JP2003506417A - Drug-carrier complex and method of using same - Google Patents

Abstract

(57) Abstract: Describes a drug-carrier complex, a drug carrier, a pharmaceutical formulation, a method for delivering a drug to an organism or tissue culture, a method for increasing the solubility of a substance, a targeting carrier, a drug delivery system, and an implant. I do. The compositions and methods of the present invention include forming a complex having a reversible association between the nucleotide and the drug. The compositions and methods of the present invention can be used to target drugs to cells, organisms or combinations of cells for the treatment of disease and for studying the mechanisms underlying the disease, and for testing drug candidates.

Description

Detailed Description of the Invention

RELATED APPLICATION This application claims the benefit of US Provisional Patent Application No. 60 / 147,919 (filed Aug. 9, 1999). All of its teachings are incorporated herein by reference.

BACKGROUND OF THE INVENTION Many drugs affect their action on cells by interacting (eg, binding) with nucleic acid sequences within the cell. The interaction of a drug with a nucleic acid sequence (eg, DNA of a cancerous cell) within a cell can arrest cell growth or result in cell death. This halts the progression of the disease state. However, many drugs used to treat diseases are either poorly soluble in aqueous solution or suitable for delivering the drug to cells or organisms (eg, mammals) in need of treatment. Either they have deleterious side effects, such as healthy cell death, due to the absence of the substance.

Many attempts have been made to overcome the problems commonly associated with drug delivery.
For example, polymeric drug carriers, most commonly water-soluble polymers with chemically associated drug molecules, often limit renal clearance in order to prolong the circulation of the drug. In particular, it has been used to increase drug accumulation in target tissues or cells and to reduce drug concentration in normal tissues. Several model and prototype carriers of this type have been developed.
Potentially, these carriers can be as small as 5-10 nanometers (nm), but larger (20-50 nm) depending on the structure and content of the drug.
Often form an aggregate. This type of carrier is intended to act essentially as a prodrug, ie as a drug substance as a result of the drug-carrier bond breaking. Some of this type of carriers target cancer cell markers. Examples of this type of drug-carrier are dextran-mitomycin conjugates; HPM with enzyme degradable peptide bonds between the drug molecule and the backbone polymer.
A-doxorubicin conjugates: There are doxorubicin-Fab conjugates that have a pH-sensitive bond between the doxorubicin molecule and the Fab. While potentially useful, this type of carrier has at least two potential drawbacks.

First, the release of drugs via the degradation of drug-carrier bonds is generally irreversible. Therefore, the drug released from the carrier circulates in the body independently of the carrier. This can reduce the efficacy of drug delivery. Release of drugs by enzyme-dependent or pH-dependent hydrolysis has been reported to improve the ratio of drug activity at the target to normal tissue. However, the expression of enzymes (such as proteases) in tumors and other pathological conditions is highly variable, which makes it difficult to predict the rate of drug release. On the other hand, enzyme-independent biodegradation can occur in both pathological and normal tissues.

The second problem relates to the exposure of drug molecules attached to the polymeric backbone to the environment. This is due to the cross-interaction of drug components with the formation of intra- and inter-molecular micelles, interaction with tissue components that alter the biodistribution of drug-carrier adducts,
As well as other undesirable effects. These actions are by "steric protection", ie by hydrophilic polymer chains (eg polyethylene glycol,
It is expected to be partially suppressed by modification of the carrier backbone with dextran or PHF (such as polyhydroxymethylethylene hydroxymethylformal). However, with sterically protected carriers, the access of the enzyme to the enzyme-sensitive drug-carrier bond may also be suppressed.

Another attempt to overcome the problems associated with drug delivery involves combining the drug with microparticles and emulsions. Microparticles and emulsions have been developed as alternatives to drug molecules not chemically bound, but rather adsorbed on or dissolved in carrier materials. However, the particles and emulsions, unless the particle (droplet) surface is modified with a hydrophilic polymer such as PEG,
It does not circulate in the body long enough and does not accumulate in the reticuloendothelial system (RES) and other organs. The overall size of sterically protected particles (droplets) is usually around 25.
larger than nm. Major problems in developing such carriers include the following facts: (1) Emulsions are generally relatively unstable in storage,
Variable (eg coalescing); (2) it is typically difficult to produce both submicron particles and emulsions on a large scale; and (3) drug molecules released from particles or droplets. Circulates independently of the carrier. Emulsions and most particles are not suitable for hydrophilic drug transport.

A specific development in drug delivery has been to use micelles developed as “self-assembling” drug carriers similar to particles and emulsions. Micelle
It is usually made from a surfactant that is a block copolymer in which one of the blocks is hydrophilic and the other is hydrophobic. The overall hydrodynamic size of the micelles is
Usually, it is 10 to 30 nm. The hydrophobic drug molecule is either incorporated into the hydrophobic core or chemically bound to one of the blocks to form the hydrophobic core. Some of the problems in developing such carriers are similar to those described above. Moreover, none of these carriers can specifically resorb the released drug, the release rate of the drug is difficult to control, and the amphipathic component can lead to toxic effects. These carriers are not suitable for transporting hydrophilic drugs.

Yet another attempt involves encapsulating the drug in the aqueous compartment of liposomes, which are vesicles that are typically in the range of about 50 nm to about 1000 nm in diameter. However, the efficacy of drug encapsulation by entrapment within liposomes and the ability to control drug delivery can be questionable. For example, drug release from liposomes is generally irreversible. Moreover, the penetration of liposomes into tumors or tumor areas with relatively low vascular permeability is often poor. Also, many problems are associated with high volume manufacturing and storage of liposome preparations, which presents significant technical challenges.

Other systems used to bind drugs for delivery to cells or organisms have similar shortcomings. Therefore, there is a need for drug delivery methods that minimize or overcome the above problems.

DISCLOSURE OF THE INVENTION The present invention relates to the field of drug delivery, and in particular to methods of forming drug-carrier complexes and for delivering and targeting drugs in organisms, tissue cultures or cells. It relates to the use of a drug-carrier complex as a pharmaceutical composition.

In one embodiment, the method of the invention comprises combining at least one nucleotide chain with a drug, thereby reversibly associating the drug and the nucleotide chain with each other to form a drug-carrier complex. Forming a drug-carrier complex by.

[0012] In another embodiment, the method of the invention comprises combining a drug with at least two nucleotide chains that hybridize to each other, thereby associating the drug with the nucleotide chains to form a water-soluble drug-carrier complex. Forming to form a drug-carrier complex.

[0013] In yet another embodiment, the method of the invention comprises combining a drug component and a nucleotide component to form a drug-carrier composition. The combined drug component and nucleotide component are lyophilized to form a drug-carrier composition.

In yet another embodiment, the method of the invention comprises lyophilizing the drug component,
Lyophilizing the nucleotide component and combining the lyophilized drug component and the lyophilized nucleotide component to form the drug-carrier composition to form the drug-carrier composition.

Another embodiment of the invention is a drug carrier comprising double-stranded nucleotides and a polymeric component covalently attached to at least one strand of said double-stranded nucleotides. The polymeric component has a water solubility of at least 1 mg / liter at 25 ° C.

A further embodiment of the present invention is a drug carrier comprising double-stranded nucleotides and an oligomeric component covalently attached to at least one strand of said double-stranded nucleotides.

In a further embodiment, the invention comprises a single-stranded nucleotide, a drug reversibly associated with said single-stranded nucleotide, and a polymer associated with said drug or said single-stranded nucleotide. It is a drug-carrier complex.

In still another embodiment, the present invention provides a drug comprising a single-stranded nucleotide, an oligomer associated with the single-stranded nucleotide, and a drug reversibly associated with the oligomer or the single-stranded nucleotide, It is a carrier complex.

In yet another embodiment, the invention is a drug carrier comprising single-stranded nucleotides and at least two polymers associated with said single-stranded nucleotides.

In another embodiment, the invention is a drug carrier comprising an oligomer, a single-stranded nucleotide entrapped by the oligomer, and a drug reversibly associated with the single-stranded nucleotide.

In yet another embodiment, the invention is a drug-carrier composition comprising a nucleotide carrier component and a drug component. The drug-carrier composition has a water content of less than about 5% by weight.

In yet another embodiment, the present invention comprises a drug-carrier composition consisting essentially of a drug component and a nucleotide component.

In a further embodiment, the invention provides a nucleotide carrier component and a reversible association with said nucleotide carrier component.
with) a pharmaceutical formulation containing a drug.

[0024] In yet another embodiment, the method of the invention comprises delivering a drug to an organism by administering the drug-carrier complex to the organism. The drug-carrier complex comprises a nucleotide carrier and a drug associated with each other.

In another embodiment, the method of the invention comprises delivering a drug to a tissue culture by administering the drug-carrier complex to the tissue culture. The drug-carrier complex comprises a nucleotide carrier and a drug reversibly associated with each other.

In yet another embodiment, the method of the present invention provides a method of administering a drug to a living organism by administering to the organism a nucleotide carrier that reversibly associates with said drug to form a drug-carrier complex. Delivery to.

In yet another embodiment, the method of the invention comprises forming a drug carrier complex comprising a drug and a nucleotide chain reversibly associated with said drug, and said drug carrier complex in an organism. Delivery of the drug to the organism by administration.

[0028] Another embodiment includes a method of delivering a drug to an organism by administering the drug-carrier complex to the organism. The drug-carrier complex comprises a drug component and a carrier component reversibly associated with each other. The drug dissociates from the drug-carrier complex,
And can reassociate with the carrier component. The degree of association may depend, for example, on the concentration of drug and carrier.

In yet another embodiment, the method of the present invention comprises reversibly associating a substance with a nucleotide carrier to increase the water solubility of the substance by forming a water-soluble complex.

In yet another embodiment, the invention provides a target comprising a nucleotide, a polymer component associated with said nucleotide, and a ligand associated with said nucleotide or said polymer component and capable of associating with a cell marker or tissue marker. It is a targeted carrier. The cell or tissue marker is selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides.

In yet another embodiment, the present invention relates to a targeted carrier comprising a nucleotide, a polymeric component associated with said nucleotide, and a ligand. The ligand is capable of associating with the nucleotide or the polymer component and with cell markers or tissue markers. The cell or tissue marker is selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides.

In a further embodiment, the present invention relates to a targeted drug-carrier complex comprising a nucleotide, a drug reversibly associated with said nucleotide, and a targeting component. The targeting moiety associates with the nucleotide or the drug. The targeting moiety comprises a ligand capable of associating with a cellular or tissue marker. The cell or tissue marker is selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides.

In yet another embodiment, the invention provides a targeted drug-carrier conjugate comprising a nucleotide, a drug reversibly associated with said nucleotide, a polymer component associated with said nucleotide or said drug, and a targeting component. Regarding the body The targeting moiety is associated with the nucleotide, the drug or the polymer. The targeting moiety comprises a ligand capable of associating with a cellular or tissue marker. The cell or tissue marker is selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides.

In a further embodiment, the invention relates to a drug delivery system comprising a matrix, nucleotides associated with or encased in the matrix, and a drug reversibly associated with the nucleotides.

Another embodiment includes an implant that includes an implant matrix, nucleotides associated with or encased in the matrix, and a drug reversibly associated with the nucleotides.

The invention described herein includes drug-carrier complexes, drug-carrier compositions, drug carriers, pharmaceutical formulations, methods of delivering drugs to organisms and tissue cultures, drugs to organisms, Provided are targeted carriers and implants for delivery to tissue culture or a combination of cells. The nucleotide based drug delivery system of the present invention has many advantages. For example, the system of the present invention can deliver a drug in a form that is not chemically modified and resorb the released drug. By using reversible drug association, the drug delivery system of the present invention can recapture the released drug. Thus, drug behavior in tissues will remain dependent on the drug release system as long as the drug release system remains functional. This offers the possibility of new opportunities in the regulation of pharmacokinetics and pharmacodynamics. In the clinical setting this is expected to result in better biological functionality and a greater margin of safety for pharmaceutical formulations and devices.

Other advantages include, for example, the relatively small size of the drug-carrier complex (eg, such as about 3 nm or about 5 nm). The drug-carrier complex of the present invention is
For example, it can be 1/5 to 1/10 of the polymer type carrier and the micelle type carrier, and 1/10 to 1/20 of the liposome. Thus, drug penetration into certain tissues, such as cancerous tissues, can be significantly efficient, especially when the endothelial and interstitial barriers are large. Also, the stability and release rate of the drug-carrier complexes of the invention can be controlled over a wide range, which may provide the opportunity to design a product according to a particular clinical purpose. Moreover, release of the drug by the drug-carrier complex of the present invention does not require interaction with enzymes, cells or other factors, thus the drug-carrier complex is more independent of biological and tissue conditions. Become. However, alternatively, the conjugates of the invention can be designed to take advantage of specific conditions of the biological or tissue status, such as pH or enzyme content. Moreover, the components of the drug-carrier complex of the present invention can be made from similar analogs of the natural components of biological systems known to be fully biodegradable and non-toxic. .

Other specific advantages of the present invention include the possibility of carrier clearance and steric protection against drug inactivation. Further, the drug-carrier complex of the present invention,
In general, it does not have problems with intramolecular or intermolecular association of drug molecules. Moreover, the methods of forming and treating the drug-carrier complexes of the present invention can be readily scaled. Alternatively, the drug-carrier complex can be lyophilized and all components of the complex can be stable in the presence of air. Moreover, no toxic detergents were used and the size of the complex is generally stable and independent of conditions and concentrations. The rate of drug release in living organisms is generally independent of the highly variable adsorption power. Ultrafiltration typically does not affect the size and structure of the drug-carrier complex.

DETAILED DESCRIPTION OF THE INVENTION The features and other details of the invention are more particularly described and pointed out in the claims either as a process of the invention or as a combination of parts of the invention. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be utilized in various embodiments without departing from the scope of the invention.

The present invention relates to the discovery that useful drug-carrier complexes can be formed by combining a nucleotide (eg, a chain of nucleotides) with the drug such that the drug and the nucleotide reversibly associate with each other. The drug-carrier complex may be used to deliver the drug to, for example, an organism, tissue culture, or individual cells. The invention further relates to the discovery that the drug-carrier complexes of the invention can be used in pharmaceutical formulations as targeting carriers, drug delivery systems, and as implants to increase drug solubility.

In one embodiment, the drug-carrier complex is formed by combining at least one nucleotide chain with the drug. The drug reversibly associates with the nucleotide component to form a drug-carrier complex. Therefore, "drug-carrier complex"("
The term "nucleotide-carrier complex", as used herein, refers to a drug and at least one nucleotide chain that is reversibly associated with each other.

The terms “associate”, “association”, or “associable” as used herein can be reversible, irreversible, or both. The association can be a physical association, a chemical association, or both. For example, the association can be a covalent bond, a hydrophobic interaction, etc.

“Reversible association,” as defined herein, is an association in which the components can be restored to their original pre-association state. For example, the reversible association of the components of the drug-carrier complex of the invention can dissociate, thereby restoring the original separate drug and nucleotide components.

In one embodiment, the amount of association of the reversible association components depends, at least in part, on the concentration of drug and carrier. In another embodiment, the components are dissociable under physiological conditions. In certain embodiments, the reversible association is an association selected from the group consisting of electrostatic bonding, hydrogen bonding, van der Waals forces, ionic interactions, or donor / acceptor bonding. The reversible association can be mediated by one or more associations between the drug and the nucleotide chain. For example, reversible association can include a combination of hydrogen and ionic bonds between the drug and the nucleotide chain. Additionally or alternatively, reversible association can be combined, for example, with covalent interactions between components, eg, drugs and nucleotides, or other non-covalent interactions.

In another particular embodiment of the drug-carrier complex of the invention, the substance comprising a metal-containing substance (eg platinum, cis-platinum, carboplatin, platinum, gold, silver) comprises: Reversibly associates with the nucleotide carrier. The association is non-covalent and reversibly releases the metal-containing biologically active drug component, which may be different from the substance originally utilized to form the drug-carrier complex. It is considered possible.

As used herein, “nucleotide”, “nucleotide chain”
Or the term "nucleotide carrier" refers to naturally occurring nucleotides (eg, base components guanine (G), thymine (T), uracil (U), cytosine (C).
, Adenine (A)) or their derivatives or structural analogs. Nucleotide chains include two or more nucleotides, such as oligonucleotides, polynucleotides, or chemical derivatives or analogs thereof. The term "oligonucleotide" generally refers to molecules having a well-defined structure and length (eg, (SEQ ID NO: 13)). The term "polynucleotide" generally refers to molecules assembled from a large number of nucleosides that vary in either sequence or length (eg, preparations of DNA or RNA obtained from cell lysates, structure A _n T _m G _{k Cl} random polymer).

In naturally occurring oligonucleotides and polynucleotides, the bases are usually linked via phosphodiester bonds. Several chemical analogs are known in which the bases are linked by non-phosphodiester bonds.

Nucleotide chains can exist in the form of straight chains or rings and can have a variety of structures, such as helical duplexes (helices), triplexes (often referred to as triplexes),
It is known to form loops, folds, crosses, or supercoils.

The invention includes, for example, linear deoxyribonucleotides, linear ribonucleotides, linear oligonucleotides containing both ribonucleotides and deoxyribonucleotides, circular DNA (eg plasmids), folded ribonucleotides ( For example, ribozyme, t-RNA), viral RNA, viral DNA,
DNA and RNA derived from cell lysates, synthetic polydeoxyribonucleotides and polyribonucleotides, chemically cross-linked double-stranded oligonucleotides, partially or fully methylated or otherwise chemically modified forms of said Any type of nucleotide chain, structure, and combinations formed therefrom, including any of

Preferred nucleotides of the invention are synthetic oligonucleotides, plasmids, R
Nucleotides with well-defined structures, such as NA transcripts, viral DNA, and viral RNA (eg, viral envelopes, complete virions, viral nucleotides in viruses). Other preferred nucleotides are
For example, to regulate the rate of biodegradation (eg, oligonucleotides containing phosphorothioate linkages) or to allow conjugation with other molecules (eg, 3'-end, 5'-end, or one Synthetic oligonucleotides in which a carboxyl group or an amino group is incorporated at the above base positions) and chemically modified synthetic oligonucleotides.

The DNA of the nucleotide chain is B DNA (Drew, HR, et al.
, Proc. Natl. Acad. Sci. U. S. A. , 78: 2179-2
183 (1981); Edwards, K .; J. , Et al. J. Mol.
Biol. , 226: 1161-1173 (1992) (both teachings are fully incorporated herein by reference)); Z DNA (Gessner, R.V.
． , Et al. J. Biol. Chem. , 264: 7921-7935 (
1989) (the teachings of which are fully incorporated herein by reference); triple-stranded DNA (Van Meervelt, L., et al., Nature, 37.
4,742-744 (1995), the teachings of which are incorporated herein by reference in its entirety); Intramolecular triplex DNA (Koshlap, KM, et al.
, Biochemistry 36: 2659 (1997)); intercalated quadruplex DNA (Kang, C., et.
al. , Proc. Natl. Acad. Sci. U. S. A. , 91: 116
36-11640 (1994); Kang, C .; , Et al. , Proc. N
atl. Acad. Sci. U. S. A. , 92: 3874-3878 (199
5) (both teachings are fully incorporated herein by reference)); quadruplex D
NA (Kang, C., et al., Nature, 356, 26-131 (
1992) (this teaching is fully incorporated herein by reference)); or bulge loop DNA (Joshua-Tor, L., et al., J. Mol.
Biol. , 225: 397-431 (1992), the teachings of which are fully incorporated herein by reference.

The nucleotides or nucleotide chains may be naturally occurring (eg isolated from cells of organisms, tissue culture cells, viruses) or synthetically produced, eg by a nucleotide synthesizer. It may have been done. The residue may be further modified after synthesizing the nucleotide chain. For example, the 5'amino group of the nucleotide chain can be modified with N-hydroxysuccinimide ester of carboxy-polyethylene glycol. Typically naturally occurring nucleosides may be used with typically non naturally occurring nucleosides to synthesize the nucleotides and nucleotide chains utilized by the invention. Methods for isolating nucleotides or nucleotide chains from cells, tissues, or organisms, and methods for synthesizing nucleotides or nucleotide chains on commercially available DNA / RNA synthesizers are well known in the art. Exemplary techniques include, for example, "Current
"Protocols in Molecular Biology" (Aus
ubel et al. , John Wiley & Sons (1999) (
This teaching is fully incorporated herein by reference))).

The term “nucleotide component” refers to the drug binding (of the drug-carrier complex of the present invention (
Drug delivery) component. The drug binding component comprises at least one nucleotide chain. The nucleotide component may include or be further associated with other components (eg, polymers, oligomers, ligands) to form drug carriers, drug delivery systems, or drug-loaded implants. In one embodiment, the nucleotide chain is an oligonucleotide chain.

The drug in the drug-carrier complex reversibly binds to the nucleotide or nucleotide chain of the invention (also referred to herein as “reversibly associated” or “reversibly associated”). It can be any substance or any structure formed by the chains. The drug can reversibly associate with a single nucleotide of one or more nucleotide chains, eg, via a donor-acceptor bond. A drug can also reversibly associate with multiple nucleotides of one or more nucleotide chains. A drug can reversibly associate with one nucleotide chain of a drug-carrier complex consisting of two nucleotide chains. Similarly, a drug can also reversibly associate with two nucleotide chains of a drug-carrier complex consisting of two nucleotide chains. Similarly, a drug can reversibly associate with a single nucleotide chain of a drug-carrier complex consisting of three nucleotide chains.

The term “drug” is used interchangeably herein with the phrase “drug ingredient”. In one embodiment, the drug of the pharmaceutical formulation is a therapeutic drug. The term "treatment", when referring to the drug used in the present invention, treats, corrects a disorder or disease (eg, a genetic disease, a viral disease such as AIDS, cancer), a correction,
Or refers to a drug used to cure. In another embodiment, the drug of the pharmaceutical formulation is a diagnostic drug (e.g., a radioactive diagnostic drug, a fluorescent diagnostic drug,
Paramagnetic diagnostic drug, superparamagnetic diagnostic drug, high x-ray density
) A diagnostic drug or a high electron density diagnostic drug). The term "diagnosis" when referring to a drug utilized in the present invention, refers to a drug utilized to determine the nature or extent of a disease, or to confirm the presence of a disorder or disease. .

In another embodiment, the drug can be, for example, an anticancer drug, an antiviral drug, an antibacterial drug, or an antiprotozoal drug. The drug is, for example, anthracycline, actinomycin, anthracenedione, bleomycin, mithramycin, chromomycin, olivomycin, protein, peptide, carbohydrate, polyamine, polycation, actinomycin D, daunorubicin, doxorubicin, idarubicin, bis. Anthracycline, mitoxantrone, bleomycin A2, distamycin, netropsin (ne
Tropsin), cisplatin, carboplatin, silver ions and particles, or gold ions and particles.

In one embodiment, the drug is an oligonucleotide drug. The term "oligonucleotide drug", as used herein, refers to a molecule containing at least two nucleotides that reversibly binds to the nucleotide chain of a drug-carrier complex. The oligonucleotide drug can be, for example, an antisense oligonucleotide or a ribozyme. Other examples of suitable drugs include metal-containing substances (eg, platinum, cisplatin, carboplatin, platinum, gold, silver) or a minor groove or major groove of the DNA or RNA helix. Ingredients such as drugs are included. In yet another embodiment, the drug contains at least one amino group. For example, the drug doxorubicin contains an amino group.

In another embodiment, as shown in FIGS. 1A and 1B, drug conjugate 10 comprises nucleotide 12, nucleotide 12 and an intercal.
drug 14).

In another embodiment, as shown in FIG. 2, drug conjugate 16 comprises a double-stranded oligonucleotide core 18 that carries a drug (not shown). The polymer 20 associates with the oligonucleotide core 18, for example by covalent bonding, thereby providing steric protection.

3A-3E represent a further embodiment of the drug-carrier complex of the present invention. In particular, Figure 3A shows drug 24 reversibly associated with double-stranded oligonucleotide 26.
The micelle 22 including is shown. Polymer 28 is a double-stranded oligonucleotide 2
Radially aligned from 6.

FIG. 3B shows a polymer-modified DNA 30 in which a drug 32 is reversibly associated with a single- or double-stranded polynucleotide or oligonucleotide 34. A polymer 36 extends from the polynucleotide or oligonucleotide 34.

FIG. 3C shows a drug-carrier complex 38 having a polymer backbone 40 attached to a plurality of oligonucleotides 42. The drug component 44 is reversibly associated with the oligonucleotide 42, the polymer 46 extends from the backbone, and the oligonucleotide 42
Is three-dimensionally protected.

FIG. 3D shows the drug-carrier complex as a modified plasmid 48. Drug 50 and polymer 52 are associated with each other and drug is reversibly associated with plasmid component 54.

FIG. 3E shows a drug-carrier complex as a gel particle 56 in which an oligonucleotide 58 and associated drug molecule 60 are encapsulated within a gel 62. Polymer 64 extends from gel 62.

The drug-carrier complex of the present invention comprises at least one drug. For example, drug-
The carrier complex comprises at least one reversibly associated oligonucleotide drug (eg, ribozyme, antisense oligonucleotide), antimicrobial agent, and metal-containing substance (eg, platinum, cisplatin, carboplatin, platinum, gold, silver). It may include a chain of nucleotides. The drug-carrier complex of the present invention can be used for delivering a drug that suppresses, inhibits, or regulates the transcription of a certain gene such as a growth-related gene in cancer.

In one embodiment, in the drug-carrier complex of the invention, the drug of the drug-carrier complex is combined with at least two nucleotide chains that hybridize to each other.

Additionally or alternatively, the second nucleotide chain is combined with the drug-carrier complex. In one embodiment, the second nucleotide chain hybridizes with at least one of the nucleotide chains of the drug-carrier complex. The second nucleotide chain is one or more nucleotides, single-stranded, double-stranded DNA, RNA, naturally-occurring or synthetic nucleotides, as in the case of the drug-carrier complex nucleotide chain described above. Can be

In another embodiment, the present invention relates to a method of forming a drug-carrier complex comprising combining a drug with at least two nucleotide chains that hybridize to each other. The drug and nucleotide can be added to the solution (eg, water) individually or together. When the drug and nucleotide are individually combined to form the drug-carrier complex, the drug may be added to the solution and then the nucleotide may be added to the solution, or the nucleotide may be added to the solution and then the drug may be added. It may be added to the solution. Alternatively, the drug and nucleotide can be added to the solution at the same time. In the absence of hybridized nucleotide chains of the drug-carrier complex, the drug is water insoluble or has a relatively low water solubility, such as a solubility of less than about 1 mg / liter at 25 ° C.

In a preferred embodiment, the dissolved drug-carrier complex is lyophilized. Freeze-drying is also called freeze-drying. Methods for lyophilizing substances that can be used to lyophilize the drug-carrier complexes of the present invention are well known in the art. The drug-carrier complex solution can be frozen (eg, by placing in a liquid nitrogen or dry ice alcohol bath) and the frozen drug-carrier complex placed in a high vacuum. Then, by evaporating (subliming) water (ice form) in vacuum without melting, components other than water (dissolved drug-carrier complex)
Remain in powder or sponge-like (dehydrated) form.

Another embodiment of the present invention is a method of forming a drug-carrier composition comprising combining a drug component and a nucleotide component. The combined drug component and nucleotide component are lyophilized to form a drug-carrier composition. In a preferred embodiment, at least one of the drug component (eg, antisense oligonucleotide, anthracycline, distamycin) and the nucleotide component (eg, single-stranded DNA or RNA, double-stranded DNA or RNA) is After dissolving in water, the ingredients are combined. The remaining ingredients can then be added to the combined drug and nucleotide components.

In yet another embodiment, the drug-carrier composition comprises lyophilizing the drug component, lyophilizing the nucleotide component, and combining the lyophilized drug component and the lyophilized nucleotide component. , A drug-carrier composition.

In a further embodiment, the invention provides a double-stranded nucleotide (eg, DNA
Or RNA) and a polymeric component covalently bound to at least one strand of double-stranded nucleotides. The polymeric component of the drug carrier has a water solubility of at least 1 mg / liter at 25 ° C.

The term “polymer” generally refers to molecules (eg, proteins) formed by the union or association of chemically similar or chemically different units (eg, amino acids, monomers such as glucose). , Polyether, polyacetal, polysaccharide). Generally, "polymer" refers to molecules that contain more than about 30 units.
The polymer can be an inorganic polymer such as, for example, siloxanes or polyphosphates and their derivatives. Alternatively, or additionally, the polymer can be organic. Organic polymers include polysaccharides, starch, cellulose, pectin, inulin, agarose, chondroitin sulfate, heparin, dextran, polypeptides (
For example, it may be a natural organic polymer such as casein, albumin, globulin, keratin, insulin, polylysine), and their derivatives. The organic polymer may be a synthetic organic polymer such as polyacetal, polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyester, polyamide, polyamine, and derivatives thereof. The organic polymer may be a semi-synthetic organic polymer such as methyl cellulose, modified starch, and derivatives thereof.

In one embodiment, the polymeric component of the drug carrier is a biocompatible polymeric component. The term "biocompatible," as used herein, refers to adverse reactions from organisms (eg, mammals), tissue cultures, or cell aggregates (
For example, a polymer that does not elicit an immune response or, if it does, does not cause untoward reactions to exceed acceptable levels. In a more preferred embodiment, the biocompatible polymer component is a polysaccharide (eg, poly α-D-glucose, polysialic acid, dextran, chondroitin sulfate, starch), a polyether, and a polyacetal (eg, poly [hydroxymethylethylene hydroxymethyl). Formal]).

[0075]   In another embodiment, the biocompatible polymer is crosslinked.

The polymeric component of the drug carrier is one or more chemically similar polymers (eg,
Polysaccharide polymer: Polysaccharide polymer, Polypeptide polymer: Polypeptide polymer)
Or a chemically different polymer (eg, a polysaccharide polymer and a polypeptide polymer; a polyether polymer and a polyacetal polymer; a polysaccharide polymer, a polypeptide polymer and a polyether polymer). In one embodiment, the polymeric component of the drug carrier comprises at least one polymer covalently attached to at least one nucleotide chain. In another embodiment, the polymer component comprises at least two chemically similar or chemically different polymers.

A further embodiment of the invention relates to a drug carrier comprising double-stranded nucleotides and an oligomeric component covalently bound to at least one strand of double-stranded nucleotides.

The term “oligomer”, as used herein, generally contains less than about 30 units, is chemically similar or is chemically different units (eg, amino acids, Molecules such as glucose, galactose, etc.) or molecules formed by the combination. Like polymers, oligomers can be, for example, inorganic or organic oligomers. The organic oligomer can be a natural organic oligomer, a synthetic oligomer, or a semi-synthetic organic oligomer.

The oligomeric component of the drug carrier can include at least one polysaccharide, or at least one oligopeptide, or a combination of both oligosaccharides and oligopeptides.
In one embodiment, the oligomer component comprises at least one oligomer covalently attached to at least one nucleotide chain. In another embodiment, the oligomer component comprises at least two oligomers. In yet another embodiment, the oligomeric component of the drug carrier comprises at least two chemically different oligomers. In yet another embodiment, the oligomeric component of the drug carrier comprises at least two chemically similar oligomers.

In another embodiment, the present invention is a drug-carrier complex comprising single-stranded nucleotide, polymer and drug. The drug is reversibly associated with single-stranded nucleotides. In one embodiment, the polymer is associated with single stranded nucleotides. In another embodiment, the polymer is associated with the drug.

In yet another embodiment, the invention is a drug-carrier complex comprising single-stranded nucleotides, an oligomer and a drug. The oligomer is associated with the single-stranded nucleotide. The oligomer is associated with the single-stranded nucleotide by covalent association. The oligomer may be associated with the drug. In one embodiment, the drug is reversibly associated with the single stranded nucleotide. In another embodiment, the drug is reversibly associated with the oligomer.

In a further embodiment, the present invention is a drug carrier comprising single-stranded nucleotides and at least two polymers associated (eg, reversibly, irreversibly) with single-stranded nucleotides. In one embodiment, the chemical association (eg, reversible, irreversible) between the polymer and the single-stranded nucleotide is a covalent bond. In another embodiment, the chemical association (eg, reversible, irreversible) between the polymer and the single-stranded nucleotide is a non-covalent bond.

In another embodiment, the present invention is a drug carrier comprising single-stranded nucleotides and at least two oligomers associated with single-stranded nucleotides. In one embodiment, the chemical association between the oligomer and the single-stranded nucleotide comprises
It is a covalent bond. In another embodiment, the chemical association between the oligomer and the single-stranded nucleotide is a non-covalent bond.

In yet another embodiment, the invention relates to a drug-carrier composition comprising a nucleotide carrier component and a drug component. The drug-carrier composition has a water content of less than about 5% by weight.

A further embodiment of the present invention relates to a drug-carrier composition consisting essentially of a drug component and a nucleotide component, essentially free of other components such as water or water vapor.

In another embodiment, the invention relates to a pharmaceutical formulation comprising a nucleotide carrier component and a drug reversibly associated with the nucleotide carrier component. The reversible association between the drug and the nucleotide carrier component is similar to the association between the drug and the nucleotide in the drug-carrier complex described above, for example, van der Waals force, electrostatic interaction, hydrogen bond, ionic bond, hydrophobic property. Interactions, or donor / acceptor bonds may be involved.

The reversible association between the drug and the nucleotide carrier component of the pharmaceutical formulation is from the group consisting of van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions, and donor / acceptor bonds. It may include at least one reversible association selected. Reversible association can also include intercalation between the drug and the nucleotide carrier component. The reversible association between the drug and the nucleotide carrier component of the pharmaceutical formulation can further include a reversible covalent bond between the drug and the nucleotide carrier component.

In one embodiment, the nucleotide carrier component of the pharmaceutical formulation is a polynucleotide carrier component. In another embodiment, the nucleotide carrier component of the pharmaceutical carrier component is an oligonucleotide carrier component.

The drug used in the pharmaceutical preparation is the same as the above-mentioned drug-carrier complex of the present invention, for example. For example, the drug in the pharmaceutical formulation can be an oligonucleotide drug (eg, an oligonucleotide, an antisense oligonucleotide, or a ribozyme). Drugs include a group consisting of an intercalator, a metal-containing substance (eg, platinum, cisplatin, carboplatin, platinum, gold, silver), a minor groove binder, or a major groove binder. More selected ingredients may be included. In yet another embodiment, the drug contains at least one amino group. For example, the drug doxorubicin contains an amino group.

In yet another embodiment, the drug of the pharmaceutical formulation is a protein or peptide containing two or more amino acids. The amino acids of the protein drug or peptide drug can be naturally occurring L-amino acids, D-amino acids, non-naturally occurring amino acids, or synthetic amino acids such as gamma amino acids and cyclic amino acids.
The protein may be post-translationally modified (eg, glycosylated, myristylated, acetylated). The N-terminal amino group, the C-terminal carboxyl group, or one or more peptide bonds within the protein can be, for example, non-amino bonds.

In yet another embodiment, the drug of the pharmaceutical formulation comprises a diagnostic label. The phrase "diagnostic label" when referring to a drug in the pharmaceutical formulation of the present invention refers to the detectable label incorporated into the drug. A label is a sample, liquid, organ,
Used to determine the concentration of drugs or drug metabolites in tissues, cell combinations, single cells, organelles, etc. Labels include, for example, radionuclides, fluorophores, chromophores, paramagnetic ions or groups, superparamagnetic nanoparticles, barium or other heavy metal ions, heavy metal (eg, gold) particles, iodine atoms, enzymes, or biotin. included. Labels are, for example, radioactivity measurements, gamma scintigraphy, positron emission tomography, nuclear magnetic resonance spectroscopy, paramagnetic resonance imaging, fluorescence spectroscopy, photoimaging, X-ray (computer-linked) tomography. It may be detectable in vitro or in vivo by radiography, electron microscopy, enzyme assays, or other respective methods. Information about the location or content of the label can be used to determine drug migration and metabolic pathways. Diagnostic labels can also be used to confirm the presence of a disorder or disease.

A further embodiment of the invention relates to a method of delivering a drug to an organism, comprising administering a nucleotide carrier-drug complex to the organism. The nucleotide carrier-drug complex comprises a nucleotide carrier and a drug in reversible association with each other. In one embodiment, the reversible association of a drug with a nucleotide carrier component used in a method of delivering a drug to an organism comprises van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions. , And a donor / acceptor bond. In another embodiment, the reversible association of the drug with the nucleotide carrier component used in the method of delivering the drug to an organism is intercalation.

In a preferred embodiment, the organism to which the drug is delivered using the method of the invention is selected from the group consisting of mammals and cells. Mammals include, for example, primates (eg, humans, rhesus monkeys), rodents (eg, hamsters, mice, rats),
Ruminants (eg sheep, horses, cows), or domestic (
For example, it can be a mammal (cat, dog). The cell can be a prokaryotic cell or a eukaryotic cell.

In one embodiment of the method of delivering a drug to an organism, the drug-carrier complex comprises
For example, it is administered to an organism by systemic administration or local administration. In another embodiment of this method, the drug-carrier complex is administered proximal to the organism. "Proximal to an organism", as used herein, means in the vicinity of an organism. For example, if the organism is a cell, the nucleotide carrier can be administered to the cell culture medium.

In a further embodiment, the present invention relates to a method of delivering a drug to a tissue culture or a combination of cells (eg, a tissue sample) comprising administering a nucleotide carrier-drug complex to the tissue culture. The nucleotide carrier-drug complex comprises a nucleotide carrier and a drug in reversible association with each other. As with the method of delivering a drug to an organism described above, the reversible association of the drug with the nucleotide carrier is
It is selected from the group consisting of van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions, and donor / acceptor bonds. The reversible association between the drug and the nucleotide carrier component in the method may be intercalation. The drug-carrier complex may be administered to a tissue culture or cell combination. Alternatively, or additionally, the nucleotide carrier can be administered proximal to the tissue culture or combination of cells.

The methods of delivering a drug to tissue culture described herein can be utilized to screen or select for a drug that has a particular effect on particular cells or tissues. For example, the drug of the nucleotide carrier-drug complex is a normal cell or a cancer cell (
For example, melanoma, breast adenocarcinoma cell line 293, tissue bioptate.
s)), viruses (eg HIV, hepatitis C), bacteria, fungi, and protozoa, and evaluated to determine whether the drug inhibits cell growth of tissue culture cells or pathogens. sell.

Another embodiment of the present invention comprises administering a drug to an organism, the method comprising administering to the organism a drug and a nucleotide carrier that reversibly associates with the drug to form a nucleotide carrier-drug complex. The method of delivery. In one embodiment, the drug and nucleotide carrier are administered to the organism at the same time. In another embodiment, the drug and nucleotide carrier are administered to the organism separately. Drugs and nucleotides,
When administered to organisms separately, the drug may be administered first, followed by the nucleotide carrier, or the nucleotide carrier may be administered first, followed by the drug.

In yet another embodiment, the present invention provides a step of forming a nucleotide carrier-drug complex comprising the drug and a nucleotide carrier reversibly associated with the drug, and the nucleotide carrier-drug complex. The present invention relates to a method for delivering a drug to an organism, which comprises the step of administering to the organism.

In yet another embodiment, the invention relates to a method of delivering a drug to an organism, comprising administering the drug-carrier complex to the organism. The drug-carrier complex is
It includes a drug component and a carrier component that are reversibly associated with each other. The drug dissociates from the drug-carrier complex and reassociates with the carrier component. The extent of association and dissociation can depend on, for example, drug and carrier concentrations and can be assessed using UV / visible spectroscopy, fluorescence spectroscopy, NMR, or other suitable method. In a preferred embodiment, the carrier component is a nucleotide carrier component. The nucleotide carrier component can be an oligonucleotide or a polynucleotide. The nucleotide carrier component can be single-stranded nucleotides, double-stranded nucleotides, DNA, RNA, naturally occurring or synthetic nucleotides.

In a preferred embodiment, the drug-carrier complex utilized in the method of delivering a drug to an organism is delivered to a combination of cells within the organism. The combination of cells may be, for example, cancer (eg breast cancer, brain cancer, prostate cancer, lung cancer), pathogenic organisms (eg bacteria, viruses, fungi) or organs (eg heart, kidneys, lungs, intestines, stomach). Can be

In one embodiment of the method of delivering a drug to an organism, the drug-carrier complex is administered to the organism and the drug-carrier complex is proximate to or within the combination of cells within the organism. Dissociate with. For example, the drug-carrier complex is administered to a human organism and dissociates in or within cancerous tissue within the human body.

The drug-carrier complex may be administered to the organism at a location remote from the cell combination of interest. Alternatively, or additionally, the drug-carrier complex may be administered to the organism at a location proximal to the cell combination of interest. For example, where the cell combination is lung cancer, administration of the drug-carrier complex via inhalation may be considered administration of the drug at a location proximal to the cell combination. Similarly, where the cell combination is cancer of the small intestine, administration of the drug-carrier complex to the abdominal cavity may be considered administration of the drug at a location proximal to the cell combination.

The nucleotide carrier-drug conjugates and pharmaceutical formulations of the invention may be administered systemically or topically, eg intravenously, intramuscularly, parenterally, orally, intranasally, by inhalation.
Alternatively, it can be administered by suppository. The nucleotide carrier-drug conjugates and pharmaceutical formulations of the invention may be administered in a single dose or for a desired effect (e.g., to sensitize cancer cells to radiosensitivity or to reduce or arrest cell proliferation, Multiple doses may be administered over the period required to achieve drug delivery to the tumor.

The nucleotide carrier-drug conjugates and pharmaceutical formulations of the present invention may be used in organisms, tissue cultures,
Alternatively, it may be mixed or combined with other pharmaceutical carriers or excipients such as sterile water, saline (such as Ringer's solution), alcohol, or talc to facilitate administration into the combination of cells. The nucleotide carrier-drug complex and the pharmaceutical preparation of the present invention are sterilized and, if desired, an auxiliary substance which does not cause a harmful reaction with the nucleotide carrier-drug conjugate and the pharmaceutical preparation, for example, a cryoprotectant, a coloring agent, or a preservative. Can be mixed with.

The actual effective amount of the nucleotide carrier-drug conjugates and pharmaceutical formulations of the present invention in a particular example is determined by the particular nucleotide carrier-drug conjugate and pharmaceutical formulation utilized,
For example, it may vary depending on the administration mode and age, weight, and disease or disorder of the organism (for example, human).

In yet another embodiment, the invention relates to a method of increasing the water solubility of a substance comprising reversibly associating the substance with a nucleotide carrier to form a water soluble complex. For example, doxorubicin, WP-631, and DMP840.
(Raghavan, KS et al., Pharm. Dev. Techn.
ol. Some known bisintercalators, such as 37: 3078-85 (1988), the teachings of which are fully incorporated herein by reference, have limited solubilities at physiological pH. Some substances containing structures that may bind to DNA (eg, highly hydrophobic intercalators) are only slightly soluble in aqueous media, thus hindering testing in cell culture. Therefore, no research has been done yet. The phrase "water-soluble" generally refers to the ability of a substance to be miscible (eg, dissolved) with a water-based solution. The water-based solution can be any solution that contains water as one of its components. For example, the water-based solution can be plasma or a physiological buffered saline solution such as phosphate buffered saline or Ringer's solution. In one embodiment,
A substance is essentially water insoluble when it is not associated with the nucleotide carrier. Water insolubility can make a substance unsuitable or inadequate for administration to an organism. Thus, increasing water solubility utilizing the methods of the present invention can be used to treat diseases and study their mechanisms, to deliver drugs to cells, organisms (eg, mammals), and tissue cultures. To increase the number of compounds that can be used as drugs.

In yet another embodiment, the invention provides a nucleotide, a polymer component associated with the nucleotide, and a nucleotide or polymer component associated with the nucleotide,
And a targeting carrier comprising a ligand capable of associating with a cell marker or a tissue marker.

The association between the ligand of the targeted carrier and the nucleotide or polymer component is
It can be a reversible association (eg van der Waals force, electrostatic interaction, hydrogen bond, ionic bond, hydrophobic interaction, or donor / acceptor bond), irreversible association, or covalent bond.

Cellular or tissue markers with which the targeting carrier ligand is associated include, for example:
It may be selected from the group consisting of proteins, polysaccharides, polypeptides, carbohydrates and lipids. The terms "protein", "polysaccharide", "polypeptide", "carbohydrate", and "lipid" refer to glycoproteins, glycolipids, lipopolysaccharides, proteoglycans,
Also referred to are lipoproteins, lipid-protein complexes, nucleosomes, and related compounds or derivatives such as lipoteichoic acid. For example, the cell or tissue marker can be a cell surface receptor such as a transmembrane receptor (eg, G protein coupled receptor, tyrosine kinase receptor, growth factor receptor). The ligand of the targeting carrier may be selected to specifically target the targeting carrier to specific cells or tissues, for example to treat a disease state or to deliver a drug to replenish a defect.

Tissue markers can be divided into, for example, two groups. One group is exclusive, or near, on the surface of pathological cells, or in the pathological extracellular matrix, or in certain combinations of cells (cell type, cell class, organ, or tissue). Molecules that are exclusively expressed can be included. Another group may include molecules that are not specific to the pathological site but are overexpressed in the pathological site.
Examples of suitable ligands associated with cell or tissue markers are platelet derived growth factor (PDGF), macrophage colony stimulating factor, and epidermal growth factor.

A typical representative of the first group is the asialofetuin receptor (
Hepatocytes), viral antigens (cells infected with herpes virus or other viruses), scavenger receptors (macrophages), HER-2 / neu (some breast cancer types). The second group includes markers typical of some types of inflammation and cancer, such as cytokine receptors, growth factor receptors, surface glycolipids and glycoproteins, integrins, selectins and the like. For example, malignant epithelial cells in primary human lung cancer co-express PDGF (ligand) and PDGF receptor (cell or tissue marker) in vivo (Antoniades, H.N.).
． , Et al. , Proceedings of the National
Academy of Sciences, 89: 3942-6 (1992).
(This teaching is fully incorporated herein by reference)); Macrophage colony stimulating factor (ligand) and its receptor (cell or tissue marker)
Is expressed in ovarian cancer and endometrial carcinoma (
Biocchi, G.M. , Et al. , Cancer, 67: 990-6 (1
991) (this teaching is fully incorporated herein by reference)); HER-
Coexpression of 2 / neu and epidermal growth factor receptor (cell or tissue marker) was observed in 65% of epithelial ovarian cancers, but only in a limited number of normal tissues from some donors. Not observed (Bast, RC, Jr., et al.
． , Cancer, 71: 1597-601 (1993) (this teaching is fully incorporated herein by reference); in lung cancer, cellular expression of Fuc-GM1 was commonly found with NCAM (Brezicka). , FT, et
al. , Tumour Biology, 13: 308-15 (1992) (
This teaching is fully incorporated herein by reference)); thrombospondin-1 co-distributes with CD51 in the majority (40-80%) of invasive lobular breast cancer cells and of these cells. It is co-distributed with CD36 in a subpopulation (30-40%) (Clezardin, P., et al., Cancer Rese.
arch, 53: 1421-30 (1993), the teachings of which are fully incorporated herein by reference); H-2 and Le (y) are 92% of cancers expressing both of these blood group antigens. Were co-expressed in the same individual colorectal cancer cells in (Cooper, HS, et al., Am. J. Pathol., 1).
38: 103-10 (1991) (this teaching is fully incorporated herein by reference); uPAR and plasminogen activator inhibitor-1 infiltrate compared to normal and benign breast tissue. Was overexpressed in primary breast cancer (Cos
tantini, V .; , Et al. , Cancer, 77: 1079-88 (
1996) (this teaching is hereby incorporated by reference in its entirety); although, in lung neoplasms, co-expression of at least two of cytokeratin, neurofilament, vimentin, and desmin is found. , In normal tissues, they have different, non-overlapping distributions (Gatter, KC, et al.
． J. of Clinical Pathology, 39: 950-4 (1
986) (this teaching is fully incorporated herein by reference); The majority of pediatric medulloblastoma cases are members of the EGFR family (EGFR, HER2, HE).
R3, and HER4) receptor proteins were expressed more than once; co-expression of HER2 and HER4 receptors occurred in 54% (Gilbert)
son, R.M. J. , Et al. , Cancer Research, 57: 3.
272-80 (1997) (this teaching is fully incorporated herein by reference)); in gastric adenocarcinoma, co-expression of multiple (3 or more) mucin core proteins is early (phase I and II). ) It occurred in 15 of 25 (60%) in advanced (stage III and IV) cancer compared to 1 in 8 (12.5%) of 8 (Ho, SB). ., Et al., Cancer Research, 55:
2681-90 (1995), the teachings of which are fully incorporated herein by reference); In colorectal cancer, EGFR-positive malignancies showed co-expression of the IL-4 receptor (Kaklamanis, L. et al. , Et al., Brit. Jo.
f Cancer, 66: 712-6 (1992) (this teaching is fully incorporated herein by reference)); in malignant salivary gland tumors, overexpression of p53 protein is associated with overexpression of c-erbB-2. It was closely correlated (Kamio
, N .; , Et al. , Virchows Archiv. , 428: 75-8
3 (1996)); epidermal growth factor receptors EGF-R and C-erbB-2 are expressed in human tumors, and in some cases, with histological grading and clinical consequences of lesions. It has been shown to be involved (Lakshmi, S
． , Et al. , Pathology, 65: 163-8 (1997).
(This teaching is fully incorporated herein by reference); Co-localization of MMP-9 with high molecular weight melanoma-associated antigen, a pericyte marker, was found in mammary ductal carcinoma (Nielsen, B.S., et al., Lab. Investiga.
77: 345-55 (1997), the teachings of which are fully incorporated herein by reference); in colon adenocarcinoma, invasion fronts.
The distribution of laminin-5-positive budding cancer cells in the front was the same as the distribution of urokinase-type plasminogen activator at the receptor (Pyke, C., et al., Cancer Research, 5).
5: 4132-9 (1995), the teachings of which are fully incorporated herein by reference), and the like.

Another embodiment of the invention is a targeted carrier comprising nucleotides and polymers. The polymeric component of the targeted carrier is a ligand capable of associating with a cellular or tissue marker. Cellular or tissue markers are selected from the group consisting of proteins, polypeptides, carbohydrates, lipids and nucleotides, and their derivatives. Cellular or tissue markers are glycolipids, glycoproteins, glycopeptides, transmembrane proteins, and extracellular matrix glycoproteins and proteoglycans, as well as other molecules present in tissues and exposed to the extracellular environment. It may be. It also includes intracellular components (eg, nucleosomes) that have been exposed to the extracellular environment in disease.

In yet another embodiment, the invention is a targeted drug-carrier complex comprising a nucleotide, a drug reversibly associated with the nucleotide, and a targeting moiety. The targeting moiety is associated with the nucleotide or drug. The targeting component comprises a ligand capable of associating with a cellular or tissue marker. The ligand can be associated with a cellular or tissue marker by, for example, covalent, non-covalent, or reversible association. The cell or tissue marker is selected from the group consisting of proteins, polypeptides, carbohydrates, lipids, and nucleotides. The drug is reversibly associated with the nucleotide. The targeting moiety is associated with either the drug or the nucleotide.

In yet another embodiment, the invention is a targeted drug-carrier complex comprising a nucleotide, a drug reversibly associated with the nucleotide, a polymeric component, and a targeting component. The polymer component is associated with nucleotides or drugs. The targeting moiety is associated with the nucleotide, drug, or polymer. The association between the targeting moiety and the drug or polymer can be, for example, a covalent bond, a non-covalent bond, or a reversible association. Targeting moieties include cell or tissue markers and ligands capable of associating with the drug. The association of the targeting moiety with the ligand can be, for example,
It can be covalent, non-covalent, or reversible. The cell or tissue marker is selected from the group consisting of proteins, polypeptides, carbohydrates, lipids, and nucleotides.

In a further embodiment, the invention provides for reversible association of a matrix with a nucleotide associated with or enclosed within the matrix (eg, van der Waals forces, electrostatic interactions). , Hydrogen bonds, ionic bonds, hydrophobic interactions, and donor / acceptor bound drugs) (FIG. 4).

In a preferred embodiment, the matrix of the drug delivery system is a gel, film or particles. The matrix provides the structural basis for the drug delivery system. Furthermore, the matrix can minimize any adverse reactions that the organism, cells, or tissue culture may have on the drug delivery system.
Preferably the matrix is biocompatible.

The matrix material is generally selected depending on the method of administration of the drug release system. In topical systems such as gels, films, patches and other systems of topical application, hydrophilic gels are frequently used as matrix materials. The gel is composed of a biocompatible polymer such as collagen, fibrin, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, polypropylene glycol, polyacrylate, or a combination thereof. Other systems, such as liniments, include emulsions, suspensions, and liposomal preparations, which may be mixed with each other or encapsulated in gels. Internal systems are often engineered based on either an injectable gel (eg, polyethylene glycol) or a biodegradable suture (eg, a copolymer of lactic acid and glycolic acid). Implantable drug delivery systems are described below as implants.

In general, it is preferred that the matrix of the drug release system remain stable as long as the drug release system remains functional, eg, the drug is released at the desired rate. In some cases, it may be desirable for the structural matrix of the drug release system to rapidly disintegrate after the drug is substantially released.
In one embodiment of the invention this effect is achieved by matrix stabilization by the drug. In a preferred embodiment, the matrix of drug release system 66 (eg, the hydrophilic polymer gel in FIG. 4) is a matrix material 68.
Is reversibly cross-linked via cross-hybridization of short oligonucleotides 70 chemically associated with (eg, covalently bound to) (FIG. 4). Preferably, the melting temperature of the double-stranded link formed as a result of hybridization is
It is around or below normal body temperature. The association of drug 72 with the double-stranded oligonucleotide stabilizes the double-stranded oligonucleotide, increasing the melting temperature and stabilizing the gel at body temperature (FIG. 4). Drug release lowers the melting temperature of the oligonucleotide link, which destabilizes the matrix (increasing the rate of matrix dissolution, biodegradation, or bioabsorption).

In yet another embodiment, the invention provides for a reversible association of an implant matrix with nucleotides associated with, or enclosed within, the matrix (eg, van der Waals forces, Implants with drugs that have electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions, and donor / acceptor bonds.

Implants that are capable of sustained drug release (implantable drug delivery systems) include, for example, long-term systemic drug delivery after a single dose, to the regional lymph nodes draining from the implantation site. It may be useful for drug delivery or post-operative local wound treatment. Implants consist of, for example, solid materials or gels and are in single block (
It may be made as a tablet, film) or may consist of a plurality of particles. Alternatively, the structural basis of the implant is sponge, foam (
It may be engineered as foam, fabric, thread or otherwise.

The implant can have any size and shape suitable for implantation. Granular, thread-like,
Alternatively, film-like implants may be more suitable for minimally invasive methods (such as implantation with needles or other surgical tools), and other types of implants may be more suitable for conventional surgery.

General requirements for implantable materials and devices are simple to apply and minimally invasive, calibrated for magnitude and course of biological effect (s). The risk of adverse reactions is minimal, the monitoring and maintenance is minimal, and in many applications it is also a requirement that complete biodegradation is achieved after the clinical objectives have been achieved. To be done. The implant should perform the correct biological function, eg, as mediated by the controlled release of the biologically active compound, and / or the structural of tissue and / or cell culture. And should be directed to functional maintenance, which may best be performed by a macromolecular or supramolecular matrix containing specialized functional domains. The matrix should be stable and biologically inert in vivo for a predetermined period of time and be fully degradable after its function is completed. Biodegradation should not produce toxic products or polymer deposits in the draining lymph nodes. Examples of implant matrix materials are silicone, copolymers of lactic acid and glycolic acid, agarose, porous metals (eg titanium), coral matrix, acrylates. Other possible materials include polyethylene glycol, polyacetals, polysaccharides, denatured or cross-linked proteins (eg albumin, gelatin) or natural proteins (eg collagen, fibrin). (Implantatio
n Biology, Greco, R.N. S. Chapter, CRC Press, Boc
a Raton, FL (1994); Holmdahl, L .; , Et al. ，
European Journal of Surgery-Supplemente
nt, 577: 56-62 (1997); Karel, I .; , Et al. , G
raefes Archive for Clinical & Experi
mental Ophthalmology, 235: 186-9 (1997)
Mikos, A .; G. , Et al. , Biotechnol. Bioeng
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Biomaterials, 14: 270-278 (1993); Galet.
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(1997); Chandrashhekar, G .; , Et al. , Journ
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materials Applications, 11: 182-257 (19)
96); Miller, B .; H. , Et al. , Journal of the
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: 72-7 (1997); Chowdhury, S .; M. , Et al. , Jo
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1996); Bourke, R .; D. , Et al. , Eye, 10: 501-
8 (1996); Rohrich, R .; J. , Et al. , Plastic
& Reconstructive Surgery, 98: 552-62 (1.
996); Gabka, C .; J. , Et al. , Seminars in S
urological Oncology, 12: 67-75 (1996); Raso.
, D. S. , Et al. , Journal of the American
Academy of Dermatology, 35: 32-6 (199
6); Yoshida, S .; H. , Et al. , Life Sciences
56: 1299-310 (1995); Sanchez-Guerrero,
J. , Et al. , New England Journal of Med
Icine, 332: 1666-70 (1995); Ahn, C .; Y. , Et
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-7 (1995); Sitting, M .; , Et al. , Biomate
Rials, 15: 451-6 (1994); Muzzarelli, R .; A.
, Et al. , Biomaterials, 14: 39-43 (1993);
Henderson, R.A. , Et al. , Spine, 18: 1268-72.
(1993); Tal, H .; , Et al. , Journal of Cli
natural Periodontology, 23: 1-6 (1996); Sm
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717-26 (1995); Nakayama, Y .; , Et al. , ASAI
O Journal, 4: M374-8 (1995); Schuman, L .; ，
et al. , Biomaterials, 16: 809-14 (1995);
Khare, A .; R. , Et al. , Biomaterials, 16 (7
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6-9 (1993); Karel, I .; , Et al. , Graefes Ar
chive for Clinical & Experimental Op
hthalology, 235: 186-9 (1997) (all these teachings are fully incorporated herein by reference).

Implants can be designed to release a dose of drug over a specified period of time. In one embodiment, the matrix is a material that is reversibly crosslinked by nucleotide / nucleotide associations. In another embodiment, release of the drug from the implant destabilizes the implant matrix.

The present invention is further described by the following examples, which are not intended to limit the present invention.

[0125]

【Example】

Example 1: Hybridization of single-stranded oligonucleotides Two types of custom-synthesized single-stranded oligonucleotides, (SEQ ID NO: 1) and t represents an amino-modified T (SEQ ID NO: 2) was purchased from a vendor. Each of these oligonucleotides was dissolved in 50 mM sodium phosphate buffer (PBS) at pH = 7 so as to have a concentration of 10 mg / ml.
Equimolar amounts of the above solutions were mixed at room temperature (25 ° C). The resulting solution (total volume 22
μl) was transferred to a 1 ml vial with a lid and the vial was heated to 95 ° C. in a 100 ml water bath for 10 minutes. The water bath was then cooled to 25 ° C. The resulting product, double-stranded oligonucleotide, was purified by size exclusion HPLC in water and lyophilized. Yield: 91%

Example 2: Formation and isolation of carrier-drug conjugates Oligonucleotides with an arbitrarily chosen sequence of 18 bases, (SEQ ID NO: 3) and a 5'amino-modified oligonucleotide complementary thereto, (SEQ ID NO: 4) was purchased from a commercial distributor. Single-stranded oligonucleotides were hybridized and lyophilized as described in Example 1 to form double-stranded oligonucleotides. The obtained double-stranded oligonucleotide was stable at room temperature (25 ° C) and body temperature (37 ° C).

Doxorubicin was dissolved in water at pH = 5, 0.2 mg / ml. The double-stranded oligonucleotide was dissolved in water at 2 mg / ml without pH adjustment. These solutions were mixed 0.1 ml each. Then, 0.3 ml of pH = 7 PBS
In addition, the resulting solution was incubated at room temperature for 10 minutes. After incubation, the reaction mixture was purified by gel chromatography on Sephadex G-25 in water. Doxorubicin eluate at 470 nm
Monitored photometrically at. Essentially all doxorubicin was present in the oligonucleotide fraction. In a control gel chromatography experiment, doxorubicin did not elute in the same fraction (see also Example 5). After gel chromatography, the doxorubicin-oligonucleotide adduct was lyophilized.
The lyophilized preparation was found to be readily soluble in water and aqueous media at physiological pH (pH = 7-8).

Example 3: Drug-Carrier Adducts with High Drug Content Essentially as described in Example 2, a ratio of doxorubicin: oligonucleotides that is approximately equivalent to one doxorubicin molecule per 4 base pairs. Ratio = 1: 5
(W / w) and the same double-stranded oligonucleotide were used to prepare high drug content drug-carrier adducts. The obtained adduct was subjected to gel chromatography (PD-
Purified by 10 columns, water) and lyophilized. Yield: 98 ± 2%.

Example 4: Lyophilization and reconstitution The adduct obtained as described in Examples 2 and 3 was used in the lyophilized state. Doxorubicin powder (Sigma Chemical Co., St. Louis, MO, St. Louis, Mo.) and lyophilized "doxorubicin for injection" were used as control preparations. Four
Each of the two preparations was made up to 0 mg doxorubicin
． Suspended in 1 ml of 50 mM PBS, pH = 7. Both oligonucleotide adducts dissolved immediately, but the two control preparations formed suspensions. 0.22 m of the resulting solution of the oligonucleotide-doxorubicin adduct solution
m PTFE membrane filter and recovered doxorubicin in the filtrate in a yield of at least 98% (by absorption at 470 nm). The suspension of the control preparation is 0.2
When filtered through a 2 mm PTFE membrane filter, the recovery of doxorubicin in the filtrate was lower than 5%. The rest was retained by the filter.

Example 5: Interaction between doxorubicin and Sephadex G-25 Sephadex G-25 (PD-10, Pharmacia (Pharmacia)
)) Was loaded on the column loaded with 0.1 ml of 0.1 mg / ml doxorubicin solution. The formation of the doxorubicin-Sephadex adduct was detected by the formation of a characteristic reddish layer. When eluted with water, the recovery of doxorubicin was 0% in the first 10 ml, followed by slow elution. When the same experiment was carried out with a doxorubicin adduct with the model carrier (Examples 2 and 3), the total amount of doxorubicin was eluted in the 2.5 ml fraction (2.5 to 5.0 ml).

In the next experiment, doxorubicin was adsorbed onto a PD-10 column and the column was washed with 10 ml H ₂ O, as described above. Then, 0.1 ml of the 1 mg / ml solution of the double-stranded oligonucleotide of Example 2 was passed through the same column. When the doxorubicin adsorbed on Sephadex G-25 was completely desorbed from the column,
Elution into the oligonucleotide fraction (2.5 to 5.0 ml).

Example 6: Electrophoresis of Doxorubicin Adducts with Model Drug-Carrier Complexes The electrophoretic mobilities of the adducts of Examples 2 and 3 were determined using a 0.8% horizontal agarose gel, 0.47 kV / m. , 0.01 M Tris-HCl buffer, pH = 8.
Doxorubicin was used as a control. The doxorubicin adduct and free doxorubicin migrated in opposite directions as measured by the fluorescence of doxorubicin.

Example 7: Modification of the carrier with polymer (steric structure protection) In order to measure the degree of conformational protection of the nucleotide core by the polymer chain, a series of double-stranded oligonucleotides were synthesized. Model antigen (fluorescein) and model protective chain (polyethylene glycol) were placed on the oligonucleotide at various intervals. Then, in order to evaluate the degree of conformational protection, fluorescence-quenched anti-fluorescein rabbit IgG (manufactured by Molecular Probes, Inc. in Oregon (
The kinetics of the interaction of the fluorescein moiety with Molecular Probes, OR)) was measured. The latter was investigated as a function of the distance between the antigen and the protective chain. Identical array Four types of amino-modified oligonucleotides having (SEQ ID NO: 5) were obtained from commercial suppliers. During the synthetic process, the oligonucleotide was further modified with fluorescein at either the 2nd, 9th, 16th or 20th position from the 5'end. In essence, these oligonucleotides (first strand) are complemented with complementary nucleotides, as described in Example 2. (SEQ ID NO: 6) (second strand) was hybridized.

The resulting double-stranded oligonucleotide was purified by SEC HPLC and, via the 5′-amino group of the first strand, N-hydroxysuccinimide ester of carboxy-polyethylene glycol (MW = 2 kDa and 20 kDa), and Modified with N-hydroxysuccinimide ester of branched "PEG2" polyethylene glycol (10 kDa). All polymers were purchased from Shearwater Polymers, Inc. The resulting conjugates were purified by SEC HPLC. A solution with almost the same oligonucleotide concentration (1 ± 0.1 nM) was added to 20 mM PBS, pH.
= 7.5. Anti-fluorescein IgG was added at a 10-fold concentration to ensure pseudo-primary conditions. Fluorescein-antibody interactions were recorded by quenching of fluorescein fluorescence by the antibody. Fluorescence was recorded at 515 nm (excitation at 490 nm). The reaction rate of fluorescence quenching depended on the position of the fluorescein moiety with respect to the polymer chain.

Polymer chains located at 16-20 bases did not attenuate the fluorescein-antibody interaction, whereas polymer chains located in the 2nd to 9th bases had a rate constant of 20. % (2 kDa polymer) down to 80% (20 kDa polymer). That is, by optimizing the distance between the polymer chains (the number of chains per base pair) and the molecular weight of the polymer, the degree of protection of the nucleotide conformation can be optimized. Based on the data described in the examples below, even 50% to 80% of the hindrance of the carrier core (protein binding kinetics (protei
n access kinetics)) and may be sufficient to delay the circulation of the carrier within a few hours.

Example 8: Sterically Protected (Polymer Modified) Model Carriers Two types of model carriers were prepared for conducting biological experiments. Array (SEQ ID NO: 7) and A complementary oligonucleotide having (SEQ ID NO: 8) (lowercase “c” indicates the base of RNA) was custom-synthesized. One of the carriers was prepared essentially as described in Example 2 with unmodified oligonucleotides. Amino-modified at the 1st, 2nd, 12th, and 19th positions from the 5'end of the oligonucleotide Another carrier was prepared using (SEQ ID NO: 9). Oligonucleotides were hybridized in water at pH = 7 with a total oligonucleotide concentration of 1 mg / ml and purified by HPLC. The amino-modified carrier was treated with a 10 kDa poly (hydroxymethylethylene hydroxymethylformal) containing 20% aldehyde groups (
PHF). The latter polymer is capable of oxidizing dextran B512 with 1.8 periodate molecules per sugar ring, followed by reduction with borohydride and subsequent periodate oxidation of the resulting glycol groups. Prepared by Aldehyde-PHF (50-fold excess) was reacted with the amino-modified carrier core in the presence of cyanoborohydride (1 mole per mole of aldehyde) overnight at room temperature. Product (oligonucleotide-
PHF conjugate) was isolated by SEC HPLC. Both carriers were freeze dried.

Example 9: Binding of drug-carrier complex to model antibody P is structurally close to the carrier of Example 8 but contains a glycol group in the polymer chain.
The HF-modified carrier was prepared using an improved technique. The amino-modified carrier was modified with 10 kDa poly (hydroxymethylethylene hydroxymethylformal) (PHF) containing 10% aldehyde groups and 10% glycol groups. The latter is an incomplete periodate oxidation of the same polymer as in Example 8 (prepared by oxidation of dextran B512 with 1.8 periodate molecules per sugar ring followed by reduction with borohydride). Prepared. 1 ml of this carrier (0.1 mg)
10 mM periodate for 5 minutes, purified on a PD-10 column, and rabbit anti-fluorescein IgG (0.01 mg) (Molecular Probes, Oregon) and 0.1 mg / ml cyanoboro. In the presence of sodium hydride,
Binding was achieved by overnight incubation at pH = 8, 25 ° C. The conjugate was separated from unreacted IgG by HPLC (yield by IgG absorption at 280 nm: 22 ± 9%). The presence of active IgG in the conjugate was determined by quenching of fluorescein fluorescence (10 nM fluorescein, pH = 8, 25 ° C.). The amount of active IgG in the carrier was calculated to be 1 ± 0.3% w / w.

Example 10: Carrier conjugate with lactose Amino-modified carrier prepared as in Example 8 was treated with 10 mM PBS.
, Trace amounts of diethylenetriaminepentaacetic acid (diethylen at pH = 8)
Modified with triaminepentaacetic anhydride. This product was conjugated with lactose by incubation in the presence of 1 mg / ml sodium cyanoborohydride at 10 mg / ml each of carrier and lactose at pH = 8 for 48 hours. The product was isolated by HPLC.

Example 11: Modification of drug-carrier adducts with PHF via (aminooxy) doxorubicin. N- (3-aminooxy-2-hydroxypropyl) -doxorubicin (AHD), which is an aminooxy derivative of doxorubicin, is prepared by converting doxorubicin into N-oxiranylmethoxyethaneamidic acid (N-oxyranylmethoxyethyleth
anamidic acid) ethyl ester and then the product
Synthesized by treating with N HCl. The product was purified by thin layer chromatography.
A carrier loaded with AHD was prepared in the same manner as in Example 2. The obtained adduct was combined with aldehyde-PHF (polymer described in Example 8) at pH = 6.5, 25 ° C.
And incubated for 24 hours. SEC HPLC (BioRad (BioR
The conjugate was isolated by ad) BioSil 125 column). By reducing the elution time of the adduct (6.2 minutes vs. 8.5 minutes for the unmodified adduct),
Polymer modification of the adduct was detected. In a control experiment, polymer incubation with a similar carrier without loading, the elution time of the latter was unchanged.

Example 12: Radioactively labeled doxorubicin loaded carrier ¹⁴ C-labeled doxorubicin (Amersham, 2.
00 GBq / mmol) 25 mCi was dissolved in 100 ml of water. 10 ml of this solution is then added to 2 mg / ml of unlabeled (“cold”) doxorubicin 0.25.
mixed with ml. The carrier of Example 8 was dissolved in water (nucleotide core: 11 mg /
ml), mixed with a calculated amount of doxorubicin solution to give an adduct with one doxorubicin molecule per 10 base pairs. After incubation for 10 minutes, the adduct was purified with Sephadex G-25 and lyophilized. Using high molecular weight DNA (Poly C / Poly G, Sigma Chemical Co., St. Louis, Mo.)
A similar complex was prepared. A similar cold (unlabeled) preparation was prepared using unlabeled doxorubicin.

Example 13 Toxicity Carrier toxicity in cell culture. The unlabeled carrier (Example 7) was examined in human epithelial cells (ATCC No. CRL1573 derived, kidney, 293 cell line) cultured in DMEM containing 5% calf serum in a substantially confluent state. 1 carrier
Various concentrations were added to the cell culture from mg / ml to 1 mg / ml. 16
After incubation for a period of time, cell viability was measured by staining with trypan blue. No cytotoxicity was detected at all carrier concentrations.

Adduct toxicity in cell culture. Carrier loaded with doxorubicin (Example 1
2) was investigated in human epithelial cells (kidney, 293 cell line) at near confluence in DMEM with 5% calf serum and in the growing cell culture. This cell line is relatively resistant to doxorubicin. After incubating for 16 hours, cell viability was measured by staining with trypan blue. No cytotoxicity was detected at concentrations of doxorubicin up to 2 mM in confluent cultured cells, whereas in proliferating cultured cells 15-20% of the cells at 2 mM were stained with trypan blue. In control experiments free doxorubicin showed essentially the same cytotoxic effect. This example demonstrates that association of doxorubicin with a nucleotide-based carrier did not increase doxorubicin toxicity in resting cells or suppress cytotoxicity in dividing cells. .

In vivo carrier toxicity. The conformationally protected model carrier of Example 8 was injected intravenously at 100 mg / kg via the tail vein of anesthetized outbred mice (n = 6, male, 31 ± 2 g). . Animals were observed for 30 days. None of the animals showed signs of toxicity. All animals survived.

Adduct toxicity in vivo. References (Chaires, JB, et al., JM
ed. Chem. 40: 261-6 (1997), the teachings of which are conformationally protected, as described in Example 8, herein incorporated by reference in its entirety. In (), the two daunorubicin molecules are αα-
The bis-intercalator WP-631 synthesized by cross-linking with dibromo-p-xylene was loaded. The content of bis-intercalator was 1 molecule per 6 base pairs of the nucleotide core. This bis-intercalator D
The NA adduct was highly stable and the release of the bis-intercalator was slow (measured with 0.9% NaCl, 10 mM PBS, pH = 7.5,
The half-life of release was 9 ± 1 hour). Male outbred mice (30 ± 2 g, n = 4 per group) were dosed intravenously with 30 mg / kg adduct and free bis-intercalator as described above. 30m in the third group
Within 15 minutes after administration of g / kg bis-doxorubicin, 100 mg / k
g unloaded carrier was injected.

The mice were observed for 30 days. Only one animal survived in the free bis-intercalator injected group, whereas all animals survived in the adduct injected group. After administration of bis-doxorubicin, all animals survived in the vehicle-treated group. These data indicate that the drug is associated with the drug-carrier complex of the invention, presumably resulting in reduced concentration of free drug in plasma and thus reduced toxicity in vivo. There is.

[0146] Example 14: Bioreactivity nonnegative carriers of the carrier (Example 8) was labeled with tritium ⁽³ H). By oxidizing carrier with periodate present in the structure 3'ribonucleotide on bases out create an aldehyde group, is reacted with ^[3 H] borohydride, tritium in the core structure of the carrier Introduced. The carrier was purified with Sephadex G-25 and 1 mg / kg was injected into normal outbred mice (male, 30 g) via the tail vein. Blood samples were collected and measured at various time points. The half-life in blood was about 30 minutes and 10 hours for the unprotected carrier and the PHF-protected carrier, respectively. In this example, it is possible to optimize the circulation time of a carrier having a nucleotide as a main raw material by protecting the three-dimensional structure,
It is shown that a carrier that circulates for a long time can be prepared by modification with a hydrophilic polymer.

Example 15: Biodistribution of doxorubicin A carrier loaded with ( ¹⁴ C) doxorubicin (Example 12) and free ( ¹⁴ C) doxorubicin (control) were treated with normal outbred mice (male, 30 g). ) Was injected at 0.3 mg / kg via the tail vein. Twenty hours after the injection, the radioactivity of the tissue was measured to examine the biodistribution. Tissue samples were disintegrated, solubilized, mixed with scintillation mixture and counted in a scintillation counter. Although the doxorubicin-DNA complex is known to be relatively unstable, significant differences in label accumulation were seen.

Administration of doxorubicin as an adduct with an unprotected oligonucleotide carrier resulted in minimal deviation from the biodistribution of free doxorubicin. Administration of doxorubicin adducts with PHF-protected carriers resulted in a 2-fold higher labeling content in skeletal muscle, ie 6 ± 2 vs 2 ± 0.4% dose / g (mean ± standard deviation). It was Doxorubicin adducts with DNA showed that the lungs (7 ± 3.1% vs 4.6 ± 1.
6% dose / g) and increased labeling distribution to the liver (12 ± 6 vs. 3 ± 0.6% dose / g). These data indicate that when a drug that binds to DNA is administered as an adduct with a carrier whose main ingredient is nucleotides, the biodistribution of the drug can change significantly. In particular, the doxorubicin-DNA association is less stable than other intercalators. Other stronger DNA binding factors (bind
er), the effect of redistribution may be more significant and may generally depend on the particular combination of nucleotide-based carrier and drug substance.

Example 16: Model carrier based on pCMV pH containing 10 mg / ml pCMV (Promega), pH
0.1 ml of a plasmid solution of PBS = 7.5 was mixed with 10 ml of a 1 mg / ml solution of bis-doxorubicin (described in Example 13). After incubating for 1 hour, the adduct was treated with SEC HPLC (BioRad BioS BioS).
il 125 column). Association of bis-doxorubicin with plasmid was detected by the appearance of absorbance at 470 nm in the excluded volume.

Example 17: Model Gel Hydrophilic polyacetal matrix material with 20% aldehyde substitution (PHF
), Molecular weight = 250 kDa was prepared as described in Example 9. 1 ml of 5
50 mg of polymer in 0 mM PBS, pH = 8 1 mg of the 3'amino-modified oligonucleotide having the sequence: was bound in the presence of sodium cyanoborohydride (1 mg / ml). Similarly, A conjugate of complementary 3'amino-modified oligonucleotides having the following sequence was prepared. Both oligonucleotides were custom made by the vendor.

The two kinds of conjugates were dissolved so as to be 50 mg / ml and then mixed. Immediately after mixing, the viscosity of the mixture increased rapidly. 0 in 10 ml DMSO
． 5 mg daunomycin was added to this mixture. Immediately thereafter, the mixture formed a gel, presumably as a result of stabilizing the intermolecular ligation of the double-stranded oligonucleotide by intercalation of daunomycin. This gel pellet (disk, d = 15 mm, h = 5 mm) was treated with 1 liter of 0.9% NaCl, pH = 7.
． Incubated in 5 with slow agitation. The release of daunomycin was recorded spectrophotometrically. The first half-release was found to be 40 ± 12 minutes.

Example 18: Oligonucleotide binding sequence to polylysine A 5'amino-modified oligonucleotide having (SEQ ID NO: 10) is hybridized with a complementary oligonucleotide, and the product is treated with 50 mM PBS.
, PH = 8.5, treated with succinic anhydride 1 mg / ml (each reagent) at room temperature for 3 hours. The product was purified by SEC on Sephadex G-25 (PD-10 column). 0.1 ml of a 2 mg / ml solution of 20 kDa polylysine hydrobromide in 1 mg / ml oligonucleotide solution in PBS, pH = 6
． Mixed with 1 ml. Then, 1 mg of ethyl- (N-dimethylaminopropyl) carbodiimide (EDC) in 0.1 ml of water was added to the mixture at 4 ° C and the mixture was incubated at 4 ° C overnight, then at 25 ° C for 3 hours. Incubated. The product was desalted on Sephadex G-25 (PD-10). SEC H
The zygote was isolated by PLC.

Example 19: Conformational Protection of Oligonucleotide-Polylysine Conjugates By reacting PHF with mercaptopropionic acid, followed by modification with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide,
N-hydroxysuccinimide ester of terminal-carboxypropylthio-PHF was prepared. The conjugate synthesized in Example 18 (approximately 0.1 mg in 1 ml of water) was mixed with 0.5 ml of 50 mM borate buffer, pH = 8. 10 mg of N-hydroxysuccinimide ester of terminal-carboxypropylthio-PHF was added to 0.
Dissolved in 1 ml DMSO. The solutions were mixed and incubated overnight at room temperature. The product was isolated by size exclusion HPLC (BioSil 125 column).

Example 20: Conformational Protected Adducts of Model Oligonucleotides An arbitrarily selected 5-amino modified 18 base phosphodiester oligonucleotide, (SEQ ID NO: 11), 0.11 mg modified with a trace amount of ethylenediaminetetraacetic anhydride, (a) a complementary oligonucleotide, (SEQ ID NO: 12), and (b) hybridized with similar complementary oligonucleotides that were amino modified at the 5'and 3'ends, and at positions 4, 9 and 15 (counting from 5 '). . Molecular weight of the hybridized amino-modified DSO was
10 kDa N-hydroxysuccinimide-polyethylene glycol 10 m
Incubated with 1 ml of g / ml solution for 8 hours at pH = 8. The product, a PEG-modified double-stranded oligonucleotide, was isolated by HPLC. PEG
Modified double-stranded oligonucleotide and unmodified double-stranded oligonucleotide,
Labeled with 111 in 0.5M citrate buffer at pH = 5.6. The labeled preparation was purified by SEC HPLC. Divide each into 4 equal doses,
Inbred mice (n = 4 in each group, male, 31 ± 2 g) were injected intravenously. Blood samples were taken immediately after injection and after 0.25, 0.5, 1, 2, 4 and 8 hours. The half-lives in blood of the model oligonucleotides in unmodified and polymer modified adducts were found to be 12 ± 5 minutes and 4.5 ± 0.7 hours, respectively.

Equivalents While the present invention has been particularly shown and described with reference to the preferred embodiments of the invention, those skilled in the art will appreciate from the spirit and scope of the invention as defined in the appended claims. It will be appreciated that various formal and detailed changes can be made to these without departing.

[Brief description of drawings]

FIG. 1A is a diagram showing the intercalation of doxorubicin (dark shade) with B-helical DNA in a space-filling model of one embodiment of the drug-carrier complex of the present invention. is there. FIG. 1B is a diagram showing intercalation of doxorubicin (dark shade) with B-helical DNA in a stick-ball model of another embodiment of the drug-carrier complex of the present invention.

FIG. 2 is a schematic diagram of a sterically protected embodiment of a drug-carrier complex of the present invention.

3A, 3B, 3C, 3D and 3E of a drug delivery complex of the invention, depicting various arrangements of drug component, polynucleotide or oligonucleotide component, and polymer component of the complex. FIG. 6 shows a further embodiment.

FIG. 4 is a schematic representation of a method of forming a drug delivery system of the present invention by combining a drug with a gel matrix that is cross-linked by mutually hybridizing nucleotide chains.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/282 A61K 31/282 4C086 31/704 31/704 4C206 31/7088 31/7088 31/7105 31 / 7105 33/24 33/24 38/00 45/00 45/00 47/48 47/48 49/00 A 49/00 C 49/04 A 49/04 A61P 31/04 51/00 31/12 A61P 31 / 04 33/02 31/12 35/00 33/02 43/00 123 35/00 G01N 33/15 Z 43/00 123 33/50 Z C12N 15/09 ZNA 33/58 A G01N 33/15 A61K 49/02 A 33/50 37/02 33/58 C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW , ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, Y U, ZA, ZW F term (reference) 2G045 AA25 AA40 BB20 BB46 BB48 CB26 DA12 DA13 FB02 FB07 4B024 AA01 AA11 CA04 HA17 4C076 AA09 AA51 AA61 AA94 AA95EE38FF FF EEEEFF EE FF EEEEEEFF FF67 GG01 GG21 4C084 AA02 AA03 AA13 AA17 BA03 MA05 MA28 MA36 MA38 NA03 NA05 NA06 NA12 NA13 NA14 NA15 ZB26 ZB33 ZB35 ZB38 4C085 HH03 HH05 HH07 HH11 JJ03 JJ11 KA01 KA09 KA10 KA11 KA27 KA28 KA29 KA30 KA36 KB07 KB78 KB91 LL18 4C086 AA01 AA02 EA10 EA16 HA01 HA12 MA01 MA05 MA28 MA36 MA67 NA03 NA05 NA06 NA12 NA13 NA15 ZB26 ZB33 ZB35 ZB38 4C206 AA01 AA02 JB16 MA01 MA05 MA48 MA56 MA58 MA87 NA03 NA05 NA06 NA12 NA13 NA15 ZB26 ZB33 ZB35 ZB38

Claims (96)

[Claims] 1. A step of combining at least one nucleotide chain with a drug, whereby the drug and the nucleotide chain are reversibly associated with each other, the drug-
A method of forming a drug-carrier complex, which comprises forming a carrier complex. 2. The method of claim 1, wherein the drug is an oligonucleotide. 3. The drug according to claim 1, which is an antisense oligonucleotide.
The method described. 4. The method of claim 1, wherein the drug is a ribozyme. 5. The method according to claim 1, wherein the drug contains a component selected from the group consisting of an intercalator, a metal-containing substance, a minor groove binder and a major groove binder. 6. The method of claim 1, wherein the drug comprises at least one amino group. 7. The method of claim 1, wherein the drug is combined with at least two nucleotide chains that hybridize to each other. 8. The method of claim 1, wherein a second nucleotide chain is combined with the drug-carrier complex such that the second nucleotide chain hybridizes to the nucleotide chain of the drug-carrier complex. . 9. A drug-carrier complex comprising the step of combining a drug with at least two nucleotide chains that hybridize to each other, whereby said drug associates with said nucleotide chain to form a water-soluble drug-carrier complex. How to form a body. 10. The method of claim 9, further comprising the step of lyophilizing the dissolved drug-carrier complex. 11. A drug-carrier comprising: a) combining a drug component and a nucleotide component; and b) lyophilizing the combined drug component and nucleotide component, thereby forming a drug-carrier composition. A method of forming a composition. 12. The method of claim 11, wherein at least one of the drug component and the nucleotide component is dissolved in water prior to combining the components. 13. A) lyophilizing the drug component; b) lyophilizing the nucleotide component; and c) combining the lyophilized drug component and the lyophilized nucleotide component, thereby providing the drug. A step of forming a carrier composition, and a method of forming a drug-carrier composition. 14. A drug comprising: a) a double-stranded nucleotide; and b) a polymer component covalently attached to at least one strand of said double-stranded nucleotide having a water solubility of at least 1 mg / liter at 25 ° C. Carrier. 15. The drug carrier according to claim 14, wherein the polymer component is a biocompatible polymer component. 16. The drug carrier according to claim 15, wherein the biocompatible polymer component is crosslinked. 17. The drug carrier according to claim 15, wherein the biocompatible polymer component is selected from the group consisting of polysaccharides, polyethers and polyacetals. 18. The drug carrier according to claim 17, wherein the polysaccharide is selected from the group consisting of chondroitin sulfate, poly α-D-glucose, polysialic acid, dextran, starch and derivatives thereof. 19. The drug carrier according to claim 17, wherein the polyacetal is selected from the group consisting of poly (hydroxymethylethylenehydroxymethylformal) and its derivatives. 20. The drug carrier of claim 14, wherein the polymeric component comprises at least one polymer covalently attached to at least one strand of the nucleotide. 21. The drug carrier according to claim 14, wherein the polymer component comprises at least two polymers. 22. The drug carrier of claim 14, wherein the polymer component comprises at least two chemically different polymers. 23. A drug carrier comprising: a) a double-stranded nucleotide; and b) an oligomeric component covalently attached to at least one strand of said double-stranded nucleotide. 24. The oligomer component contains at least one member selected from the group consisting of oligosaccharides and oligopeptides.
The described drug carrier. 25. The oligomeric component comprises at least one of the nucleotides.
3. Containing at least one oligomer covalently attached to one chain.
3. The drug carrier according to item 3. 26. The drug carrier according to claim 23, wherein the oligomer component comprises at least two oligomers. 27. The drug carrier according to claim 26, wherein the oligomer component comprises at least two chemically different oligomers. 28. a) a single-stranded nucleotide; b) a drug reversibly associated with the single-stranded nucleotide; c) a polymer associated with the single-stranded nucleotide or the drug, A drug-carrier complex comprising: 29. The drug-carrier complex of claim 28, wherein the polymer is covalently associated with the single-stranded nucleotide. 30. The polymer is associated with the drug.
The described drug-carrier complex. 31. a) a single-stranded nucleotide; b) an oligomer associated with said single-stranded nucleotide; c) a drug reversibly associated with the oligomer or the single-stranded nucleotide, A drug-carrier complex comprising: 32. The drug-carrier complex of claim 31, wherein the oligomer is covalently associated with the single-stranded nucleotide. 33. The oligomer according to claim 3, wherein the oligomer is associated with the drug.
1. The drug-carrier complex according to 1. 34. A drug-carrier comprising: a) a single stranded nucleotide; and b) at least two polymers associated with said single stranded nucleotide. 35. a) an oligomer; b) a single-stranded nucleotide encapsulated by said oligomer; and c) a drug reversibly associated with the single-stranded nucleotide, A drug-carrier complex comprising: 36. a) a nucleotide carrier component; and b) drug components, A drug-carrier composition comprising: and a water content of less than about 5% by weight. 37. A drug-carrier composition consisting essentially of a drug component and a nucleotide component. 38. a) a nucleotide carrier component; and b) a drug that is reversibly associated with the nucleotide carrier component, A pharmaceutical formulation comprising: 39. The pharmaceutical preparation according to claim 38, wherein the drug is an oligonucleotide. 40. The pharmaceutical preparation according to claim 38, wherein the drug is an antisense nucleotide. 41. The pharmaceutical preparation according to claim 38, wherein the drug is a ribozyme. 42. The pharmaceutical preparation according to claim 38, wherein the drug contains a component selected from the group consisting of an intercalator, a metal, a minor groove binder, and a major groove binder. 43. The pharmaceutical preparation according to claim 38, wherein the drug contains an amino group. 44. The reversible association between the drug and the nucleotide carrier component is from the group consisting of van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions and donor / acceptor bonds. 39. A pharmaceutical formulation according to claim 38, which comprises at least one member selected. 45. The pharmaceutical preparation according to claim 44, further comprising a reversible covalent bond between the drug and the nucleotide carrier component. 46. The pharmaceutical formulation of claim 38, wherein the reversible association between the drug and the nucleotide carrier component is intercalation. 47. The pharmaceutical preparation according to claim 38, wherein the nucleotide carrier component is a polynucleotide carrier component. 48. The pharmaceutical preparation according to claim 38, wherein the nucleotide carrier component is an oligonucleotide carrier component. 49. The pharmaceutical preparation according to claim 38, wherein the drug is a therapeutic drug. 50. The pharmaceutical preparation according to claim 38, wherein the drug is a diagnostic drug. 51. The diagnostic drug is a radioactive diagnostic drug, a fluorescent diagnostic drug, a paramagnetic diagnostic drug, a superparamagnetic diagnostic drug, a high x-ray density diagnostic drug and a high electron density diagnostic drug. The pharmaceutical preparation according to claim 50, which is selected from the group consisting of: 52. The pharmaceutical preparation according to claim 38, wherein the drug is a protein. 53. The pharmaceutical preparation according to claim 38, wherein the drug is a chemical selected from the group consisting of intercalators, metals, minor groove binders, and major groove binders. 54. The pharmaceutical preparation according to claim 38, wherein the drug comprises a diagnostic label. 55. A method of delivering a drug to an organism, comprising the step of administering to the organism a drug-carrier complex comprising a drug and a nucleotide carrier reversibly associated with each other. 56. The reversible association of the drug with the nucleotide carrier is selected from the group consisting of van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions and donor / acceptor bonds. Item 55. The method according to Item 55. 57. The method of claim 55, wherein the reversible association between the drug and the nucleotide carrier component is intercalation. 58. The method of claim 55, wherein the organism is selected from the group consisting of mammals and cells. 59. The method of claim 55, wherein the nucleotide carrier is administered to the organism. 60. The method of claim 55, wherein the nucleotide carrier is administered proximal to the organism. 61. A method of delivering a drug to a tissue culture comprising administering to the tissue culture a drug-carrier complex comprising a drug and a nucleotide carrier reversibly associated with each other. 62. The reversible association of the drug with the nucleotide carrier is selected from the group consisting of Van der Waals forces, electrostatic interactions, hydrogen bonds, ionic bonds, hydrophobic interactions and donor / acceptor bonds. Item 61. The method according to Item 61. 63. The method of claim 61, wherein the reversible association between the drug and the nucleotide carrier component is intercalation. 64. The method of claim 61, wherein the nucleotide carrier is administered to the tissue culture. 65. The method of claim 61, wherein the nucleotide carrier is administered proximal to the tissue culture. 66. A method of delivering a drug to an organism, comprising the step of administering to the organism a drug and a nucleotide chain that reversibly associates with the drug to form a drug-carrier complex. 67. The method of claim 66, wherein the drug and the nucleotide chain are administered to the organism at the same time. 68. The method of claim 66, wherein the drug and the nucleotide chain are administered to the organism separately. 69. A) forming a drug-carrier complex comprising a drug and a nucleotide chain reversibly associated with the drug; and b) administering the drug-carrier complex to an organism. A method of delivering a drug to an organism, comprising: 70. A method of delivering a drug to an organism, comprising the step of administering a drug-carrier complex comprising a drug component and a carrier component to the organism, wherein the drug component and the carrier component are reversible with each other. A method wherein the drug is associated, whereby the drug can dissociate from the drug-carrier complex and reassociate with the carrier component. 71. The method of claim 70, wherein the carrier is a nucleotide carrier. 72. The method of claim 70, wherein said drug-carrier complex is delivered to a combination of cells in said organism. 73. The method of claim 72, wherein the combination of cells is a tumor. 74. The method of claim 72, wherein the combination of cells is pathogenic. 75. The method of claim 72, wherein the combination of cells is a tissue culture. 76. The method of claim 72, wherein the combination of cells is an organ. 77. The method of claim 70, wherein the drug-carrier complex dissociates within the organism near or within a combination of cells. 78. The method of claim 77, wherein the drug-carrier complex is administered to the organism at a location remote from the cell combination. 79. A step of reversibly associating a substance with a nucleotide carrier, whereby the substance and the nucleotide carrier form a water-soluble complex.
A method of increasing the water solubility of a substance. 80. The method of claim 79, wherein the substance is essentially insoluble in water. 81. a) a nucleotide; b) a polymeric component associated with said nucleotide; and c) associated with said nucleotide or said polymeric component and selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides. A targeting carrier comprising a ligand capable of associating with a cell marker or a tissue marker. 82. a) a nucleotide; and b) a polymeric component associated with said nucleotide which is a ligand capable of associating with a cell or tissue marker selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides. A targeting carrier consisting of 83. a) a nucleotide; b) a drug reversibly associated with the nucleotide; and c) a ligand capable of associating with a cell or tissue marker selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleotides. A targeting drug-carrier complex comprising: the nucleotide or a targeting moiety associated with the drug. 84. a) a nucleotide; b) a drug reversibly associated with the nucleotide; c) a polymeric component associated with the nucleotide or the drug; and d) selected from the group consisting of proteins, polypeptides, carbohydrates and lipids. A targeting drug-carrier complex comprising a nucleotide, a targeting moiety associated with the drug or the polymer, comprising a ligand capable of associating with a cell marker or a tissue marker. 85. A drug delivery system comprising: a) a matrix; b) nucleotides associated with or encapsulated in the matrix; and c) a drug reversibly associated with the nucleotides. 86. The drug delivery system of claim 85, wherein the matrix is a gel. 87. The drug delivery system of claim 85, wherein the matrix is a film. 88. The drug delivery system of claim 85, wherein the matrix is particles or vesicles. 89. The drug delivery system of claim 85, wherein the matrix is a material that is reversibly crosslinked via nucleotide / nucleotide associations. 90. The drug delivery system of claim 85, wherein drug release destabilizes the matrix. 91. An implant comprising: a) an implant matrix; b) nucleotides associated with or encapsulated in the matrix; and c) a drug reversibly associated with the nucleotides. 92. The implant of claim 91, wherein the matrix is a biocompatible gel. 93. The matrix of claim 91, which is a biocompatible film.
The implant described. 94. The matrix is biocompatible particles, vesicles, foams,
93. The implant of claim 91, which is selected from the group consisting of fabrics, sutures, and sponges. 95. The implant of claim 91, wherein the matrix is a material that is reversibly crosslinked via nucleotide / nucleotide associations. 96. The implant of claim 91, wherein drug release destabilizes the implant matrix.