functional group (PEGD ₅₀₀₀ ) and inhibited non-specific uptake into cultured cells (FIG. 17) . Optimal inhibition is achieved with a combination of three components.
[356] Example 49 Galactose-Mediated Uptake into Liver Cancer Cells
[357] HepG2 cells are plated at 24-well plates at 50,000 cells / well. 1 μg pCMV-Luc is contacted with an equal volume of compound 12 and modified as described below. Modification with a PEG-containing complexing agent is carried out in a 2: 1 CD: PEG ratio, where CD represents cyclodextrin in compound 12.
[358] Compound 12 / pcMV-Luc Particulate Mixture-No Modification
[359] Glu-PEG-Pep-Ad Glucose-PEG ₃₄₀₀ -CAEAEAEAE-Ad, 2 CD: 1 PEG
[360] Gal-PEG-Pep-Ad Glucose-PEG ₃₄₀₀ -CAEAEAEAE-Ad, 2 CD: 1
[361] PEG
[362] PEG-Pep-AdPEG ₅₀₀₀ -CAEAEAEAE-Ad, 2 CD: 1 PEG
[363] 200 μl Optimem is added to each transfection mixture and transferred to each well of cells. After 4 hours of transfection, 800 μl complete medium is added to each well. Medium is removed, cells are washed with PBS and 1 ml complete medium is added to each well 24 hours after transfection. After 48 hours of transfection, cells are washed with PBS, lysed and analyzed for luciferase activity. The transfection process described above proceeds in the presence of 1 mM glucose or 1 mM galactose as a competitive inhibitor.
[364] Results: The particulate mixture modified with Glu-PEG-Pep-Ad or PEG-Pep-Ad has a negative zeta potential and therefore does not transfect cells. However, the polyplex modified with Gal-PEG-Pep-Ad shows enhanced transfection inhibited in the presence of free galactose, thus illustrating galactose-mediated transfection into liver cancer cells (FIG. 18).
[365] Example 50 Synthesis of Diamantane Compound
[366] References: Breslow, et al. JACS (1996) v 118 p8495-8496.
[367] Zhang et al. JACS (1993) v115 p9353-9354
[368] Anhydrous pyridine (5 ml) is placed in a reactor equipped with a small magnetic rod and cooled in a water bath. Methyldichlorophosphate (1.0 mL) is added dropwise. The mixture is kept cold for an additional 15 minutes during which time a precipitate of N-methylpyridinium dichlorophosphate is formed.
[369] Adamantane ethanol dissolved in 5 ml pyridine is added to the reactor, which is sealed after the reaction mixture is frozen. The resulting mixture is stirred overnight at room temperature. The sealed reactor is then opened and the resulting mixture is placed in 10% sodium bicarbonate (50 mL). The resulting solution is evaporated in vacuo. 800 ml water is added to the remaining solids and the product is extracted with 150 ml ether. The aqueous phase is acidified to pH 1.4 with 2N HCl and then extracted with 3 × 150 mL CHCl ₃ : nBuOH (7: 3). The organic layer is washed with water and the mixed solvent is evaporated in vacuo to give a solid phase. The solid is recrystallized from acetone / hexane to give a white solid in 27% yield. Electrospray mass spectroscopy identified the product as a pure requisite.
[370] Example 51 Synthesis of Diamantane-PEG ₅₀₀₀
[371]
[372] Dichloromethane is dried overnight in CaH ₂ under reflux and freshly evaporated prior to use in the reaction. Bis (2- (1-adamantyl) ethyl phosphate (in Example 51) dissolved in 0,4 mL dichloromethane in a stirred solution of PEG-epoxide (MW 5000) dissolved in fresh distilled dichloromethane (0.2 mL). The resulting diamantane compound) is added slowly, the resulting solution is stirred for 4 days at 35 ° C. The solvent is removed under vacuum until dry, 6 ml of water is added to the resulting solid, where a precipitate is made. The resulting mixture is stirred at room temperature for 30 minutes and then centrifuged to remove solids (unreacted diamantane compound) The supernatant is dialyzed overnight on a 3500 MWCO membrane in water and freeze-dried, where 99 Yield of white solid in% yield was identified by Maldi Tof analysis as desired product.
[373] Example 52 Competitive Substitution Experiment between Ad-PEG ₃₄₀₀ and Diamantane-PEG ₅₀₀₀
[374] Competitive uptake experiments are performed by adding diAdPEG ₅₀₀₀ solution to a preformed composition of AdPEG ₃₄₀₀ , polymer, DNA. The salt solution is then added and the particle size is measured as a function of time.
[375] The initial composition is formed by addition of a solution of compound 12 (16.6 μl water + 2.61 μl compound 12 (5 mg / ml) + 2.37 μl AdPEG ₃₄₀₀ (12.5 mg / ml) to DNA solution (20 μl DNA, 0.1 mg / ml)). Characteristics of such composition solutions are as follows:
[376] [DNA] = 0.05 mg / mL
[377] AdPEG _3400: Mole ratio of CD = 1: 1
[378] Charge ratio = 3 +/-
[379] Total tissue volume = 40 μl
[380] The composition is incubated 10 minutes before the addition of di-AdPEG5K solution (10 mg / ml). The volume of the solution is determined such that the molar ratio between diAdPEG ₅₀₀₀ and AdPEG ₃₄₀₀ is 1: 1, 1: 2, 1: 4 or 1: 6. For example, if the molar ratio is 1: 2, 2.38 μl of diAdPEG ₅₀₀₀ solution are added.
[381] After an additional 30 minutes of incubation, 1.2 ml water is added to dilute enough to be read by the DLS apparatus. Particle size is measured for 10 minutes, after which 600 μl 1 × PBS is quickly mixed into the composition solution. In addition, the particle size is measured every 1 minute for 30 minutes.
[382] For comparison, two different composition solutions are prepared. In one case, diAdPEG ₅₀₀₀ is not added. In other cases, AdPEG ₃₄₀₀ is not added. Under these conditions, the particulate mixture is not stabilized with the use of AdPEG ₃₄₀₀ . The salt increases the average particle size from 70 nm to 350 nm over the course of 30 minutes. However, diAdPEG ₅₀₀₀ alone shows stabilization in salts. Particle size remains constant after addition of the salt solution. This is also valid when diAdPEG5K is present in the 1/6 content of AdPEG ₃₄₀₀ . The results are shown in FIG.
[383] Example 53 pH Sensitive Adamantane-PEG Modifiers
[384]
[385] The binding constant between the encapsulating compound guest and host decreases as the guest / host is charged. For example, adamantanecarboxylic acid in protonated form (neutral form) has a binding constant of 500,000, while adamantanecarboxylic acid in unprotonated form (negative form) has a binding constant of ˜30,000. It can be used to incorporate pH-sensitive reactions in materials containing inclusion compounds. For example, it can be transformed into an adamantane-PEG (Ad-PEG) compound having a secondary amine in close proximity to adamantane. Ad-PEG compounds have high affinity at physiological pH but are more readily released at acidic pH as in cellular endosomes. Promoted unpackaging in the endosome promotes DNA release and cellular internalization of the polyplex.
[386] Synthesis of pH-Sensitive Hydrolysable Adamantane-PEG Modifiers
[387] PEG5K-NH ₂ (132 mg, 0.0264 mmol) is dissolved in water and cooled to 0 ° C. To the mixture is added NaOH solution (5N, 0.053 mL, 0.264 mmol, 10 eq) and 1-adamantyl fluoroformate (52 mg, 0.264 mmol, 10 eq) THF solution (3 mL). The mixture is stirred at this temperature for 5 minutes, then warmed to room temperature and stirred for 2 hours. THF is removed under vacuum. Insoluble solids are removed by centrifugation. The remaining aqueous solution is transferred to a Spectra / Por MWCO 3,500 membrane and dialyzed in water for one day. After freeze drying, adamantane carbamate-PEG5K (80 mg) is obtained. The structure of the compound is confirmed by ¹ H NMR, HPLC, MALDI TOF MS.
[388] Synthesis of Hydrolyzable Adamantane-Schiff Base-PEG
[389] PEG5K-ALD and 1-adamantanemethylamine (1 eq) are mixed in methanol. A few drops of formic acid are added to the mixture as a catalyst for the formation of the Schiff base. The mixture is stirred at 60 ° C. for 12 h, after which the solvent is evaporated in vacuo. The mixture is dialyzed in water to give the desired adamantane-Schiff base-PEG5K.
[390] Example 55 Synthesis of Adamantane-PEG-Transferrin (Ad-PEG-Tf) (FIG. 20)
[391] 1. Transferrin coupling through carbohydrate functional groups
[392] FMOC-NH-PEG _5000- NHS (Shearwater Polymers, 0.2 mmol, 1 g) is added to a round bottom flask equipped with a magnetic rod. To this was added tert-butyl carbazate (Aldrich, 1.6 mmol, 0.2112 g) dissolved in 7 mL dichloromethane / ethyl acetate (1: 1). The next day, the solvent is removed under vacuum. The FMOC group was removed by dissolving the resulting solid for 5 hours in 10 ml of 20% piperidine dissolved in dimethylformamide. The solvent is removed in vacuo and the residue is dissolved again in water. The resulting solution is centrifuged to remove undissolved FMOC groups and then dialyzed overnight at Pierce's Slide-A-Lyzer, 3500 MWCO. The solution is lyophilized to give 790 mg H ₂ N-PEG ₅₀₀₀ -NH-NH-CO-OtBu.
[393] Thereafter, N-hydroxysuccinimide (Aldrich, 0.24 mmol, 27.3 mg) and adamantanecarboxylic acid (Aldrich, 0.39 mmol, 71.2 mg) were dissolved in 7 ml dichloromethane H ₂ N-PEG ₅₀₀₀ -NH-. To NH-CO-OtBu (2) (0.16 mmol, 790 mg). To the resulting solution is added 1,3-dicyclohexylcarbodiimide (Aldrich, 1.6 mmol, 0.326 g) dissolved in 3 ml dichloromethane. The resulting solution is stirred overnight at room temperature. The next day, the resulting solid is filtered on a thin glass frit and the filtrate is concentrated in a rotary evaporator under vacuum. The residue is dissolved in 10 ml water and centrifuged to remove unreacted adamantanecarboxylic acid. The solvent is removed under vacuum and the residue is dissolved again in 6 ml of 4M HCl dissolved in dioxane to deprotect the t-butoxycarbonyl group. The resulting solution is stirred at room temperature for 4 hours. The solvent is then removed under vacuum and the residue is dissolved again in water. The resulting solution was dialyzed overnight in Slide-A-Lyzer, 3500 MWCO, Pierce and lyophilized to yield 635 mg Ad-PEG ₅₀₀₀ -NH-NH ₂ .
[394] Step 2: Transferrin-PEG-Ad conjugate synthesis
[395] A 100 mg (1.28 μmol) human transferrin (iron deficient) (Sigam-Aldrich) solution dissolved in 1 ml of 30 mM sodium acetate buffer (pH 5) is gel filtered on a Sephadex G-25 (Supelco) column. The resulting 4 mL solution (monitoring: absorbance at 280 nm) containing transferrin was cooled to 0 ° C. and 80 μl of 30 mM sodium acetate buffer (pH 5) containing 4 mg (19 μmol) sodium peroxide was added. do. The mixture is kept in a dark water bath for 2 hours. For removal of low molecular weight products, additional gel filtration (Sephadex G-25, 30 mM sodium acetate buffer, pH 5) is performed. Here a solution containing 85 mg (1.09 μmol) transferrin oxide is obtained. The modified transferrin solution is added immediately to a solution containing 54.5 mg (10.9 μmol) Ad-PEG ₅₀₀₀ -NH-NH ₂ dissolved in 1 ml of 100 mM sodium acetate (pH 5). The resulting solution is stirred overnight at room temperature. The pH is then brought to 7.5 by the addition of 1M sodium bicarbonate and four portions of 9.5 mg (150 μmol) sodium cyanoborohydride are added at 1 hour intervals. After 18 hours, pegylated transferrin is purified and concentrated on Centricon YM-50,000 NMWI apparatus (Millipore).
[396] Step 3: Iron-Dripping of Transferrin-PEG-Ad Synthesized by Transferrin Oxidation
[397] 40 mg apo-transferrin-based compound (apo-transferrin or apo-transferrin-PEC-Ad) is dissolved in 700 μl dH ₂ O. To the solution is added 200 μl of 5 mM iron citrate and 100 μl of 84 mg / ml NaHCO ₃ . The solution is left for 2-3 hours and then dialyzed in PBS overnight. The iron-dropping efficiency measures the ratio of absorbance (from oxidized iron) at 465 nm to absorbance (from tryptophan residues in protein) and measures the A ₄₆₅ / of commercially available holotransferin. Calculate normalized to A ₂₈₀ ratio. The iron drop efficiency for transferrin, freshly oxidized transferrin in transferrin, oxidation buffer (sodium acetate, pH 5) is measured and shown in FIG. 21. Oxidation of transferrin reduces the iron loading efficiency of the protein.
[398] Step 4: Binding Affinity of Transferrin-PEG-Ad (synthesized by transferrin oxidation) to transferrin receptor in PC3 cells
[399] PC3 cells are incubated with 250 nM fluresin-transferrin (FITC-TF) containing varying amounts of unlabeled transferrin and transferrin-PEG-Ad. FITC-hTF cell binding is assessed by FACS analysis. Unlabeled transferrin competes effectively with FITC-hTF, while transferrin-PEG-Ad competes very poorly with FITC-hTF, which is believed to be due to reduced affinity for the receptor. The results are shown in FIG.
[400] Example 56 Transferrin Coupling via Lysine Group (FIG. 23).
[401] Step 1: Synthesis of VS-PEG ₃₄₀₀ -Ad
[402] Vinylsulfone-PEG _3400- NHS (Shearwater Polymers, 0.147 mmol, 0.5 g) is added to a round bottom flask equipped with a magnetic rod and dissolved in 5 ml DMSO. To this is added adamantanemethylamine (Aldrich, 0.147 mmol, 0.0243 g). The resulting solution is stirred at room temperature for 1 hour. The solvent is removed in vacuo and the residue is dissolved again in water. The resulting mixture is dialyzed overnight in a 1000 MWCO membrane (Spectra Por). The solution is lyophilized to give 0.49 g vinylsulfone-PEG ₃₄₀₀ -Ad.
[403] Step 2: Synthesis of Transferrin-PEG-Ad (Tf-PEG-Ad) Conjugates
[404] 250 mg (3.21 μmol) human transferrin (iron poor) (Sigam-Aldrich) solution dissolved in 10 mL of 0.1 M sodium tetraborate buffer (pH 9.4) was added 109 mg (32.1 μmol) vinylsulfone-PEG _3400- Ad. do. The resulting solution is stirred at room temperature for 2 hours. PEGylated transferrin is purified from unreacted vinylsulfone-PEG ₃₄₀₀ -Ad using Centricon YM-50,000 NMWI device (Millipore) and unreacted transferrin using hydrophobic interaction column butyl-650S (Tosoh Biosep) (Confirmed by HPLC and MALDI-TOF analysis).
[405] Step 3: Iron-Dripping of Transferrin-PEG-Ad Synthesized by Coupling with Lysine Groups
[406] Apo-transferrin and Tf-PEG-Ad are iron-loaded according to the procedure described in Example 55. The degree of iron-dropping is quantified as described above. The iron-dropping efficiency of Tf-PEG-Ad synthesized by binding via lysine groups is almost 100%.
[407] Example 57 Binding Affinity of Transferrin-PEG-Ad (synthesized by binding via lysine) to transferrin receptor in PC3 cells
[408] PC3 is plated in 6-well plates at 125,000 cells / ml. After 24 hours, cells were treated at various concentrations of hTf, hTf-PEG-Ad (synthesized by the oxidation of hTf), hTf-PEG-Ad (synthesized and purified by the VS-lysine reaction), hTf- (PEG-Ad) ₂ (250 nMFITC-Tf mixed with (synthesized and purified by VS-lysine reaction)). Absorption after 20 minutes of exposure is measured by FACS. Unlike Tf-PEG-Ad synthesized by transferrin oxidation, Tf-PEG-Ad compound synthesized by lysine binding effectively competes with FITC-Tf for receptors on the PC3 cell surface. The results are shown in FIG.
[409] Example 58 Zeta Potential of Tf-Modified Polyplexes
[410] An eastern volume aliquot of compound 12 is added to an aliquot of plasmid DNA (2 μg DNA, 0.1 mg / ml in water) at a 3 +/− charge ratio to form a particulate mixture. Holo-transferrin or holo-Tf-PEG-Ad (17 mg / ml in water) is then added to the particulate mixture. The particles are diluted with the addition of 1.2 ml of water and the zeta potential is measured with a ZetaPals dynamic light scattering instrument (Brookhaven Instrument). The results are shown in FIG. Unmodified holo-transferrin binds to the particulate mixture in an electrostatic interaction. When 2 nmol Tf / μg DNA is added, the particulate mixture is close to neutral. Holo-transferrin-PEG-Ad (Tf-PEG-Ad in FIG. 25) is likely to bind to the particulate mixture by both electrostatic interaction and inclusion compound interaction. Thus, there is more binding between the Holo-Tf-PEG-Ad and the particles, as evidenced by the continuous reduction in zeta potential of the modified particles at higher concentrations of Holo-Tf-PEG-Ad. At 2 nmol Tf / μg, the particulate mixture modified with Holo-Tf-PEG-Ad is negatively charged (zeta potential ˜7 mV).
[411] Example 59 Synthesis of Ad-Phos-PEG ₅₀₀₀ -galactose
[412]
[413] The compound numbers below refer to the above formulas.
[414] I. Synthesis of Adamantanephosphonic acid. 2. Dibenzyl phosphite (0.712 g, 2.71 mmol) is injected into a solution of argon protected 1-adamantanemethylamine (0.493 g, 2.98 mmol) dissolved in dry CCl ₄ . White precipitate is observed immediately after addition of dibenzyl phosphite. The solution is stirred for 12 hours. To the mixture is added CH ₂ Cl ₂ (30 mL). The organic phase is washed twice (2x40 mL) with diluted acidic water (pH 4). The organic phase is then dried over MgSO ₄ . The solvent is evaporated in vacuo. The resulting white solid is crystallized with a solvent mixture of CH ₂ Cl ₂ and hexanes. Needle-shaped crystals (0.69 g) (1) are obtained in 60% yield. The crystals were subjected to hydrogenation for 16 hours using 10% Pd / C (200 mg) in ethanol (40 mL) at a pressure of 15 psi. The catalyst is removed by filtration. The filtrate solvent is removed in vacuo. Quantitative calculation of (2) is achieved. The resulting compound 2 is used without further purification.
[415] II. Synthesis of NH ₂ -PEG ₅₀₀₀ -galactose (4). FMOC-PEG _3400- NHS (Shearwater, 760 mg, 0.152 mmol) is dissolved in DMSO (3.7 mL). To the solution is added a solution of galactosamine (385 mg, 1.52 mmol) and diisopropylethylamine (0.264 mL, 1.52 mmol) dissolved in DMSO (14 mL). The solution is stirred for 20 minutes and then dialyzed for 24 hours in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution is then freeze dried to afford 745 mg FMOC-NH-PEG ₅₀₀ -galactose (3). (3) is dissolved in DMF (12 ml) containing piperidine (3 ml). The solution is stirred for 16 hours. The DMF is then removed under high vacuum. 40 ml water is added to the resulting solid. White solids are removed by centrifugation. The aqueous solution is dialyzed for 24 hours in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution is lyophilized to give 625 mg NH ₂ -PEG ₅₀₀ -galactose (4).
[416] III. Synthesis of Adamantane-Phos-PEG ₅₀₀ -galactose (5). (4) (63 mg, 0.013 mmol) is dissolved in imidazole buffer solution (1 mL, 0.1 N, pH = 6.5). To the solution was added a solution (2) dissolved in CH ₃ CN (4 mL), and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 100 mg, 40 eq.) Was added. Add. The solution is stirred for 16 h at room temperature. The solution is dialyzed in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.) and then lyophilized to afford (5).
[417] Example 60 Synthesis of Ad-Glu-Glu-PEG ₅₀₀ -galactose
[418]
[419] The compound numbers below refer to the above formulas.
[420] 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). To the mixture is added Z-Glu (Bn) -OSu (4.68 g, 10 mmol) dissolved in THF (30 mL). To the mixture is further added 30 ml THF, 20 ml CH ₃ CN, 10 ml 2N NaOH. The solution is stirred for 16 h at room temperature. THF and CH ₃ CN are evaporated under high vacuum. The pH is adjusted to 3 by addition of 1N HCl to the aqueous mixture. Precipitation is observed. The mixture is extracted with chloroform (3x30 mL). The organic phase is dried over MgSO ₄ . MgSO ₄ is removed by filtration. The organic solvent is evaporated to give a white sticky solid 6. (6) is used in the next step without further purification.
[421] (6) (3.51 g, 6.1 mmol) is dissolved in dry THF (40 mL). To this solution at 0 ° C. under argon, 1-adamantanemethylamine (1.007 g, 6.1 mmol), 1-hydroxybenzotriazole (0.93 g, 6.1 mmol), DCC (1.32 g, 6.4 mmol), diisopropyl Ethylamine (1.06 mL, 6.1 mmol) is added. The mixture is then warmed to room temperature and stirred overnight. The precipitate is filtered off. THF is removed under vacuum to give a yellow solid. The yellow solid is crystallized in methanol to give plate crystal 6 (2.1 g, 49%). (6) is then dissolved in 40 ml methanol and shaken in a hydrogenation reactor at 25-30 psi pressure in the presence of 200 mg 10% Pd / C. The catalyst is filtered after 24 hours. H-Glu-Glu-Ad (7) is obtained in quantitative yield after the removal of methanol under vacuum. (7) is used without further purification.
[422] Ⅱ. Synthesis of Ad-Glu-Glu-PEG ₅₀₀ -galactose (9). Vinylsulfone (VS) -PEG ₅₀₀ -NHS (Shearwater, 423 mg, 0.085 mmol) and galactosamine (216 mg, 0.85 mmol) are added to a PBS solution (2.25 mL, 1x, pH 7.2). The solution is stirred for 1 hour and then dialyzed for 24 hours in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution is then freeze dried. Product (8) is analyzed by MALDI-TOF and HPLC. (8) is dissolved in borax buffer solution (6 mL, 0.1 N, pH 9.4). Compound (7) (121 mg) is dissolved in DMSO solution (2 mL) and then added to the polymer solution. The mixture is stirred at 35 ° C. for 16 hours and subsequently at 50 ° C. for 7 hours. Monitor this reaction by HPLC. The polymer is dialyzed using a 3500 MWCO membrane and lyophilized to give 419 mg Ad-Glu-Glu-PEG ₅₀₀ -galactose (9) in 90% yield.
[423] Example 61 Synthesis of Ad-Glu-Glu-PEG ₅₀₀₀
[424]
[425] 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 is stirred at room temperature for 24 hours. The solution is then dialyzed for 24 hours in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution is lyophilized to give 276 mg Ad-Glu-Glu-mPEG ₅₀₀₀ (10). (10) is confirmed by MALDI-TOF MS, HPLC, ¹ H NMR.
[426] Example 62 Formulation of Transferrin with PEG-Modified Polyplex
[427] The polyplex (polymer to DNA charge ratio of 3 +/-) modified with Tf-PEG-Ad (or Tf- (PEG-Ad) ₂ ) and PEG-Ad (or PEG-Glu-Glu-Ad) is You can organize together. All components of equal volume are used. Tf-PEG-Ad (or Tf- (PEG-Ad) ₂ ) dissolved in water is added to the (12) solution dissolved in water. An aliquot of PEG-Ad (or PEG-Glu-Glu-Ad) is added to the mixed solution. Thereafter, a trimolecular mixture of polymers is added to the DNA solution. The solution is gently mixed by pipetting and particle size, zeta potential, salt stability are measured as described above. The zeta potential of the particles is Tf-PEG-Ad (or Tf- (PEG-Ad) ₂ ) vs. It can be adjusted by changing the relative proportion of PEG-Ad (or PEG-Glu-Glu-Ad). Some examples of zeta potential change and particle size as a function of particle deformation are shown in FIGS. 26, 27, 28.
[428] Example 63 adamantane-anionic peptide-PEG ₃₄₀₀ -galactose / glucose (Ad-pep-PEG-gal / glu).
[429] Anionic peptides (SEQ ID NO: E-A-E-A-E-A-E-A-C) are synthesized by the Biopolymer Synthesis Facility (Beckman Institute, California Institute of Technology) using an automatic sequencing device. Prior to cleaving the peptide from the resin, adamantane-carboxylic acid (ACA, Aldrich) is conjugated to the N-terminus of the peptide by DCC binding chemistry. The resulting peptide (ACA-E-A-E-A-E-A-E-A-C, MW 1084) is cleaved from the resin and analyzed by Maldi-TOF.
[430] Galactose- and glucose-PEG ₃₄₀₀ -vinyl sulfone (gal / glu-PEG _3400- VS) is composed of NHS-PEG _3400- VS (Shearwater Polymers) and 20 equivalents of glucosamine or galactosamine (Sigma) at phosphate buffered saline, pH 7.2. Is reacted at room temperature for 2 hours to obtain approximately 95% yield. The solution is dialyzed extensively in water and then lyophilized. Thiol of the anionic peptide (2 equivalents) is reacted with galactose-PEG ₃₄₀₀ -VS or glucose-PEG ₃₄₀₀ -VS in 50 mM sodium boronate buffer (pH 9.5) containing 10 mM TCEP. The solution is acidified and the precipitated peptide (insoluble below pH 9.0) is removed by centrifugation. Supernatants are harvested, extensive dialysis and lyophilized. The desired product is confirmed by Maldi-TOF assay (schematic shown below).
[431]
[432] Example 64 Synthesis of Naphthalene-PEG ₅₀₀₀
[433]
[434] 500 mg PEG ₅₀₀₀ -NHS (0.1 mmol, Shearwater Polymers) is added to a glass vial equipped with a magnetic rod. To this was added 146 μl 1-naphthalenemethylamine (1 mmol, 10 eq, Aldrich) dissolved in 8 mL dichloromethane and the solution stirred for 16 h. The solvent is then removed under vacuum. 20 ml water is added to the mixture. Insoluble residue is removed by centrifugation. The aqueous solution is dialyzed for 24 hours on a Spectra / Por 3500 MWCO dialysis membrane. The solution was lyophilized to give naphthalene-PEG ₅₀₀₀ as a white downy solid. The product is analyzed by ¹ H NMR, MALDI TOF MS, reverse phase HPLC. Naphthalene-PEG ₃₄₀₀ is synthesized by a similar protocol (56% yield; confirmed by Maldi-TOF analysis).
[435] Example 65 Synthesis of Naphthalene-PEG ₅₀₀₀ -galactose
[436]
[437] Vinylsulfone (VS) -PEG _5000- NHS (Shearwater, 423 mg, 0.085 mmol) and galactosamine (216 mg, 0.85 mmol) were added to PBS solution (2.25 mL, 1 ×, pH 7.2). The solution is stirred for 1 hour and then dialyzed for 24 hours in water (4 × 4 L) using a 3500 MWCO membrane (Spectra / Por 7, Spectrum Lab, Inc.). The solution is then freeze dried to yield vinylsulfone-PEG ₅₀₀₀ -galactose. The product is analyzed by MALDI-TOF and HPLC. 300 mg (0.06 mmol) of vinylsulfone-PEG ₅₀₀₀ -galactose are dissolved in borax buffer solution (3 mL, 0.1 N, pH 9.4). 1-naphthalenemethylamine (8.8 μl, 0.06 mmol) is dissolved in DMSO solution (3 mL) and then added to the polymer solution. The mixture is stirred at 55 ° C. for 36 h. The polymer is dialyzed using a 3500 MWCO membrane and lyophilized to give naphthalene-PEG ₅₀₀ -galactose.
[438] As will be appreciated, the foregoing description and examples are intended to detail the specific embodiments. It will be apparent to those skilled in the art that various modifications and changes can be made without departing from the spirit and scope of the invention. All patents, periodicals and other documents mentioned herein are incorporated by reference in their entirety.

权利要求:
Claims (28)
[1" claim-type="Currently amended] Adamantane Derivatives of Formula

here,
J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
,
Peptide or peptide residue, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
Ad is adamantyl;
R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
PEG is -O (CH ₂ CH ₂ O) _z -wherein z is 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 (= 0) O- or a carbohydrate moiety;
a is 0 or 1;
b is 0 or 1;
d is 0 to 6;
e is 1 to 6;
n is 0 to 6;
y is 0 or 1;
x is 0 or 1.
[2" claim-type="Currently amended] A composition containing a complexing agent having a particulate mixture of a polymer and a therapeutic drug and a functional group and an encapsulation complex of the polymer.
[3" claim-type="Currently amended] 3. The composition of claim 2, wherein the polymer has a host functional group and the complexing agent has a guest functional group.
[4" claim-type="Currently amended] 3. The composition of claim 2, wherein the polymer has a guest functional group and the complexing agent has a host functional group.
[5" claim-type="Currently amended] 3. The composition of claim 2, wherein the polymer forms a mixture with a complexing agent that retains the host and guest functional groups and retains the guest and host functional groups.
[6" claim-type="Currently amended] 6. The host functional group according to claim 3, 4 or 5, wherein the functional group is cyclodextrin, carcerond, cavitanal, crown ether, cryptand, cucurbituril, calicsarren (calixarene), spherand (spherand) or a mixture thereof.
[7" claim-type="Currently amended] 6. A composition according to claim 3, 4 or 5, wherein the complexing agent further comprises a spacer functional group.
[8" claim-type="Currently amended] The composition of claim 3, 4 or 5, wherein the encapsulation guest is selected from adamantane, diadamantane, naphthalene, cholesterol.
[9" claim-type="Currently amended] 10. The composition of claim 8, wherein the host functional group is cyclodextrin and the encapsulating guest is adamantane or diadamantane.
[10" claim-type="Currently amended] The method according to claim 2, 3, 4 or 5, wherein the functional group is a ligand, a nuclear batch signal, an endosome releasing peptide, an endosome releasing polymer, a second therapeutic agent, a stabilizing polymer / hydrophilic polymer or mixtures thereof, And the spacer functional group is selected from direct bonds, phosphate groups, polyethylene glycols, short anionic peptide sequences.
[11" claim-type="Currently amended] The method of claim 2, 3, 4 or 5, wherein the therapeutic drug is an antibiotic, steroid, polynucleotide, small molecule drug, virus, plasmid, peptide, peptide fragment, chelating agent, biologically active macromolecule, mixtures thereof The composition, characterized in that selected from.
[12" claim-type="Currently amended] 12. The composition of claim 11, wherein the therapeutic agent is a polynucleotide.
[13" claim-type="Currently amended] A method of delivering a therapeutic drug, characterized in that a therapeutically effective amount of the composition of claim 2, 3 or 5 is administered to a person in need thereof.
[14" claim-type="Currently amended] In a method of making a composition, the method consists of combining a therapeutic agent, a polymer having a host or guest functional group, a complexing agent to form a composition, wherein the polymer and the therapeutic agent form a particulate mixture and And the complexing agent forms an inclusion complex.
[15" claim-type="Currently amended] 15. The method of claim 14, wherein the therapeutic agent and polymer are first combined to form a particulate mixture, and then the particulate mixture is combined with a complexing agent such that the polymer and the complexing agent form an inclusion complex.
[16" claim-type="Currently amended] 15. The method of claim 14, wherein the polymer and the complexing agent are first combined to form an encapsulation complex, and then the encapsulation complex is combined with the therapeutic agent such that the polymer and the therapeutic agent form a particulate mixture.
[17" claim-type="Currently amended] A composition containing a particulate mixture of a cyclodextrin comprising a polymer and a therapeutic drug and a complexing agent comprising the cyclodextrin polymer and an inclusion guest selected from adamantane, diamantane, and a functional group.
[18" claim-type="Currently amended] 18. The composition of claim 17, wherein the therapeutic drug is selected from antibiotics, steroids, polynucleotides, small molecule drugs, viruses, plasmids, peptides, peptide fragments, chelating agents, biologically active macromolecules, mixtures thereof.
[19" claim-type="Currently amended] 19. The composition of claim 18, wherein the therapeutic drug is a polynucleotide.
[20" claim-type="Currently amended] 18. The composition of claim 17, wherein the complexing agent is an adamantane derivative of the formula:

here,
J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
,
Peptide or peptide residue, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
Ad is adamantyl;
R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
PEG is -O (CH ₂ CH ₂ O) _z -wherein z is 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 (= 0) O- or a carbohydrate moiety;
a is 0 or 1;
b is 0 or 1;
d is 0 to 6;
e is 1 to 6;
n is 0 to 6;
y is 0 or 1;
x is 0 or 1.
[21" claim-type="Currently amended] A method of delivering a therapeutic drug, the method comprising administering a therapeutically effective amount of the composition of claim 17 to a person in need thereof.
[22" claim-type="Currently amended] A chemical formula

here,
J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
,
Peptide or peptide residue, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
Y is an additional host or guest functional group;
R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
PEG is -O (CH ₂ CH ₂ O) _z -wherein z is 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 (= 0) O- or a carbohydrate moiety;
a is 0 or 1;
b is 0 or 1;
d is 0 to 6;
e is 1 to 6;
n is 0 to 6;
y is 0 or 1;
x is 0 or 1.
[23" claim-type="Currently amended] The compound of claim 22, wherein the host or guest is selected from adamantyl, naphthyl, cholesterol, cyclodextrin, mixtures thereof.
[24" claim-type="Currently amended] A chemical formula

here,
J is -NH-, -C (= 0) NH- (CH ₂ ) _d- , -NH-C (= 0)-(CH ₂ ) _d- , -CH ₂ SS-, -C (= 0) O -(CH ₂ ) _e -OP (= O) (O- (CH ₂ ) _e -Ad) O-,
,
Peptide or peptide residue, or -NH- (C = 0) -CH (R ¹ ) -NH- (C = 0) -CH (R ¹ ) -NH-;
Y is an additional host or guest functional group;
R ¹ is — (CH ₂ ) _a —CO ₂ H, ester or salt thereof; Or-(CH ₂ ) _a -CONH ₂ ;
PEG is —O (CH ₂ CH ₂ O) ₂ —, where z is 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 (= 0) O- or a carbohydrate moiety;
a is 0 or 1;
b is 0 or 1;
d is 0 to 6;
e is 1 to 6;
n is 0 to 6;
q is 1 to 5;
w is 1 to 5;
y is 0 or 1;
x is 0 or 1;
z is 1 to 5.
[25" claim-type="Currently amended] The compound of claim 24, wherein the host or guest is selected from adamantyl, naphthyl, cholesterol, cyclodextrin, mixtures thereof.
[26" claim-type="Currently amended] A composition comprising a particulate mixture of a cyclodextrin comprising a polymer and a therapeutic drug and an inclusion complex of the cyclodextrin polymer and a complexing agent comprising an encapsulating guest, wherein the complexing agent is a compound of claim 23 or 25. Composition.
[27" claim-type="Currently amended] 27. The composition of claim 26, wherein the therapeutic drug is selected from antibiotics, steroids, polynucleotides, small molecule drugs, viruses, plasmids, peptides, peptide fragments, chelating agents, biologically active macromolecules, mixtures thereof.
[28" claim-type="Currently amended] The composition of claim 27, wherein the therapeutic agent is a polynucleotide.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-12-19|Priority to US25634100P

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2000-12-19|Priority to US25634400P

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2000-12-19|Priority to US60/256,344

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2000-12-19|Priority to US60/256,341

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2001-05-29|Priority to US29354301P

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2001-05-29|Priority to US60/293,543

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2001-12-19|Application filed by 캘리포니아 인스티튜트 오브 테크놀로지, 인설트 테라페틱스, 인코퍼레이티드

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2001-12-19|Priority to PCT/US2001/048620

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2003-10-17|Publication of KR20030081354A

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2009-04-22|Application granted

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2009-04-22|Publication of KR100894186B1

优先权:

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申请号 | 申请日 | 专利标题

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US25634100P| true| 2000-12-19|2000-12-19|

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US25634400P| true| 2000-12-19|2000-12-19|

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US60/256,344|2000-12-19|

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US60/256,341|2000-12-19|

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US29354301P| true| 2001-05-29|2001-05-29|

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US60/293,543|2001-05-29|

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PCT/US2001/048620|WO2002049676A2|2000-12-19|2001-12-19|Compositions containing inclusion complexes|