KR20030068034A - Amphiphilic macrocyclic derivatives and their analogues - Google Patents

Abstract

A soluble amphiphilic macrocycle analogue having a lipophilic group attached to one side of the monomer constituting the macrocycle and a hydrophilic group attached to the other. Such amphiphilic macrocycle derivatives can be self-assembled in aqueous solvents to form micelles or vesicles and used as hosts for solubilization and / or stabilization of various compounds. Embodiments of the present invention utilize macrocyclic oligosaccharides and preferably use cyclodextrins as macrocycle derivatives for modification.

Description

Amphiphilic macrocyclic derivatives and their analogues

A representative example of a macrocyclic oligosaccharide is cyclodextrin, which is a cyclic oligosaccharide consisting of D-glucose residues linked together by α- (1-4) bonds. The most representative examples of cyclodextrins include 6, 7, or 8 α- (1-4) -binding D-glucopyranosyl units joined together to form a cylindrical molecule, each of α-, β- , And γ-cyclodextrin. Due to the conformation of the glucopyranose unit, all secondary hydroxyl groups are located on one edge of the cylinder and all primary hydroxyl groups are on the other. Inside the cylinder (cavity) of the molecule, the hydrogen atoms and glycosidic oxygen atoms align and become hydrophobic.

The cylindrical structure can be used as a host for trapping various compounds, usually organic compounds, within their cavities in the food, pharmaceutical and chemical industries. Cyclodextrins have been used to form a capture complex comprising hydrophobic molecules in which hydrophobic molecules are encapsulated within the coexistent hydrophobic pupil of the cyclodextrin macrocycle. This molecular encapsulation process increases the water solubility of the collected molecules and imparts other properties such as increased stability and lower volatility. It also makes it possible to regulate the availability of molecules such as bioavailability of drugs. See Uekama et al., CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3, 1-40 (1987).

Problems exist with the use of unmodified cyclodextrins to form capture complexes for the pharmaceutical industry. For example, low cost beta-cyclodextrins that are widely used are limited in their use due to their relatively low water solubility. R. B. Friedman, in US Pat. No. 4,920,214, discloses how water solubility of cyclodextrins can be significantly increased by modifying with alkylene carbonate to form hydroxyethyl ether. However, low solubility is still a problem for many unmodified cyclodextrins.

Another limitation in using cyclodextrin as a host of molecules is that the hydrophobic molecules that can be trapped are limited by the size of the central cavity. Several attempts have been made to modify the cyclodextrin structure to encapsulate other molecules of any size. To impart lipophilic properties, the 2- and 3-positions (secondary-hydroxyl side) of the glucose unit are transformed into lipophilic groups, while the 6-position (primary-hydroxyl side) is the same as the amino group. Transform into polar groups. Such derivatives are described by Skiba et al. In US Pat. No. 5,718,905, which together with other amphiphiles form monolayers, nanoparticles, and mixed concentration (solution) phases.

Similar derivatives with lipophilic substituents on the secondary side are described in various reports (P. Zhang et al., Journal of Physical Organic Chemistry 1992, 5, 518-528; A. Gulik et al., Langmuir 1998, 14, 1050-1057). D. Duchene and D. Wouessidjewe, Proc. Int. Symp. Cyclodextrins 8th, 1996, 423-430). Such derivatives have the property of forming aggregates of nanoparticles that can capture hydrophobic or hydrophilic guest molecules to a greater or smaller extent. However, the captured guest is released as soon as the solution medium and the nanoparticles come into contact (E. Lemos-Senna et al., Proc. Int. Symp. Cyclodextrins, 8th, 1996, 431-434). Such a system can capture both water soluble and insoluble drugs (M. Skiba et al., International Journal of Pharmaceutics, 1996, 129, 113-121). The self-assembly properties of amphiphilic cyclodextrins have been reviewed by Coleman et al. In Molecular Engineering for Advanced Materials, 1995, 77-97, Kluwer Academic Publishers (J. becher and K. Schaumberg eds). Cyclodextrins have also been modified by lipophilic groups at the 6-position (C.-C. Ling, R. Darcy and W. Risse, J. Chem. Soc. Chem. Commun., 1993, 438-440 Reference). Djedaini-Pilard et al. Describe in US Pat. No. 5,821,349 a cyclodextrin modified at the 6-position with an alkylamino group in order to integrate the collected hydrophobic guest molecules into other organic surfactant systems only. Amphiphilic cyclodextrins described to date are insoluble in water and are unable to form sufficiently stable micelles or vesicles with retainable structural properties of the molecules trapped in micelles or vesicles even after dilution in solution medium.

It is a first object of the present invention to form micelles and vesicles spontaneously in an aqueous solvent by molecular self-assembly to have a structure capable of retaining molecules trapped in micelles or vesicles even after dilution in the solution medium. To modify the macrocycle derivatives exemplified by cyclodextrins and other macrocyclic oligosaccharides that are advantageous for delivery. A second object of the present invention is to modify the surface of the micelles or vesicles of the present invention to facilitate specific attachment of the micelles or vesicles to specific cell membrane structures, thereby facilitating targeting and intercellular delivery of the captured therapeutic drug molecules. will be.

The present invention relates to soluble macrocycle derivatives of the kind that form micelles and vesicles used for encapsulation of molecules.

The present invention relates to a soluble amphiphilic macrocycle derivative in which a lipophilic group is attached to one side of the monomer constituting the macrocycle and a hydrophilic group is attached to the other.

1 is a chemical formula of a representative macrocyclic oligosaccharide, β-cyclodextrin,

2 is a schematic diagram of a modular design of macro-amphiphiles of cylindrical (a) and wedge (b) based on the core of the macrocycle,

3 is an electron micrograph of HE-SC ₁₆ -CD vesicles,

4 is an electron micrograph of HE-SC ₁₂ -CD vesicles,

5 shows elution of carboxyfluorescein (CF) trapped in HE-SC ₁₂ -CD, HE-SC ₁₆ -CD vesicles,

6 shows the release of CF from HE-SC ₁₆ -CD vesicles, and

FIG. 7 compares the transfection capacity of DOTAP with oligoethyleneoxy (hydroxyethyl) cyclodextrin (FE) -SC ₆ , -SC ₆ NH ₂ , -SC ₁₆ and -SC ₁₆ NH ₂ .

According to the present invention, a soluble amphiphilic derivative having a lipophilic group attached to one side of a unit constituting the macrocycle and a hydrophilic group attached to the opposite side of the macrocycle, wherein at least two hydrophilic groups are used for the macrocycle. Attached to one side of each unit forming; And by attaching at least one lipophilic group to the opposite side of each unit forming a macrocycle, thereby providing a soluble amphiphilic derivative wherein the number of hydrophilic groups present is always greater than the number of lipophilic groups.

The derivative is preferably an oligosaccharide derivative, more preferably a cyclodextrin derivative. However, in another embodiment, the oligosaccharide derivative is no longer a saccharide when too much derived, but still retains the basic cyclic structure that can be used as the derivative according to the present invention. These "non-oligosaccharide" molecules can be modified to include a relatively large number of lipophilic groups and hydrophilic groups, using the same chemical methods used to modify the oligosaccharide derivatives as described above, which are further described below. It explains in detail.

Modifications and effects of macrocycle derivatives, in summary, are more than the number of lipophilic groups in which one or more lipophilic groups are attached to one side of the derivative forming the macrocycle and the number of hydrophilic groups on the opposite side of the unit is present. It can be explained that what can be achieved in many cases. As a result, a unit is provided, which in turn provides a macrocycle having amphipathic characteristics. However, also due to the relative number of hydrophilic and lipophilic groups, amphiphilic macrocycles are soluble in aqueous solvents. Such soluble amphiphilic macrocycles have never been described in the prior art. A representative advantage of the amphiphile according to the invention is therefore that stable macrocycle aggregates can be self-assembled in an aqueous solvent (these aggregates are described in more detail below) and solubilization and / or stabilization of guest molecules is possible. .

According to one specific preferred embodiment, the lipophilic group is attached at the 6-position of the cyclodextrin molecule and the hydrophilic group is attached at the 2- and 3-positions. Depending on the number and effective size of the 6-position lipophilic groups and the number and effective size of the 2- and 3-positions, the resulting wedge or cylindrical macrocyclic amphiphiles (FIG. 2) Self-assemble into micelles or bilayer vesicles in aqueous solution. These micelles can encapsulate hydrophobic molecules, while bilayer vesicles can encapsulate hydrophobic or hydrophilic molecules.

The advantage of these macrocyclic cyclodextrin derivatives is that, as described above, they aggregate simultaneously to form very stable micelles or vesicles that are distinct from conventional liposomes, moreover they are water soluble. The unique aggregation properties of these derivatives can be usefully used to encapsulate drugs containing biological macromolecules such as proteins and DNA to enhance delivery of these therapeutic agents to individual sites of action.

In another embodiment, aggregates of macrocycle derivatives encapsulate other molecules.

In another embodiment, the aggregate of macrocycle derivatives encapsulate a therapeutic molecule for human or animal.

According to a preferred embodiment of the present invention, there is provided an amphiphilic macrocycle derivative characterized in that the cyclodextrin derivative of the following formula.

Wherein n is 5-11 or more and represents the number of modified glucose units in the macrocycle, which may be the same or different depending on the X- and R-groups.

X ₁ , X ₂ and X ₃ each independently provide a linking group; In another embodiment they are each independently a simple covalent bond or a dendrimer group; And according to another embodiment, an atom or radical having two or more valences, O, S, Se, N, P, CH ₂ , CH ₂ O, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfoxide May be a seed.

R ₁ independently provides a group that is totally lipophilic; Examples of R ₁ are H, saturated or unsaturated aliphatic or aromatic carbon or silicon radicals or halides thereof, and when R ₁ is a straight or branched aliphatic chain, the number of carbons can be between 2-18. R ₁ may be a cyclic aliphatic structure such as hexyl or cholesteryl. Examples of R ₁ are benzyl and pyridyl.

R ₂ and R ₃ independently provide groups which are generally polar and / or capable of hydrogen bonding. Examples of R ₂ and R ₃ are H, (CH ₂ ) _2-4 OH, CH ₂ CH (OH) CH ₂ OH, CH ₂ CH (OH) CH ₂ NH ₂ , CH ₂ CH ₂ NH ₂ ; Cations such as protonated amino groups; Anions such as sulfate and sulfonato; Pharmaceutically acceptable ions; There is a group that is totally hydrophilic.

R ₂ and R ₃ may be dendrimers and may include polymer groups such as poly (ethyleneimine) (PEI), polyamides, polyamino acids such as polylysine; Or groups having polar as well as non-immunogenic properties such as poly (ethylene glycol) or sialylGalGlcNAc; Antigenic groups, such as antenna oligosaccharides, intended to promote the production of antibodies; Or a group such as lactosyl, which may be attached for the purpose of facilitating the attachment of an amphiphile or its complex with a guest molecule to a particular cell or a particular protein. On the second side of the modified cyclodextrins modified with other groups known in the art as specific ligands for cellular receptors, such as folic acid, galactose, biotin, lipopolysaccharide, gangliosides, sialo-gangliosides, glycosphingolipids, etc. By attachment, targeting ligands may appear on the outer surface of micelles or vesicles of the invention. The groups can be grouped to promote "cognition" by other molecules, including multifunctional interactions. If these groups are polymers or dendrimers, they may be conjugated to the amphiphiles, for example by active polymerization; Or where the amphiphile is a copolymer, it can be cross-linked by a divalent or polyvalent reagent such as, for example, an activated diacid compound or diepoxide, or copolymerized into a polylactic or glycolic acid substrate.

Coupling of vesicles or micelles with antibodies of the invention may be another pathway to target specific cell types. Synthetic procedures for antibody coupling are known in the art and can be applied to the modified cyclodextrins of the present invention, which method provides a free amino group for biotinylation, or N-letters, in a second aspect. Providing a free carboxyl group for peptide coupling of the antibody via rutaryl detergent dialysis, or a maleimide for sulfhydryl antibody coupling, or pyridyl for sulfhydryl and maleimide antibody coupling Dithiopropionate is provided, or similar methods known in the art can be used.

In another embodiment, the macrocycle derivative is in bis (cyclodextrin amphiphilic) form wherein two amphiphilic cyclodextrins of the form are linked by one or more lipophilic groups by sharing the R ₁ group It provides a 'bola amphiphile' characterized in that it comprises two polar CD molecules, thus (R ₂ , R ₃ ) -macrocycle- (R ₁ ) -macrocycle- (R ₂ , R ₃ ) In this case, the connection group X can be known. In another embodiment, the bis-amphiphile comprises a bolazin in which a common set of lipophilic groups (R ₁ ) and a common macrocyclic molecule connect two sets of polar head groups (R ₂ , R ₃ ). As an amphoteric form, it is (R ₂ , R ₃ ) (R ₁ ) -macrocycle- (R ₂ , R ₃ ), wherein the linking group X is known. The advantage of the vololatic amphiphile, which comprises a polar group at each end, is that the vesicles are assembled into a single layer of molecules.

In another embodiment, group X ₁ or group X ₂ and X ₃ , or group R ₁ or group R ₂ and R ₃ are chemical precursor groups having a multivalent binder or by reaction of chemical precursor groups through a catalyst By independent reactions, they can be linked intermolecularly to each other as separate sets. Examples of catalysts may be photochemical irradiation and the like.

In another embodiment, group X ₁ or group X ₂ and X ₃ , or group R ₁ or group R ₂ and R ₃ are chemical precursor groups having a multivalent binder or by reaction of chemical precursor groups with a catalyst By reaction of, it is possible to provide an oligomeric cyclodextrin which is oligomerized by being intermolecularly linked to each other as an independent set.

In another embodiment, an amphiphilic macrocycle derivative is provided wherein the unit forming the macrocycle is a monosaccharide unit forming an oligosaccharide macrocycle having the formula:

Wherein n is 3-11 or more and represents the number of modified monosaccharide units in the macrocycle, which may be the same or different depending on the X- and R-groups, and X ₁ , X ₂ , and X ₃ , R ₁ , R ₂ and R ₃ have the same meanings as described above.

Examples of the macrocycle derivatives include modified units constituting the macrocycle, each independently, L-glucose, or mannose, galactose, glucose, altose, idose, rhamnose (R ₁ X ₁ = CH ₃ ), or Being an aglycone derivative of D- or L-hexose such as arabinose (R ₁ X ₁ = H); Or wherein the macrocycle is a disaccharide or oligomer such as lactose.

R ₂ and R ₃ may be dendrimers and may include polymer groups such as poly (ethyleneimine) (PEI), polyamides, polyamino acids such as polylysine; Or groups having polar as well as non-immunogenic properties such as poly (ethylene glycol) or sialylGalGlcNAc; Antigenic groups, such as antenna oligosaccharides, intended to promote the production of antibodies; Or a group such as lactosyl, which may be attached for the purpose of facilitating attachment to a specific cell or a specific protein of an amphiphile or a complex with a guest molecule thereof. Other similar groups known in the art as specific ligands for cellular receptors, such as folic acid, galactose, biotin, lipopolysaccharide, gangliosides, sialo-gangliosides, glycosphingolipids, etc., the polarity of modified oligosaccharides or oligosaccharide analogs By attaching to cotton, targeting ligands may appear on the outer surface of the micelles or vesicles of the invention. The groups can be grouped to promote "cognition" by other molecules, including multifunctional interactions. If these groups are polymers or dendrimers, they may be conjugated to the amphiphiles, for example by active polymerization; Or where the amphiphile is a copolymer, it can be cross-linked by a divalent or polyvalent reagent such as, for example, an activated diacid compound or diepoxide, or copolymerized into a polylactic or glycolic acid substrate.

Coupling of vesicles or micelles with antibodies of the invention may be another pathway to target specific cell types. Synthetic procedures for antibody coupling are known in the art and can be applied to the modified oligosaccharides or analogs of the invention, which methods are polar in nature, providing free amino groups for biotinylation, or N-letters. Providing a free carboxyl group for peptide coupling of the antibody via rutaryl detergent dialysis, or a maleimide for sulfhydryl antibody coupling, or pyridyl for sulfhydryl and maleimide antibody coupling Dithiopropionate is provided, or similar methods known in the art can be used.

In another embodiment, the amphiphile is in bis (amphiphilic) form, in which two macrocyclic molecules of the form share an R ₁ group, thereby connecting two polarities linked by one or more lipophilic groups It provides a 'bola amphiphile' characterized in that it comprises a macrocycle molecule, and thus, (R ₂ , R ₃ ) -macrocycle- (R ₁ ) -macrocycle- (R ₂ , R ₃ ), In this case, the connection group X can be known. In another embodiment, the bis-amphiphile comprises a bolazin in which a common set of lipophilic groups (R ₁ ) and a common macrocyclic molecule connect two sets of polar head groups (R ₂ , R ₃ ). As an amphoteric form, it is (R ₂ , R ₃ ) (R ₁ ) -macrocycle- (R ₂ , R ₃ ), wherein the linking group X is known. The molecules of the monolayer can be assembled to form vesicles.

In another embodiment, group X ₁ or group X ₂ and X ₃ , or group R ₁ or group R ₂ and R ₃ are chemical precursor groups having a multivalent binder or by reaction of chemical precursor groups through a catalyst By independent reactions, they can be linked intermolecularly to each other as separate sets. Examples of catalysts may be photochemical irradiation and the like.

In another embodiment, group X ₁ or group X ₂ and X ₃ , or group R ₁ or group R ₂ and R ₃ are chemical precursor groups having a multivalent binder or by reaction of chemical precursor groups with a catalyst By the reaction of, it is possible to provide an oligomerized amphiphile by being intermolecularly linked to each other as an independent set.

Macrocycle derivative, characterized in that the unit forming the macrocycle has the following general formula.

Wherein n is 2-11 or more and represents the number of cyclic units forming a macrocycle, which may be the same or different,

If either K, L, or M is zero (thus providing the open-chain units rather than the cyclic as part of the macrocycle), the rest are each independently a simple chemical bond (thus a five-membered ring as in furanose sugars). Providing units); Or at least one of the atoms or radicals having two or more valences, which may also be located at any position not occupied by the moiety participating in the linkage with adjacent units forming a macrocycle.

Y is a group connecting macromolecular units, which may be the same or different, and includes, for example, an oxygen, sulfur, selenium, nitrogen, phosphorus, carbon or silicon radical having a valence of 2-4; Or OCH ₂ as in (1-2) -binding-fructofuranooligosaccharides; or OCH ₂ CH (OH) as in (1-6) -binding-furanooligosaccharides; or (1-5) -binding- OCH (CH ₂ OH) as in furanooligosaccharides.

X ₁ , X ' ₁ , X ₂ , X' ₂ , X ₃ , X ' ₃ , X ₄ , X' ₄ , X ₅ , X ₆ are each independently 0 or provide a linking group for the R group; It may be a simple covalent bond or a dendrimer group; Other examples may be atoms or radicals having two or more valences, CH ₂ , CH ₂ O, O, S, Se, N, P, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfoxide .

When one or more (but not all) of R ₁ , R ′ ₁ , R ₄ , and R ′ ₄ are each independently 0, the remainder is a group that is wholly lipophilic; Examples are H, saturated or unsaturated aliphatic or aromatic carbon or silicon radicals or halides thereof. When R ₁ -R ′ ₄ is a straight or branched aliphatic chain, n is preferably greater than 1 and the carbon number is 2-18. R ₁ -R ′ ₄ may be a cyclic aliphatic structure such as hexyl or cholesteryl, examples of aromatic groups include benzyl and pyridyl.

When one or more (but not all) of R ₂ , R ' ₂ , R ₃ , and R' ₃ are each independently 0, the remainder is a mainly polar and / or hydrogen-bondable group. Examples of R ₂ and R ₃ include H, (CH ₂ ) _2-4 OH, CH ₂ CH (OH) CH ₂ OH, CH ₂ CH (OH) CH ₂ NH ₂ , CH ₂ CH ₂ NH ₂ , and protons. Cations such as added amino groups; Anions such as sulfates and sulfonates; Pharmaceutically acceptable ions; Overall hydrophilic groups.

R ₂ , R ' ₂ , R ₃ , R' ₃ may be dendrimers and may include polymer groups such as poly (ethyleneimine) (PEI), polyamides, polyamino acids such as polylysine; Or groups having polar as well as non-immunogenic properties such as poly (ethylene glycol) or sialylGalGlcNAc; Antigenic groups, such as antenna oligosaccharides, intended to promote the production of antibodies; Or a group such as lactosyl, which may be attached for the purpose of facilitating attachment to a specific cell or a specific protein of an amphiphile or a complex with a guest molecule thereof. Polar aspects of oligosaccharides or oligosaccharide analogs modified with similar other groups known in the art as specific ligands for cellular receptors, such as folic acid, galactose, biotin, lipopolysaccharide, gangliosides, sialo-gangliosides, glycosphingolipids, etc. By attaching to the targeting ligand can appear on the outer surface of the micelles or vesicles of the present invention. The groups can be grouped to promote "cognition" by other molecules, including multifunctional interactions. If these groups are polymers or dendrimers, they may be conjugated to the amphiphiles, for example by active polymerization; Or where the amphiphile is a copolymer, it can be cross-linked by a divalent or polyvalent reagent such as, for example, an activated diacid compound or diepoxide, or copolymerized into a polylactic or glycolic acid substrate.

Coupling of vesicles or micelles with antibodies of the invention may be another pathway to target specific cell types. Synthetic procedures for antibody coupling are known in the art and can be applied to the modified oligosaccharides or analogs of the present invention, which methods are polar in nature, providing free amino groups for biotinylation, or N-letters. Providing a free carboxyl group for peptide coupling of the antibody via rutaryl detergent dialysis, or a maleimide for sulfhydryl antibody coupling, or pyridyl for sulfhydryl and maleimide antibody coupling Dithiopropionate is provided, or similar methods known in the art can be used.

R ₅ , R ₆ are groups which may be polar or lipophilic, preferably H.

Examples of such amphiphiles include disaccharides in which two or more monocyclic units forming a macrocycle are (1-1)-or (1-2)-or (1-3)-or (1-6) -linked disaccharides. Derived from or from disaccharide sucrose, or one or more monomers (cyclic or open-chain) constituting the macrocycle are carbohydrate analogs (physically or pharmaceutically, not natural carbohydrates or derivatives thereof for this purpose) As defined as a molecule capable of functioning usefully) or furanose sugar or sialic acid or fructose.

In another embodiment, the amphiphile is in bis (amphoteric) form, in which two macrocyclic molecules of the form share one group of R ₁ , R ₁ ', R ₄ , R ₄ ' It provides a 'bola amphiphile' characterized in that it comprises two polar macrocycle molecules connected by the above lipophilic groups, and thus, (R ₂ , R ₂ ', R ₃ , R ₃ ') -macro Cycle- (R ₁ , R ₁ ′, R ₄ , R ₄ ′) -macrocycle- (R ₂ , R ₂ ′, R ₃ , R ₃ ′), wherein the linking group X is known do. In another embodiment, the bis-amphoteric has a set of common lipophilic groups (R ₁ , R ₁ ′, R ₄ , R ₄ ′) and two sets of polar headgroups (R) with a common macrocyclic molecule. ₂ , R ₂ ', R ₃ , R ₃ ') is a volaic amphiphilic form connecting (R ₂ , R ₂ ', R ₃ , R ₃ ') (R ₁ , R ₁ ', R ₄ , R ₄ ′) -macrocycle- (R ₂ , R ₂ ′, R ₃ , R ₃ ′), wherein the linking group X is known. The molecules of the monolayer can be assembled to form vesicles.

In another embodiment, group X ₁ , X ' ₁ , X ₄ , X' ₄ , or group X ₂ , X ' ₂ , X ₃ , X' ₃ , or group R ₁ , R ₁ ', R ₄ , R ₄ 'or group R ₂ , R ₂ ', R ₃ , R ₃ 'may be reacted by a chemical precursor group via a catalyst (e.g. light irradiation) or by reaction of a chemical precursor group with a polyvalent binder. As a separate set, it may be intramolecularly linked to each other.

In another embodiment, group X ₁ , X ' ₁ , X ₄ , X' ₄ , or group X ₂ , X ' ₂ , X ₃ , X' ₃ , or group R ₁ , R ₁ ', R ₄ , R ₄ 'or groups R ₂ , R ₂ ', R ₃ , R ₃ 'are intermolecularly linked to each other as independent sets by reaction of chemical precursor groups with a catalyst or by reaction of chemical precursor groups with a polyvalent binder To provide oligomerized amphiphiles.

In another embodiment, the amphiphilic molecule (any of the above molecular forms or embodiments) is self-assembled in an aqueous solvent. After self-assembly, the resulting micelles or vesicles are partially separated from aqueous solvents by physical or chemical means, from alcohols or other polar solvents such as, for example, dimethyl formamide, dimethyl sulfoxide, tetramethylurea, dimethyl carbonate or polymers. To another phase, such as an aqueous phase, or to an emulsion, to a substrate in the form of a gel, or to a lyophilised suspension.

In another embodiment, the assembly of amphiphilic molecules consists of two or more of the above-described molecular forms or embodiments, such that the complementary properties of each amphiphile, eg, the property of controlling the colloidal stability of the assembly, or Molecular assemblies having the properties of cell-adhering with one another can be provided.

In another embodiment, an amphiphilic molecule can be mixed with other molecules, preferably with other amphiphiles, such as ceramides or glycerides, to control the properties of the assembly, for example to control its colloidal stability and the like.

In another embodiment, the amphiphile complexes with a therapeutic molecule for solubilization or stabilization, or for formulation into pharmaceutical compositions useful for the treatment of diseases of humans or animals.

In another embodiment, the drug forming a complex with an amphiphile is lipophilic or polar. The drug may be bound to the cavity of the macrocycle, to the lipophilic interior of the assembly, to the aqueous internal region of the amphiphilic assembly. Examples of drugs that can form complexes with the amphiphiles or that can be trapped within the lipophilic interior of the assembly or within the aqueous internal region of the lipophilic assembly include antitumor agents (paclitaxel, doxorubicin). , Cisplatin and the like); Anti-inflammatory agents (diclofenac, rofecoxib, celecoxib, etc.); Antifungal agents such as amphotericin B; Peptides, proteins, and analogs thereof, including those to which non-peptide groups such as carbohydrates, heme, and fatty acids are attached; Oligosaccharide analogues such as oligosaccharides, and sialyl Lewis analogs; Oligonucleotides and their analogs; Plasmid DNA; And complexes with gene transfer agents of DNA or oligonucleotides, and the like.

In another embodiment, an amphiphile comprises a molecule or atom, eg, a peptide antigen or an antibody, used for analysis or diagnosis; Or complexes with molecules used as radiation sensitizers, such as porphyrin.

In another embodiment, an amphiphile forms a complex with a molecule that functions as a prodrug such as a precursor of nitric oxide.

In another embodiment, the amphiphilic complex can be covalently bound to the polymer; Or the polymer may be conjugated to the amphiphilic molecule of the complex, for example by active polymerization; Or amphiphiles may be copolymers, for example, amphiphiles may be crosslinked by divalent or polyvalent reagents such as activated diacid compounds or diepoxides, or polylactic acid or polyglycolic acid It may be copolymerized into a substrate of.

In another embodiment, the guest molecule is covalently attached to the amphiphile. In other words, the guest molecule functions as the R-group specified above, thereby providing a precursor of the guest molecule in the active form, for example, providing a prodrug that is biodegradable to release the active form of the drug. .

In another embodiment, the amphiphilic-drug complex is prepared by sonication. The advantage here is that the composite is more easily absorbed by forming smaller particles.

In a preferred embodiment, the average particle diameter of the aggregates formed by the amphiphiles of the present invention is in the range of 50-500 nm.

In another embodiment, the amphiphile or complex thereof is a diluent, carrier, preservative (including antioxidant), binder, excipient, flavoring agent, thickener, lubricant, dispersant, wetting agent, compatible with the amphiphile or complex or aggregates thereof. In the form of a pharmaceutical formulation comprising a pharmaceutically acceptable component such as a surfactant, or a tonicity agent.

In another embodiment, the amphiphile or complex is dispersed in a suitable solvent, buffer, isotonic solution, emulsion, gel or liquidated suspension.

In another embodiment, an amphiphilic or complex is administered orally, topically, orally, in a dosage unit formulation that preferably is administered parenterally, but usually comprises a pharmaceutically acceptable non-toxic carrier, adjuvant and carrier. It may also be administered by an optional route of administration, including intranasal, intraocular, vaginal, rectal, or inhalation spray. The term parenteral, as used herein, includes subcutaneous injection, intravenous injection, intramuscular injection, intrascleral injection, spinal cavity injection, intraperitoneal injection or infusion technique.

The present invention provides an amphiphilic or amphiphilic-drug complex of a pharmaceutical formulation that exhibits sustained release of the drug. Such formulations are generally known and biodegradable multiples in which the active ingredient (here an amphiphilic or a complex thereof) is dispersed or covalently linked by unstable bonds or stored in reservoir form between polymer membranes. Designs comprising acids, or polyesters, or inert polymers. Sustained release is obtained through diffusion of the active ingredient through the polymer matrix or by hydrolysis of the covalent bonds present. Sustained release can also be achieved by delivery of the active ingredient through an osmotic pump, where the amphiphile can act as an osmotic inducer that provides the potential for the ingress of water.

Macrocyclic oligosaccharide molecules have lipophilic groups with lipophilic groups on one side of the macrocycle and polar hydrophilic groups on the other side. The relative effective volume of the combination of lipophilic and polar groups on either side of the molecule determines the morphology of the amphiphile (FIG. 2), which in turn determines the geometry of the self-assembly of the molecule (J. Israelachvili). , Intermolecular and Surface Forces, 2nd Edn., Academic Press, 1991, Chapter 17). Molecules with relatively small or less lipophilic groups and more or larger polar groups tend to be wedge-shaped and tend to form micelles, where the larger polar ends of the molecules point outward toward the solvent and the smaller lipophilic ends Is inward away from the solvent. In contrast, derivatives with similar effective volumes of lipophilic and polar ends are cylindrical and tend to form bilayers, which aggregate into vesicles with one or more bilayer walls (large lipophilic ends and small polarities). Molecules with terminals form inverted micelle phases in nonpolar solvents). This modification enables the capture of guest molecules into the lipophilic and water soluble interior of the molecular assembly as well as in the macrocycle cavity.

The invention will be more readily understood from the description of the following examples with reference to the accompanying drawings.

Example 1 illustrates the introduction of lipophilic groups into one side (primary side) of the cyclodextrin molecule. Examples 2 and 3 illustrate the introduction of a hydroxyethyl (oligoethyleneoxy) group as a polar group to the other side of the molecule. Example 4 was prepared before the concentration of amphiphilic cyclodextrin; Preparation of a complex with a hydrophilic (water soluble) host molecule, carboxyfluorine; And confirming that the life of the capture is at least 3 days. Example 5 illustrates the formation of a complex with a lipophilic guest molecule, azadipyrromethene. Example 6 describes the preparation of polyamino (polycationic) cyclodextrin amphiphiles. Example 7 describes the synthesis of cyclodextrin bola amphiphiles. Example 8 illustrates the use of cyclodextrin amphiphiles in the delivery of guest molecules (plasmid DNA) into cells, as measured by transfection results.

Example 1

A solution of hexanethiol (11 g, 93 mmol) dissolved in anhydrous dimethylformamide was stirred while removing moisture by adding potassium tert-butoxide (10.5 g, 93 mmol) under a nitrogen atmosphere. After 30 minutes, heptakis (6-bromo-6-deoxy) -β-cyclodextrin (7 g, 4.4 mmol) (Gadelle and Defaye, Angewandte Chemie, Int. Ed. Engl., 1991, 30, 78) Prepared according to the method described). The reaction mixture was stirred at 80 ° C. (5 days), then cooled and added to excess water. The resulting precipitate was filtered, washed repeatedly with water and methanol and finally stirred in hexane, filtered and vacuum dried (10 hours). The yield was 5.7 g (70%) and the melting point was 278 DEG C (decomposition).

¹ H NMR (270 MHz, CDCl ₃ ): δ 6.72 (d, J = 6.4 Hz, OH-2), 5.25 (s, OH-3), 4.97 (d, J = 3.1 Hz, H-1), 4.03- 3.89 (m, H-3, H-5), 3.74 (m, H-2), 3.49 (m, H-4), 3.07 (m, H-6a), 2.89 (m, H-6b), 2.61 (t, SCH ₂ ), 1.57-1.29 (m, CH ₂ ′), 0.89 (t, CH ₃ ) ppm.

¹³ C NMR (270 MHz, DMSO-d ₆ ): δ 105.9 (C-1), 88.7 (C-4), 76.6, 76.2, 75.5 (C-3, C-2, C-5), 37.4-32.0 ( C), 26.0 (C-6) ppm of the alkyl chain.

Elemental Analysis: Calculation of (C ₁₂ H ₂₂ O ₄ S) ₇ C 54.94, H 8.45, S 12.22; Experimental values C 55.9, H 8.95, S 12.35%

Example 2

Preparation of Heptakis (6-dodecylthio-2-oligoethyleneoxy) -β-cyclodextrin (HE-SC ₁₂ )

Heptakis (6-dodecylthio) -β-cyclodextrin (500 mg, 200 mmol), 50 mg K ₂ CO ₃ and 1.00 g (56 equiv) of ethylene carbonate were mixed in 5 mL of tetramethylurea. The K ₂ CO ₃ was not completely dissolved. The reaction mixture was stirred at 150 ° C. for 4 hours. Later in this time, TLC (silica, CHCl ₃ / MeOH / H ₂ O 50/10/1) resulted in a complete reaction with R _f of the starting material to zero and a single product with R _f of 0.5 It can be seen that formed. CO ₂ emissions also stopped. After cooling the reaction mixture to room temperature, the solvent was removed by rotary evaporator at 100 ° C. The crude product was separated into a brown viscous oil, taken up in 2 mL of methanol and purified by size exclusion chromatography using a lipophilic Sephadex LH 20-100 8 g column using methanol as eluent. A yellow waxy product (560 mg, 184 mmol, 89% yield) was obtained.

¹ H-NMR (CDCl ₃ ): δ 5.05 (br, 7H, H-1), 3.4-4.0 (m, 84H, H-2, H-3, H-4, H-5 and 14 x OCH ₂ CH ₂ 0), 3.00 (m, 14H, H-6), 2.60 (m, 14H, SCH ₂ ), 1.60 (m, 14H, CH ₂ ), 1.27 (br s, 126H, CH ₂ ), 0.89 (t, 21H, CH ₃ ) ppm.

¹³ C-NMR (CDCl ₃ ): δ 13.9 (CH ₃ ), 22.4 (CH ₂ ), 28.8 (CH ₂ ), 29.2 (CH ₂ ), 29.5 ((CH ₂ ) _n ), 31.7 (CH ₂ ), 33.4 (CH ₂ S), 33.4 (C-6), 61.2 (CH ₂ OH), 70.5-72.0 (C-2, C-3, C-5), 72.2 (CH ₂ O), 81.0 (C-4) , 100.7 (C-1) ppm.

Elemental Analysis: Calculated Value for (C ₂₂ H ₄₂ O ₆ S) ₇ , C 60.83, H 9.68, S 7.37; Found C 60.12, H 9.38, S 7.62%.

Electrospray MS: m / z from 2890 to 3067 (MNa ⁺ ) for deca (ethyleneoxy) product

Example 3

Preparation of heptakis (6-hexadecylthio-2-oligoethyleneoxy) -β-cyclodextrin (HE-SC ₁₆ )

Heptakis (6-hexadecylthio) -β-cyclo dissolved in 6 mL of tetramethylurea, as described in the synthesis of heptakis (2,3-hydroxyethyl, 6-thiododecyl) -β-cyclodextrin The product was obtained from 600 mg (213 mmol) of dextrin, 60 mg of K ₂ CO ₃ and 1.05 g (56 equiv) of ethylene carbonate. The crude product was purified by crystallization from 25 mL of methanol containing 20% acetone to separate as a light brown powder in 71% yield.

¹ H-NMR (CDCl ₃ ): δ 5.05 (br, 7H, H-1), 3.4-4.0 (m, 84H, H-2, H-3, H-4, H-5 and 14 x OCH ₂ CH ₂ O), 3.00 (m, 14H, H-6), 2.60 (m, 14H, SCH ₂ ), 2.00 (br, OH), 1.57 (m, 14H CH ₂ ), 1.30 (br s, 182H, CH ₂ ), 0.88 (t, 21H, CH ₃ ) ppm.

¹³ C-NMR (CDCl ₃ ): δ 14.1 (CH ₃ ), 22.7 (CH ₂ ), 29.2 (CH ₂ ), 29.4 (CH ₂ ), 29.5 (CH ₂ ), 29.7 (CH ₂ ), 29.8 ((CH ₂ ) _n ), 32.0 (CH ₂ ), 33.7 (CH ₂ S), 34.1 (C-6), 61.5 (CH ₂ OH), 71.0-72.5 (C-2, C-3, C-5), 72.6 (CH ₂ O), 81.2 (C-4), 100.9 (C-1) ppm.

Elemental Analysis: Calculated Value for (C ₂₄ H ₅₀ O ₆ S) ₇ C 63.67, H 10.20, S 6.53; Found C 62.90, H 9.47, S 6.77%.

Electrospray MS: m / z from 3196 to 3458 (MNa ⁺ ) for octa (ethyleneoxy) product

The properties of heptakis (6-dodecylthio-2-oligoethyleneoxy) -β-cyclodextrin and heptakis (6-hexadecylthio-2-oligoethyleneoxy) -β-cyclodextrin dissolved in water are as follows. .

The amphiphilic cyclodextrin is dispersed in water by sonication of a thin film (cast by slowly evaporating a solution of cyclodextrin dissolved in chloroform) in an sonication bath. HE-SC ₁₂ is sonicated at room temperature for 2 hours and HE-SC ₁₆ is sonicated at 50 ° C. for 2 hours. Dynamic light dispersion reveals the presence of vesicles having an average diameter of 170 nm. Cyclodextrin vesicles of 50-300 nm diameter were also identified using transmission electron microscopy using uranyl acetate as a backstain (FIG. 1). Continued sonication of the HE-SC ₁₂ solution (9 hours) yields a monodisperse solution of spherical vesicles with an average diameter of 60 nm (FIG. 2). Thus, in order to obtain a size suitable for the capture of a particular molecule or for a specific therapeutic use, the particle size can be adjusted according to the sonication time.

Heptakis (6-hexadecylthio-2-oligoethyleneoxy) -β-cyclodextrin was analyzed using differential scanning calorimetry. Heating scans in differential scanning calorimetry (DSC) showed a reproducible endothermic phase transition of 10% (w / w) aqueous dispersion. The transition occurred at about 48-49 ° C. and the enthalpy of the transition reached 59 kJ / mol cyclodextrin. This conventional L _β- L transition was confirmed by measuring the fluorescence polarization of diphenylhexatriene in the presence of the vesicle solution by the standard method described in RRC New, Liposomes: a practical approach, Oxford University Press, 1990. . Thus, vesicles of amphiphilic cyclodextrins depend on molecular structure and undergo thermal transitions that can affect important variables such as vesicle stability and bilayer permeability.

Example 4

Preparation of Vesicles of Amphiphilic Cyclodextrins Containing Carboxyfluorescein

The vesicles of heptakis (6-dodecylthio-2-oligoethyleneoxy) -β-cyclodextrin and heptakis (6-dodecylthio-2-oligoethyleneoxy) -βcyclodextrin are carboxyfluorescein (CF) It was prepared by sonication in a buffer solution. The capture of CF in the inner aqueous region of the cyclodextrin vesicles was confirmed by two independent experiments (i) and (ii) as follows; And in experiment (iii), the duration of capture was found to be more than 3 days.

(i) A small alicut of cyclodextrin vesicle solution (5-20 mM) was diluted 1000-fold, resulting in non-captured CF with incidental strong CF fluorescence immediately measured when diluted. The fluorescence of the trapped CF is negligible due to self-quenching. Next, the vesicles in the dilution solution were solubilized by adding 0.1% w / w of synthetic detergent Triton X-100, which resulted in an increase in CF fluorescence as the captured CF was released and diluted. . In this concentration range (approximately 20 mM), the fluorescence intensity of CF correlates linearly with that concentration, and the incremental change in fluorescence upon addition of Triton X-100 results in the volume of captured vesicles It is a direct measure of the percentage. The captured volumes reached 7.7 +/- 1.9% and 11.4 +/- 2.7% for the two independent preparations of HE-SC ₁₆ and 5.0 +/- 2.4% and 7.2 for the two independent preparations of HE-SC ₁₂ . +/- 5.3%.

(ii) CF trapped in the vesicles was separated from free (non-captured) CF by gel filtration using Sephadex G25. Independent turbidity measurements indicate that vesicles of HE-SC ₁₂ and HE-SC ₁₆ elute much faster than free CF. The peak of captured CF coincided with the elution of the vesicles (FIG. 4). This confirms the presence of an aqueous internal region in the vesicles. Furthermore, as expected, the amount of CF trapped in the cyclodextrin vesicles correlated with the cyclodextrin concentration.

(iii) Immediate release of CF from the vesicles of HE-SC ₁₆ (separated from free CF by gel filtration) was measured over time. At room temperature, leakage of CF was limited and vesicles maintained at least 75% CF after 3 days.

These experiments show that macrocyclic oligosaccharide vesicles can be encapsulated and maintain significant amounts of hydrophilic guest molecules in the region.

Example 5

Encapsulation of lipophilic (water insoluble) azadipyromemethene in vesicles of HE-SC ₁₆ cyclodextrin

Azadipyrromethene (fixed concentration) and HE-CD (variable concentration) solutions were prepared as follows: For solutions containing 0.05 mg / ml HE-CD, the HE-CD (25 mg / dissolved in chloroform) 20 μl ml solution), HE-CD-F (fluorescent label with methyl anthranilate) (10 μl 0.5 mg / ml solution dissolved in chloroform) and the azadipyrromethene (100 μl 20 mM solution dissolved in methanol) The combined solution was combined in a small bayer and the solvent was evaporated under a nitrogen stream. Subsequently, HEPES buffer (10 mM, 1 ml) was added followed by sonication (60 ° C., 1 hour). The fluorescence of the cyclodextrin and the absorbance of the dissolved (chemically bound) azadipyrromethene were measured and then measured again after one week. Table 1 (encapsulating azadipyrromethene into the vesicles of the HE-SC ₁₆ amphiphile) shows that lipophilic guests are chemically bound within the vesicle bilayer and / or cyclodextrin molecular cavities to effectively dissolve in water It is displayed.

Example 6

Synthesis of heptakis [2- (ω-amino-oligoethyleneoxy) -6-deoxy-6-hexylthio] -β-cyclodextrin.

(i) Preparation of heptakis [2- (ω-azido-oligoethyleneoxy) -6-deoxy-6-hexylthio] -β-cyclodextrin

Heptakis [6-deoxy-6-hexylthio-2- (ω-iodo-oligoethyleneoxy)]-β- dissolved in anhydrous dimethylformamide (25 ml) with sodium azide (625 mg, 9.5 mmol). Cyclodextrin (620 mg, 0.19 mmol) (prepared by the method described in the literature Mazzaglia et al., Eur. J. Org. Chem., 2001, 1715-1721) was stirred at 100 ° C. for 6 days. The reaction mixture was cooled down and the undissolved sodium azide was filtered off, then the solvent was evaporated in vacuo. After the organic residue was dissolved in chloroform, the insoluble matter was filtered off. The chloroform was evaporated to give crystalline product (300 mg, 60% yield).

¹ H-NMR (CDCl ₃ ): δ 5.07 (br, H-1), 3.5-4.2 (m, H-2, H-3, H-5, OCH ₂ ), 3.2-3.5 (m, H-4 , CH N ₃ ), 2.7-2.9 (m, H-6), 2.59 (m, SCH ₂ ), 1.57 (m, CH ₂ ), 1.29 (m, CH ₂ ), 0.89 (t, CH ₃ ) ppm.

¹³ C-NMR (CDCl ₃ ): d 14.1 (CH ₃ ), 22.6 (CH ₂ ), 28.7 (CH ₂ ), 29.7 (CH ₂ ), 31.6 (CH ₂ ), 33.8 (CH ₂ S, C-6) , 50.8 (CH ₂ N ₃ ), 70.0-71.9 (C-3, C-5, OCH ₂ ), 80.9 (C-2, C-4), 101.2 (C-1) ppm.

Elemental Analysis: Calculated Value for (C ₁₆ H ₂₉ O ₅ SN ₃ ) ₇ , C 51.18, H 7.78, N 11.19, S 8.54; Found C 50.07, H 7.67, N 10.14, S 7.69%.

(i) Preparation of heptakis [2- (ω-amino-oligoethyleneoxy) -6-deoxy-6-hexylthio] -β-cyclodextrin

Heptakis [2- (ω-azido-oligoethyleneoxy) -6-deoxy-6-hexylthio]-dissolved in anhydrous dimethylformamide (20 ml) with triphenylphosphine (1.4 g, 5.3 mmol). β-cyclodextrin (vacuum drying) was stirred at room temperature under nitrogen (5 hours). The reaction solution was then kept at 50 ° C. with a dropwise addition of concentrated ammonium hydroxide solution (8 ml) over 30 minutes. The reduction reaction was terminated after 24 hours at 45 ℃, and confirmed by thin layer chromatography (silica, CHCl ₃ -MeOH 5: 1) showed the absence of the starting material. The reaction mixture was concentrated in vacuo to a small volume and then water was added to give a precipitate of phosphorus compound which was filtered off. The pH of the filtrate was adjusted to 2 by the addition of HCl (1M), followed by evaporation in vacuo to afford the crude product, which was then extracted with boiling hexane in a Soxhlet extractor to remove residual phosphorus compounds. The yield of polyamine hydrochloride salt was 385 mg (56%).

¹ H-NMR (DMSO-d ₆ ): δ 8.2 (br s, NH ₃ ), 5.09 (br, H-1), 3.45-4.00 (m, H-2, H-3, H-5, OCH ₂ ), 3.36 (m, H-4), 2.98 (m, H-6), 2.58 (m, SCH ₂ ), 1.55 (m, CH ₂ ), 1.25-1.32 (m, CH ₂ ), 0.85 (t, CH ₃ ) ppm.

¹³ C-NMR (DMSO-d ₆ ) 14.1 (CH ₃ ), 22.9 (CH ₂ ), 28.9 (CH ₂ ), 29.6 (CH ₂ ), 31.3 (CH ₂ ), 33.7 (C-6, SCH ₂ ), 39.9 (CH ₂ NH ₃ ), 70.5-73.0 (C-3, C-5, OCH ₂ ), 80.1 (C-2, C-4), 101.7 (C-1) ppm.

Elemental Analysis: Calculated for (C ₁₆ H ₃₂ O ₅ NSCl) ₇ , C 49.87, H 8.36, N 3.63, S 8.31, Cl 9.18; Experimental value c 48.94, H 7.58, N 3.80, S 8.03, Cl 8.21%

Example 7

Synthesis of Heptakis [6- (12'-amino-dodecanoylamino) -6-deoxy-2-oligoethyleneoxy] -β-cyclodextrin

(i) Heptakis (6-azido-6-deoxy-2-oligoethyleneoxy) -β-cyclodextrin

Heptakis (6-azido-6-deoxy) -β-cyclodextrin (2 g, 1.5 mmol) (described in Parrot-Lopez et al., J. Am. Chem. Soc., 1992, 114, 5479-5480) Method) was dissolved in tetramethylurea (23 ml), followed by addition of potassium carbonate (0.2 g) and ethylene carbonate (6.7 g, 76 mmol). After heating the reaction mixture to 150 ° C. (4 h), TLC analysis at this time (silica, CHCl ₃ -MeOH 5: 1) indicated that the reaction was complete. After evaporation of the solvent in vacuo, the residue was dried in vacuo overnight and the product was purified by size exclusion chromatography (Sephadex LH-20, MeOH).

¹ NMR (DMSO-d ₆ ): δ 3.20-3.80 (m, H-2, H-3, H-4, H-5, OCH ₂ ), 4.53 (br, H-1) ppm.

MALDI-MS: m / z from 1774 to 1950 (MNa ⁺ ) for deca (ethyleneoxy) product

(ii) Preparation of Heptakis [6- (12'-amino-dodecanoylamino) -6-deoxy-2-oligoethyleneoxy] -β-cyclodextrin trifluoroacetic acid salt

Heptakis (6-azido-6-deoxy-2-oligoethyleneoxy) -β-cyclodextrin (0.183 g, 0.01 mmol) dissolved in methanol (10 ml) with triphenylphosphine (0.56 g, 2.13 mmol) ) Was stirred at room temperature (2 hours). A concentrated aqueous ammonia solution (40 ml) was added and stirring continued (22 hours). The solution was evaporated in vacuo and the residue was stirred with water (10 min). Acidified to pH 1 with hydrochloric acid (1 molar concentration), filtered and the filtrate was evaporated in vacuo. The residue was stirred with hexane (10 ml), filtered, redissolved in water (50 ml), concentrated in vacuo and purified by size exclusion chromatography (Sephadex G-25, water). The reduction product, heptakis (6-amino-6-deoxy-2-oligoethyleneoxy) -β-cyclodextrin (180 mg, dissolved in DMF (10 ml) and N-ethylmorpholine (85 μl, 0.08 mmol) 0.08 mmol) was treated after 1 hour with an active amino acid solution prepared as follows: 12'-N- dissolved in anhydrous DMF (10 ml) with dicyclohexylcarbodiimide (165 mg, 0.08 mmol) and 4A molecular sieve. tert-Butyloxycarbonylamino-dodecanoic acid (250 mg, 0.08 mmol) was stirred at 0 ° C. (1 hour) and then at room temperature (1 hour). The combined solution was stirred at room temperature (4 hours), filtered through Celite 520 and evaporated under vacuum to give a brown residue. The residue was dissolved in methanol and purified by size exclusion chromatography (Sephadex LH-20, methanol). The product was dissolved in methanol (10 ml), trifluoroacetic acid (2 ml) was added, followed by stirring at room temperature (1 hour) and the solution was evaporated to give the product of trifluoroacetic acid salt. MALDI-MS (free amine): The m / z series were 2805 to 2981 (MNa ⁺ ) for the deca (ethyleneoxy) product.

Example 8

DNA Encapsulation, and Cell Transfection

The amphiphilic cyclodextrin vesicles were prepared as follows: The CD was dissolved in chloroform; The solvent was removed under a nitrogen atmosphere to hydrate with twice distilled deionized water if the membrane remained. Large amounts of pre-generated vesicles and DNA solution are mixed or anhydrous CD membranes are reformed with DNA solution using an optimal mass ratio of CD: DNA of 10: 1 to encapsulate DNA (pCMVluc plasmid) and sonicated for size reduction Was done. Transfection studies were performed in Day1 COS-7 cells. CD-DNA complexes were added to the cells at a dose of 1 g of DNA per well for 4 hours in the presence of serum-free Opti-MEM, then serum-containing medium was added, and then the cells were further incubated for 20 hours. After replacing the medium with a new one, the cells were expressed for an additional 24 hours, and then the degree of luciferase expression was measured using the Promega Luciferase Assay Kit, and the Biorad Dc Protein Test Kit (Biorad). Dc Protein Assay Kit) was used to standardize on proteins. The results (FIG. 7) show that the CD causes a significant increase in transfection compared to unbound DNA, and can be close to commercially available vector DOTAP in terms of efficiency. Thus, the amphipathic CD can carry drugs, eg DNA, into biological cells.

It is believed that those skilled in the art can, to the maximum extent, utilize the present invention based on the above detailed description disclosed herein. Therefore, the specific embodiments are merely illustrative and are not intended to limit the remaining parts which are not disclosed in any case.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Claims (59)

As a soluble amphiphilic macrocycle derivative having a lipophilic group attached to one side of the monomer constituting the macrocycle, and a hydrophilic group attached to the other side, Two or more hydrophilic groups are attached to one side of each unit forming a macrocycle; And A macrocycle derivative, characterized in that the number of hydrophilic groups present is always greater than the number of lipophilic groups, with one or more lipophilic groups attached to the opposite side of each unit forming a macrocycle. The macrocycle derivative according to claim 1, wherein the units constituting the macrocycle have the following general formula. Wherein n is 2-11 or more and represents the number of cyclic units forming a macrocycle, which may be the same or different, If one of K, L and M is 0, the others are each independently a simple chemical bond; Or one or more of the atoms or radicals having two or more valences, and they can also be located at any position not occupied by the moiety participating in the linkage with adjacent units forming a macrocycle; Y is a group connecting the units forming a macrocycle, which may be the same or different, X ₁ , X ' ₁ , X ₂ , X' ₂ , X ₃ , X ' ₃ , X ₄ , X' ₄ , X ₅ , and X ₆ are each independently 0 or provide a linking group, When one or more (but not all) of R ₁ , R ′ ₁ , R ₄ , and R ′ ₄ are each independently 0, the remainder is a predominantly lipophilic group; When one or more (but not all) of R ₂ , R ' ₂ , R ₃ , and R' ₃ are each independently 0, the remainder is a mainly polar and / or hydrogen-bondable group; And R ₅ , R ₆ are polar or lipophilic groups. 4. The Y group according to claim 2, wherein the Y group is selected from the group comprising oxygen, sulfur, selenium, nitrogen, phosphorus, carbon or silicon radicals having a valence of 2-4, or is OCH _2, or Or OCH ₂ CH (OH), or OCH (CH ₂ OH). The compound according to any one of claims 2 to 4, wherein X ₁ , X ' ₁ , X ₂ , X' ₂ , X ₃ , X ' ₃ , X ₄ , X' ₄ , X ₅ , and X ₆ are each independently. Or a simple covalent bond or an atom or radical having two or more valences. The group of claim 4, wherein the radical is a group consisting of CH ₂ , CH ₂ O, O, S, Se, N, P, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfoxide, polymer, dendrimer Derivatives, characterized in that independently selected from. 6. The compound of claim 2, wherein R ₁ , R ′ ₁ , R ₄ , R ′ ₄ are independent from the group consisting of H, saturated or unsaturated aliphatic or aromatic carbon or silicon radicals or halides thereof. Derivatives, characterized in that selected. 8. The derivative of claim 7, wherein R ₁ is a straight or branched aliphatic chain having 2 to 18 carbon atoms. 8. The derivative of claim 7, wherein the cyclic aliphatic structure is a hexyl or cholesteryl group. 8. The derivative of claim 7, wherein the aromatic group is a benzyl group. The compound of claim ₂ , wherein R ₂ , R ′ ₂ , R ₃ , R ′ ₃ are each independently H, (CH ₂ ) _2-4 OH, CH ₂ CH (OH) CH _2. OH, CH ₂ CH (OH) CH ₂ NH ₂ , CH ₂ CH ₂ NH ₂ , cation, anion, pharmaceutically acceptable ions; Hydrophilic groups throughout; Derivatives selected from the group consisting of polymers and dendrimers. The method of claim 10, wherein the polymer comprises: poly (ethyleneimide) polyamide, polyamino acid, non-immunogenic polar group; Antigenic group; And a group promoting adhesion to a specific cell or protein. 12. The derivative of claim 11 wherein the non-immunogenic group is poly (ethylene glycol). 12. The derivative according to claim 11, wherein said non-immunogenic group is sialylGalGlcNAc. The derivative of claim 11, wherein the antigenic group is an antenna oligosaccharide. The derivative of claim 11, wherein the group promoting adhesion is selected from the group consisting of folic acid, galactose, biotin, lipopolysaccharide, ganglioside, sialo-ganglioside, and glycosphingolipid. The amphiphilic macrocycle derivative according to any one of claims 2 to 15, wherein the monomer forming the macrocycle is a monosaccharide unit forming an oligosaccharide macrocycle having the following formula. Wherein n is 3-11 or more and represents the number of modified monosaccharide units in the macrocycle, which may be the same or different depending on the X- and R-groups; X ₁ , X ₂ and X ₃ each independently provide a linking group; R ₁ independently provides a group that is totally lipophilic; R ₂ and R ₃ independently provide a group which is fully polar and / or capable of hydrogen bonding. 19. The derivative of claim 18, wherein the modified units constituting the macrocycle are independently aglycone derivatives of D- or L-hexose or disaccharides. 18. The derivative of claim 17, wherein the hexose unit is selected from the group consisting of mannose, galactose, glucose, altrose, idose, rhamnose, arabinose or disaccharide units. 19. The derivative of claim 18, wherein the macrocycle unit is L-glucose. (none) Amphiphilic macrocycle derivative, characterized in that the cyclodextrin derivative of the formula: Wherein n is 5-11 or more and represents the number of modified glucose units in the macrocycle, which may be the same or different depending on the X- and R-groups, X ₁ , X ₂ and X ₃ each independently provide a linking group; R ₁ independently provides a group that is totally lipophilic; R ₂ and R ₃ independently provide groups which are generally polar and / or capable of hydrogen bonding. 22. The derivative of claim 21, wherein the lipophilic group is attached at the 6-position of the unit constituting the cyclodextrin macrocycle, and the polar hydrophilic group is attached at the 2- and 3-positions. A derivative according to claim 21, wherein X ₁ , X ₂ , X ₃ are each independently a simple covalent bond or an atom or radical having two or more valences. The method of claim 22, wherein the radical is selected from the group consisting of O, S, Se, N, P, CH ₂ , CH ₂ O, carbonyl, ester, amido, amino, phosphate, sulfonyl, sulfoxide Derivatives. 22. The derivative according to claim 21, wherein R ₁ is selected from the group consisting of H, saturated or unsaturated aliphatic or aromatic carbon or silicon radicals or halides thereof. 26. The compound of any of claims 21-25, wherein R ₂ and R ₃ are dendrimers; Polymers; Groups having polar as well as non-immunogenic properties; Antigenic groups intended to promote the production of antibodies; Or a group which can be attached for the purpose of facilitating the attachment of an amphiphile to a particular cell or to a particular protein. 27. The derivative of claim 26, wherein the polymer is selected from the group comprising poly (ethyleneimide) polyamides, polyamino acids, non-immunogenic polar groups. 27. The derivative of claim 26, wherein said non-immunogenic group is poly (ethylene glycol). 27. The derivative of claim 26, wherein said non-immunogenic group is sialylGalGlcNAc. 27. The derivative of claim 26, wherein said antigenic group is an antenna oligosaccharide. 27. The derivative of claim 26, wherein said group promoting adhesion is selected from the group consisting of folic acid, galactose, biotin, lipopolysaccharide, ganglioside, sialo-ganglioside, glycosphingolipid. 31. The macrocycle derivative according to any one of claims 21 to 30, wherein the macrocycle derivative is in the form of a bis (cyclodextrin amphiphilic compound) in which two amphiphilic cyclodextrins share an R ₁ group. Providing a 'bola amphiphile' comprising two polar groups linked by a sex group, and thus, (R ₂ , R ₃ ) -macrocycle- (R ₁ ) -macrocycle- (R ₂ , R ₃ ) Wherein the linking group X is known. The macrocycle derivative according to any of claims 21 to 30, wherein the common lipophilic group (R ₁ ) set and the common macrocycle molecule connect two sets of polar headgroups (R ₂ , R ₃ ). Derivative, characterized in that the form of the vololar amphiphile, which is (R ₂ , R ₃ ) (R ₁ ) -macrocycle- (R ₂ , R ₃ ), wherein the linking group X is known. 32. The method according to any one of claims 21 to 31, wherein group X ₁ or groups X ₂ and X ₃ , or group R ₁ or groups R ₂ and R ₃ are reacted by a chemical precursor group through a catalyst, or Derivatives, characterized in that they can be intramolecularly linked to one another as a separate set by reaction of chemical precursor groups with a multivalent binder. 32. The method according to any of claims 21 to 31, wherein group X ₁ or groups X ₂ and X ₃ , or group R ₁ or groups R ₂ and R ₃ are reacted by a chemical precursor group with a catalyst, or A derivative characterized in that, by reaction of a group of chemical precursors with a multivalent binder, intermolecularly linked to each other as an independent set to provide an oligomerized amphiphilic cyclodextrin. 36. The derivative according to any one of claims 1 to 35, wherein the amphiphilic macrocycle is self-assembled in an aqueous solvent. 37. The derivative of claim 36, wherein the assembly of the amphiphilic macrocycle may comprise one or more of the molecular forms or embodiments described in any of the preceding claims. 38. The derivative of any of claims 1 to 37, wherein the amphiphilic macrocycle can be mixed with other molecules. 39. The derivative of claim 38, wherein said other molecule is selected to modulate the properties of the macrocycle assembly. 40. The derivative of claim 39, wherein said regulated molecule is ceramide or glyceride. 41. The derivative of any of claims 36-40, wherein the amphiphilic macrocyclic assembly forms a complex with a guest molecule. 42. The derivative of claim 41, wherein the guest molecule forms a complex with an amphiphilic macrocycle for formulation into a pharmaceutical composition useful for the treatment of a disease of a human or animal. 43. The derivative of claim 41 or 42, wherein said guest molecule complexed with an amphiphilic macrocycle has lipophilic properties. 43. The derivative of claim 41 or 42, wherein said guest molecule forming a complex with an amphiphilic macrocycle has a polarity. 45. The guest molecule of any of claims 41 to 44 wherein the guest molecule is bound to the cavity of each unit of the macrocycle, to the lipophilic interior of the assembly, to the aqueous internal region of the amphiphilic assembly, or to an amphiphilic compound. Derivative, characterized in that to form a complex. 46. The derivative according to any one of claims 36 to 45, wherein the amphiphilic assembly forms a complex with a molecule or atom used for analysis or diagnosis. The method of claim 46, wherein the amphiphilic assembly comprises a peptide antigen or an antibody; Or a complex formed with a molecule used as a radiation-sensitizer. 47. The derivative of claim 46, wherein said amphiphile forms a complex with a molecule that functions as a prodrug. 49. The derivative of claim 48, wherein said prodrug is a precursor of nitric oxide. 50. The derivative of any of claims 41 to 49, wherein the amphiphilic assembly can be attached to a polymer. The derivative of claim 36, wherein the amphiphilic assembly comprises units of copolymer. 53. The derivative of claim 51 wherein the amphiphilic complex is copolymerized into a polylactic acid or polyglycolic acid substrate. 53. The derivative of any of claims 41-52, wherein the guest molecule functions as an R-group as specified in claim 21, thereby providing a precursor of the guest molecule in active form. 55. The derivative of any of claims 41 to 53, wherein the guest molecule is a therapeutic molecule. 55. The derivative according to any one of claims 36 to 54, wherein the amphiphile or complex thereof is present as a pharmaceutical formulation comprising a pharmaceutically acceptable component. 55. The pharmaceutically acceptable component of claim 54, wherein the pharmaceutically acceptable component is a diluent, carrier, preservative (including antioxidant), binder, excipient, flavor, thickener, lubricant, dispersant, wetting agent, surfactant compatible with the amphiphile or complex Or derivatives comprising at least one of isotonic agents. 56. The derivative of claim 55, wherein the amphiphile or complex is dispersed in a suitable solvent, buffer, isotonic solution, emulsion, gel or lyophilised suspension. 57. The method of claim 41 to 56 wherein the amphiphile or complex is preferably a dosage unit formulation comprising a pharmaceutically acceptable non-toxic carrier, adjuvant and carrier, preferably parenteral, oral, topical, A derivative characterized by being administered by a route of administration including intranasal, intraocular, vaginal, rectal, or inhalation spray. The derivative of claim 36, wherein the amphiphilic or amphiphilic-therapeutic molecular complex comprises a pharmaceutical agent exhibiting sustained release of the drug.