JP2016130238A - Chronic obstructive pulmonary disease (copd) treatment with epigenetic control carrier having antioxidative function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel therapeutic agent which can treat oxidative damage due to reactive oxygen species (ROS) and epigenetic abnormality simultaneously, particularly useful for the treatment of chronic obstructive pulmonary disease (COPD).SOLUTION: The present invention provides a complex for co-deliverying an antioxidant and a gene to an object, comprising a carrier capable of co-deliverying an antioxidant and a gene, an antioxidant, and a plasmid DNA. The carrier is preferably liposome, biodegradable polymer, or lipid-coated biodegradable polymer.SELECTED DRAWING: None

Description

  The present application relates to a complex for co-delivery of an antioxidant and a gene to a subject. The complex includes a carrier capable of co-delivery of a gene with an antioxidant, an antioxidant, and plasmid DNA. The application also relates to complexing agents capable of co-delivery of genes with antioxidants. The application further relates to a pharmaceutical composition comprising the complex.

  Since the 1960s, it has been recognized that histone acetylation and close remodeling of chromatin structure are related to gene induction, but the molecular mechanism by which genes are transcriptionally activated by transcription factors and histone acetylation. It has only recently been understood. Changes in chromatin structure are important for gene expression control, and such a mechanism for controlling gene expression by structural change of chromatin is called an epigenetic mechanism. The chromatin structure is changed by an acquired chemical modification such as histone acetylation or DNA / histone methylation as described above, and this acquired modification can be caused by various factors.

  In recent years, it has been reported that epigenetic abnormalities are related to the onset of disease and pathological deterioration, and it is necessary to take into account normalizing epigenetic abnormalities in the treatment of diseases.

On the other hand, reactive oxygen species (ROS) are produced from 0.4 to 4% of oxygen consumed in mitochondria that perform energy metabolism essential for biological activities. ROS includes superoxide (O2 · ⁻ ), hydrogen peroxide (H ₂ O ₂ ), peroxynitrite ion (ONOO ⁻ ), and very reactive hydroxyl radicals (· OH). ROS is used for biological defense and intracellular signal transmission, and plays an important role in the living body. On the other hand, it is highly reactive to oxidatively denature biomolecules such as DNA and lipids. It is known to cause various cell damage. Thus, the ROS in the biological function is a “double-edged blade”. Normally, the ROS that has finished its role is quickly reduced and erased by an antioxidant that eliminates ROS present in the living body. However, when excessively generated ROS impairs homeostasis, ROS induces organ-level damage and causes serious diseases such as arteriosclerosis and renal failure.

  Thus, it is clear that the epigenetics and reactive oxygen species (ROS) mentioned above are involved in pathology, but in recent years it has begun to be reported that these two are related to each other. The usefulness of simultaneously improving genetic abnormalities has been suggested.

For example, it is known that amyloid beta (Aβ), which is considered to be one of the causes of Alzheimer's disease, aggregates due to oxidative crosslinking with hydrogen peroxide and is involved in pathological progression. At the same time, it has been reported that hydrogen peroxide contributes to hypomethylation of the promoter region of the gene responsible for the production of Aβ precursors, thereby causing an increase in Aβ production. In addition, in human colon cancer cell SNU-407 exposed to excessive hydrogen peroxide, the tumor suppressor gene is increased by increasing methylation of the promoter region of Runt domain transcription factor 3 (RUNX3), which is a tumor suppressor gene. Is involved in the progression of colorectal cancer through an epigenetic mechanism along with oxidative damage by ROS. In chronic obstructive pulmonary disease (COPD), ONOO ⁻ directly nitrates histone deacetylase (HDAC2), or ROS such as H ₂ O ₂ ubiquitinates and phosphorylates via the PI3kσ-Akt process. Proteasome degradation is promoted by applying acquired modifications. This decreases the expression and activity of HDAC2, resulting in increased expression of inflammatory genes, resulting in a chronic inflammatory response (Non-Patent Documents 1 and 2).

  Thus, it has been found that ROS not only causes disease by directly causing oxidative damage, but also promotes disease by causing epigenetic abnormalities. Because both affect each other and cause the disease, it is highly likely that the treatment with only one of the two will cause the disease again, and treating oxidative damage and epigenetic abnormalities due to ROS at the same time is a radical treatment. Is considered important.

Malhotra, D., et al., J. Clin. Invest., 121 (11): 4289-4302, 2011 P. J. Barnes, et al., Eur. Respir. J., 25: 552-563, 2005

  As described above, there is a demand for the development of a new treatment method that enables simultaneous treatment of oxidative damage caused by reactive oxygen species (ROS) and epigenetic abnormalities.

  For example, inflammatory diseases are intractable diseases that lead to abnormal production of inflammatory cytokines due to overexpression of inflammatory genes and tissue destruction due to chronic inflammatory reactions. Among them, chronic obstructive pulmonary disease (COPD) is estimated to have 210 million affected people, and is one of the four major adult diseases in the world after cancer, myocardial infarction, and stroke. For the disease groups other than COPD, prevention and treatment have progressed and the mortality rate has been halved in the past quarter century, whereas the mortality rate of COPD has never decreased, and has continued to increase. In COPD, smoking is the main onset mechanism, but air pollution is considered to have an impact, and it is highly likely that patients will continue to increase in the future. Although the estimated number of patients in Japan is said to be 7 million, the number of patients actually receiving treatment is about 500,000, and the increase in the number of patients will surely occur. There are concerns that it will rise to the third leading cause of death in 2030. Currently, there is no radical treatment for COPD, and progress is prevented by coping therapy such as smoking cessation, oxygen therapy, and steroid administration. Furthermore, in Japan, where the super-aging population is advancing, the number of COPD patients is expected to increase at an accelerating rate, and the development of new treatments is urgently needed.

  The present application provides for the removal of reactive oxygen species and the improvement of epigenetic abnormalities for diseases having pathological mechanisms based on epigenetics, that is, the improvement of abnormal expression of enzymes or proteins involved in epigenetics control, An agent capable of simultaneously performing the above is provided. The present application provides a complex for co-delivery of an antioxidant and a gene to a subject. The complex includes a carrier capable of co-delivery of a gene with an antioxidant, an antioxidant, and plasmid DNA. The plasmid DNA may include DNA encoding an enzyme or protein involved in epigenetic control, or DNA encoding a molecule that suppresses the expression or activity of the enzyme or protein. The application also provides a pharmaceutical composition comprising the complex. The application further provides a complexing agent capable of co-delivery of an antioxidant and a gene.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research on a therapeutic strategy for a disease in which epigenetics and reactive oxygen species are pathologically related to chronic obstructive pulmonary disease (COPD) as a model. As a pathological mechanism of COPD, an epigenetic abnormality of histone deacetylase (HDAC2) dysfunction due to reactive oxygen species has been proposed. Based on this, the present inventors examined a strategy for simultaneously treating two types of targets, namely, suppression of acquired modification of HDAC2 by elimination of reactive oxygen species and recovery of the expression level of HDAC2 for the treatment of COPD. Therefore, a complex comprising an antioxidant for eliminating reactive oxygen species, a plasmid DNA encoding HDAC2 for restoring the expression level of HDAC2, and a carrier capable of co-delivery of the antioxidant and the plasmid is used. As a result, excellent anti-inflammatory effects and improved steroid refractory were observed in the COPD model. Based on this finding, the present invention has been completed.

That is, in one aspect, the present invention may be as follows.
[Aspect 1]
A complex for co-delivery of an antioxidant and a gene to a subject, the complex comprising a carrier capable of co-delivery of the antioxidant and the gene, an antioxidant, and plasmid DNA.
[Aspect 2]
The complex according to aspect 1, wherein the carrier capable of co-delivery of an antioxidant and a gene is a liposome, a biodegradable polymer or a lipid-coated biodegradable polymer.
[Aspect 3]
The liposome is a cationic liposome, and the cationic liposome is:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
At least one cationic lipid selected from the group consisting of:
Diacylphosphatidylcholine;
Lysophosphatidylcholine;
Diacylphosphatidylglycerol;
Diacylphosphatidic acid;
Diacylphosphatidylserine;
Diacylphosphatidylethanolamine;
Sphingomyelin;
Ceramide;
Diacylglycerol; and cholesterol;
At least one non-cationic lipid selected from the group consisting of (wherein the acyl group contained in the non-cationic lipid is a saturated or unsaturated acyl group having 12 to 20 carbon atoms), The complex according to aspect 2.
[Aspect 4]
The composite according to embodiment 2, wherein the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan.
[Aspect 5]
The lipid-coated biodegradable polymer is a biodegradable polymer coated with a cationic lipid, where the cationic lipid is:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
Selected from the group consisting of
And here, the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan.
The composite according to claim 2.
[Aspect 6]
The antioxidant is selected from the group consisting of porphyrin antioxidants, phthalocyanine antioxidants, ascorbic acid, glutathione, uric acid, melatonin, urobilinogen, tocopherols, tocotrienols, carotenoids, xanthophylls, polyphenols, flavonoids 6. The complex according to any one of aspects 1 to 5.
[Aspect 7]
The antioxidant is a porphyrin antioxidant, and the porphyrin antioxidant is
Cationic metalloporphyrin complex represented by the following formula (I):

(Where
M represents a metal atom for forming a complex;
Ar ₁ , Ar ₂ , Ar ₃ , and Ar ₄ each independently represent an unsubstituted or optionally substituted carbocyclic or heterocyclic aromatic group, and Ar ₁ , Ar ₂ , At least one of Ar ₃ and Ar ₄ is an aromatic group having a cationic group); and a cationic metalloporphyrin dimer represented by the following formula (II):

(Where
M represents a metal atom for forming a complex;
Ar ₁ and Ar ₁ ′ represent pyridine, wherein 1-position of pyridine is bonded to X and 4-position is bonded to porphyrin,
Ar ₂ , Ar ₃ , and Ar ₄ , and Ar ₂ ′, Ar ₃ ′, and Ar ₄ ′ are each independently an unsubstituted or optionally substituted carbocyclic or heterocyclic fragrance Indicates a group,
X is —CH ₂ -phenyl-CH ₂ — or represented by any of the following formulas (III) to (V):
_{-C 2 H 4 - (NH-} CH 2 CH 2) a - ···· (III)
(Wherein, a represents an integer of 1 to 10)
_{-C 2 H 4 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 - ··· (IV)
_{-C 3 H 6 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 CH 2 - ··· (V)
(In the formula, b represents an integer of 3 to 5))
The complex according to any one of aspects 1 to 5, which is selected from the group consisting of:
[Aspect 8]
Plasmid DNA is as follows:
DNA encoding an enzyme or protein involved in the control of epigenetics;
DNA encoding an antisense nucleic acid or double-stranded RNA that suppresses the expression of an enzyme or protein involved in the control of epigenetics;
DNA encoding an antibody or fragment thereof that binds to an enzyme or protein involved in the control of epigenetics;
The complex according to any one of aspects 1 to 7, which comprises DNA selected from the group consisting of:
[Aspect 9]
A pharmaceutical composition comprising the complex according to any one of aspects 1 to 8.
[Aspect 10]
A pharmaceutical composition for treating COPD, comprising the complex according to any one of aspects 1 to 8, wherein the plasmid DNA is a plasmid DNA encoding histone deacetylase 2 (HDAC2). The said pharmaceutical composition.
[Aspect 11]
A pharmaceutical composition for treating COPD, comprising a complex composed of a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA,
Here, the carrier capable of co-delivery of the antioxidant and the gene is a liposome composed of didodecyldimethylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE),
The antioxidant is represented by the following formula (VI):

A plasmid Mn porphyrin dimer, and the plasmid DNA is a plasmid DNA encoding HDAC2.
Said pharmaceutical composition.
[Aspect 12]
A pharmaceutical composition for treating COPD, comprising a complex composed of a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA,
Here, the carrier capable of co-delivery of the antioxidant and the gene is polylactic acid particles coated with 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP),
The antioxidant is represented by the following formula (VI):

A cationic Mn porphyrin dimer represented by: the antioxidant is present in the polylactic acid particle injection,
The plasmid DNA is a plasmid DNA encoding HDAC2, and the plasmid DNA is attached to the surface of polylactic acid particles coated with lipids.
Said pharmaceutical composition.
[Aspect 13]
A complexing agent for complexing an antioxidant and plasmid DNA, selected from the group consisting of cationic liposomes and biodegradable polymers;
Here cationic liposomes are:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
At least one cationic lipid selected from the group consisting of:
Diacylphosphatidylcholine;
Lysophosphatidylcholine;
Diacylphosphatidylglycerol;
Diacylphosphatidic acid;
Diacylphosphatidylserine;
Diacylphosphatidylethanolamine;
Sphingomyelin;
Ceramide;
Diacylglycerol; and cholesterol;
Composed of at least one non-cationic lipid selected from the group consisting of (wherein the acyl group contained in the non-cationic lipid is a saturated or unsaturated acyl group having 12 to 20 carbon atoms) And the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan,
The complexing agent.

  The complex of the present invention can simultaneously eliminate an active enzyme species by an antioxidant and improve an epigenetic abnormality by a plasmid DNA in a subject. Therefore, the complex of the present invention is useful for radical treatment based on epigenetics for diseases in which epigenetics and reactive oxygen species are pathologically related.

FIG. 1 is a schematic diagram showing two therapeutic targets by the complex of the present invention using COPD as a model. (A) is a schematic diagram showing the pathological mechanism of COPD. (B) is a schematic diagram showing two targets for COPD treatment with the complex of the present invention. FIG. 2 is a graph showing the anti-inflammatory effect and the steroid resistance-suppressing effect of the DNA complexed lipid-coated MnPD-containing PLA nanoparticles in the COPD model. * Indicates that there is a significant difference (p <0.05).

  The present invention will be specifically described below, but the present invention is not limited thereto. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

Complexes for co-delivery of an antioxidant and a gene to a subject In one embodiment, the application provides a complex for co-delivery of an antioxidant and a gene to a subject, wherein the co-delivery of the antioxidant and the gene The complex comprising a possible carrier, an antioxidant, and plasmid DNA.

  As a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA, those described in detail below can be used.

  The procedure for producing a plasmid composed of a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA is not particularly limited as long as a complex containing these components is formed. After forming a complex with any two components first, the remaining one component may be further added to form a complex. Alternatively, all three components may be mixed simultaneously to form a complex. Conditions for forming the complex can be appropriately selected by those skilled in the art.

  For example, when a carrier capable of co-delivery of an antioxidant and a gene is a liposome, a lipid containing the liposome and an antioxidant are mixed first to prepare a liposome containing the antioxidant, and then plasmid DNA May be combined.

  Alternatively, when the carrier capable of co-delivery of the antioxidant and the gene is polylactic acid, the antioxidant and the plasmid are mixed in advance, and then the polylactic acid is added thereto and stirred to form the complex. It may be formed.

  When the carrier capable of co-delivery of an antioxidant and a gene is a biodegradable polymer such as lipid-coated polylactic acid, the antioxidant and the biodegradable polymer are mixed to form particles, and the lipid After the coating, a complex may be prepared by further complexing plasmid DNA.

Carriers capable of co-delivery of genes with antioxidants / antioxidants and complexing agents for complexing plasmid DNA Anti-oxidants and carriers capable of co-delivery of genes are used for both antioxidants and genes. There is no particular limitation as long as it is a carrier that can be retained when administered to a subject and can deliver both an antioxidant and a gene into the cells of the subject. Carriers capable of co-delivery of an antioxidant and a gene include, for example, liposomes, biodegradable polymers or lipid-coated biodegradable polymers.

  The liposome used as a carrier capable of co-delivery of an antioxidant and a gene is preferably a cationic liposome. The cationic liposome may be composed of at least one cationic lipid. Alternatively, the cationic liposome may be composed of at least one cationic lipid and at least one non-cationic lipid.

The cationic lipid constituting the cationic liposome is not particularly limited, but the following:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
It may be selected from the group consisting of: A preferred cationic lipid is didodecyldimethylammonium bromide (DDAB).

The non-cationic lipid constituting the cationic liposome is not particularly limited, but the following:
Diacylphosphatidylcholine;
Lysophosphatidylcholine;
Diacylphosphatidylglycerol;
Diacylphosphatidic acid;
Diacylphosphatidylserine;
Diacylphosphatidylethanolamine;
Sphingomyelin;
Ceramide;
Diacylglycerol; and cholesterol;
The acyl group contained in the non-cationic lipid is a saturated or unsaturated acyl group having 12 to 20 carbon atoms. For example, the acyl group contained in the non-cationic lipid includes dodecanoyl group (lauroyl group), tetradecanoyl group (myristoyl group), pentadecanoyl group, hexadecanoyl group (palmitoyl group), 9-hexadecenoyl group (palmiyl). Trenoyl group), heptadecanoyl group (margaroyl group), octadecanoyl group (stearoyl group), 9-octadecenoyl group (oleoyl group), 11-octadecenoyl group (vaccenoyl group), 9,12-octadecadienoyl group ( Linoleoyl group), 9,12,15-octadecantrienoyl group (9,12,15-linolenoyl group), 6,9,12-octadecatrienoyl group (6,9,12-linolenoyl group), eicosanoyl group ( Arachidinoyl group), 8,11-eicosadienoyl group, 5,8,1 - eicosa triethanolamine alkanoyl group, 5,8,11 eicosatetraenoic enoyl group (arachidonoyl group), and the like. Preferred non-cationic lipids include dioleoylphosphatidylethanolamine (DOPE) and / or dimyristoyl phosphatidylcholine (DMPC).

  The biodegradable polymer used as a carrier capable of co-delivery of an antioxidant and a gene is not particularly limited, but polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, And a biodegradable polymer selected from the group consisting of chitosan.

  The lipid-coated biodegradable polymer used as a carrier capable of co-delivery of an antioxidant and a gene is not particularly limited, but is a biodegradable polymer coated with a cationic lipid. Here, as the cationic lipid and the biodegradable polymer, those described above for the liposome and the biodegradable polymer can be used.

  The carrier capable of co-delivery of the antioxidant and the gene is also understood as a complexing agent for complexing the antioxidant and the plasmid DNA.

Antioxidant Antioxidant contained in the complex of the present application is not particularly limited as long as it is a compound having antioxidant activity. For example, porphyrin antioxidant, phthalocyanine antioxidant, ascorbic acid, glutathione, uric acid, It may be an antioxidant selected from the group consisting of melatonin, urobilinogen, tocopherols, tocotrienols, carotenoids, xanthophylls, polyphenols, flavonoids.

  Examples of the porphyrin antioxidant include a cationic metal porphyrin complex or a cationic metal porphyrin dimer. In a preferred embodiment, the porphyrin antioxidant is a cationic metal porphyrin dimer.

  The cationic metalloporphyrin complex has the following formula (I):

The compound represented by these may be sufficient.

  The central metal M in the formula (I) is not particularly limited, but is preferably a transition metal. For example, the central metal M can be selected from the group consisting of manganese atoms, nickel atoms, iron atoms, copper atoms, cobalt atoms, and zinc atoms. A more preferred central metal is a manganese atom.

Ar ₁ , Ar ₂ , Ar ₃ , and Ar ₄ in the formula (I) are each independently an unsubstituted or optionally substituted carbocyclic or heterocyclic aromatic group. The aromatic group may be monocyclic, polycyclic, or condensed cyclic. Examples of the carbocyclic aromatic group include monocyclic, polycyclic, or condensed cyclic carbocyclic aromatic groups having 6 to 36 carbon atoms, preferably 6 to 20 carbon atoms. This includes, for example, groups derived from benzene rings, naphthalene rings and the like. Heterocyclic aromatic groups are derived from 5- to 10-membered monocyclic, polycyclic, or condensed heterocyclic rings having one or two abnormal nitrogen, oxygen or sulfur atoms. It is a group. Examples thereof include groups derived from pyridine ring, pyrimidine ring, azole ring and the like. Preferable aromatic groups include a phenyl group and a 4-pyridyl group.

At least one of Ar ₁ , Ar ₂ , Ar ₃ , and Ar _{4 in} the formula (I) is an aromatic group having a cationic group. The cationic group of the aromatic group is cationized with a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably about 1 to 5 carbon atoms. And quaternary ammonium groups are preferred. These cationic groups may be directly bonded to an aromatic group, but are linear or branched having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably about 1 to 5 carbon atoms. Or a cationic group may be substituted as a substituent of the aromatic group, but a hetero atom of the aromatic group, preferably a nitrogen atom is a carbon atom. It may be cationized by a linear or branched alkyl group having 1 to 20, preferably 1 to 10 carbon atoms, more preferably about 1 to 5 carbon atoms. Examples of such aromatic groups include 4-N, N, N-tri-lower alkylamino such as 4-N, N, N-trimethylaminophenyl group and 4-N, N, N-triethylaminophenyl group. Examples thereof include N-lower alkyl-4-pyridyl groups such as phenyl group, N-methyl-4-pyridyl group, and N-ethyl-4-pyridyl group, and N, N′-disubstituted imidazole group. In addition, the aromatic group having such a cationic group is a group bonded directly to a carbocyclic aromatic group such as a phenyl group or a naphthyl group or through an alkylene group, an amide group, or an ester group. It may be.

  These aromatic groups may have a substituent. As the substituent that the aromatic group may have, a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, an amino group, Examples thereof include an amino group substituted with the aforementioned alkyl group, an alkoxy group containing the aforementioned alkyl group, and the like.

At least one of Ar ₁ , Ar ₂ , Ar ₃ , and Ar _{4 in} the formula (I) is an aromatic group having a cationic group, but preferably two or more of these aromatic groups are It has a cationic group. For example, Ar ₁ , Ar ₂ , Ar ₃ , and Ar ₄ are all N-lower alkyl-4-pyridyl groups such as N-methyl-4-pyridyl group, 4-N, N, N-trimethylaminophenyl group. A compound that is a 4-N, N, N-tri-lower alkylaminophenyl group, an N, N′-disubstituted imidazole group, such as Ar ₁ , Ar ₂ , and Ar ₃ are N-methyl-4-pyridyl groups, etc. A 4-N, N, N-tri-lower alkylaminophenyl group such as an N-lower alkyl-4-pyridyl group, a 4-N, N, N-trimethylaminophenyl group, or an N, N′-disubstituted imidazole group. , Ar ₄ compounds wherein a phenyl group; a N- lower alkyl-4-pyridyl group, 4-N, N, N- trimethyl-aminophenyl group, such as Ar ₁ and Ar ₂ N- methyl-4-pyridyl group 4-N, N, N- tri (lower alkyl) aminophenyl group or N, a N'- disubstituted imidazole group, the compound Ar ₃ and Ar ₄ is a phenyl group,; Ar ₁ and Ar ₃ are N- methyl N-lower alkyl-4-pyridyl group such as -4-pyridyl group, 4-N, N, N-tri-lower alkylaminophenyl group such as 4-N, N, N-trimethylaminophenyl group, or N, N A compound having a '-disubstituted imidazole group and Ar ₂ and Ar ₄ being a phenyl group; and the like.

  The cationic metalloporphyrin dimer has the following formula (II):

The compound represented by these may be sufficient.

  The central metal M in the formula (II) is not particularly limited, but is preferably a transition metal. For example, the central metal M can be selected from the group consisting of manganese atoms, nickel atoms, iron atoms, copper atoms, cobalt atoms, and zinc atoms. A more preferred central metal is a manganese atom.

Ar ₁ and Ar ₁ ′ in the formula (II) represent pyridine, where the 1-position of pyridine is bonded to X and the 4-position is bonded to porphyrin.

In the formula (II), r ₂ , Ar ₃ , and Ar ₄ and Ar ₂ ′, Ar ₃ ′, and Ar ₄ ′ are each independently an unsubstituted or optionally substituted carbocycle. Is a formula or heterocyclic aromatic group. The aromatic group may be monocyclic, polycyclic, or condensed cyclic. Examples of the carbocyclic aromatic group include monocyclic, polycyclic, or condensed cyclic carbocyclic aromatic groups having 6 to 36 carbon atoms, preferably 6 to 20 carbon atoms. This includes, for example, groups derived from benzene rings, naphthalene rings and the like. Heterocyclic aromatic groups are derived from 5- to 10-membered monocyclic, polycyclic, or condensed heterocyclic rings having one or two abnormal nitrogen, oxygen or sulfur atoms. It is a group. Examples thereof include groups derived from pyridine ring, pyrimidine ring, azole ring and the like. Preferable aromatic groups include a phenyl group and a 4-pyridyl group.

  These aromatic groups may have a substituent. As the substituent that the aromatic group may have, a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, an amino group, Examples thereof include an amino group substituted with the aforementioned alkyl group, an alkoxy group containing the aforementioned alkyl group, and the like.

In the formula (II), X is —CH ₂ -phenyl-CH ₂ — or represented by any one of the following formulas (III) to (V):
_{-C 2 H 4 - (NH-} CH 2 CH 2) a - ···· (III)
(Wherein, a represents an integer of 1 to 10)
_{-C 2 H 4 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 - ··· (IV)
_{-C 3 H 6 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 CH 2 - ··· (V)
(Wherein b represents an integer of 3 to 5)
Preferably X is —CH ₂ -phenyl-CH ₂ —.

  A preferred porphyrin antioxidant is represented by the following formula (VI):

It is a cationic manganese porphyrin dimer having

Plasmid DNA
The plasmid DNA contained in the complex of the present application is not particularly limited, but is capable of modulating the expression level or activity of an enzyme or protein in a subject to which the complex of the present application is applied. Examples of such plasmid DNA include DNA encoding an enzyme or protein; DNA encoding an antisense nucleic acid or double-stranded RNA that suppresses expression of the enzyme or protein in a subject; or an antibody that binds to the enzyme or protein or Plasmid DNA containing the DNA encoding the fragment.

  Plasmid DNA containing DNA encoding an enzyme or protein expresses the enzyme or protein in a subject to which the complex of the present application is applied. Thereby, the expression level of the enzyme or protein can be increased.

  Plasmid DNA containing an antisense nucleic acid or DNA encoding a double-stranded RNA that suppresses the expression of an enzyme or protein in the subject expresses the antisense nucleic acid or double-stranded RNA in the subject to which the complex of the present application is applied. Thereby, the expression of the enzyme or protein can be suppressed based on the action of the antisense nucleic acid or the action of RNA interference.

DNA encoding an antibody or fragment thereof that binds to an enzyme or protein expresses an antibody or fragment thereof that binds to the enzyme or protein in a subject to which the complex of the present application has been applied. Thereby, the activity of the enzyme or protein is suppressed or enhanced. Antibodies or fragments thereof that bind to specific enzymes or proteins can be produced by methods well known to those skilled in the art, and DNAs that encode them can be appropriately identified by those skilled in the art. Antibody fragments include, but are not limited to, Fab, F (ab ′) ₂ , Fab ′, Fv, scFv.

  In a preferred embodiment, the plasmid DNA contained in the complex of the present application is capable of modulating the expression level or activity of an enzyme or protein involved in the control of epigenetics. Such plasmid DNA is, for example, DNA encoding an enzyme or protein involved in epigenetic control; DNA encoding an antisense nucleic acid or double-stranded RNA that suppresses expression of the enzyme or protein in a subject; or DNA encoding an antibody or fragment thereof that binds to the enzyme or protein. Enzymes or proteins involved in epigenetic control include, for example, DNA methylases, methylated DNA binding proteins, histone modifying enzymes, chromatin structure factors, chromatin remodeling factors, chromatin insulators, and the like. In a preferred embodiment, the enzyme or protein involved in epigenetic regulation is histone deacetylase 2 (HDAC2).

  In a particularly preferred embodiment, the plasmid DNA contained in the complex of the present application is a plasmid DNA encoding HDAC2.

In another aspect, the present application relates to a pharmaceutical composition comprising a complex for co-delivery of an antioxidant and a gene to a subject.

  The pharmaceutical composition of the present invention can be administered by oral administration or parenteral administration such as intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, or rectal administration. Formulations suitable for oral administration include tablets, capsules, powders, granules, solutions, syrups and the like. As a formulation suitable for parenteral administration, the pharmaceutical composition of the present invention may be prepared as a sterile solution or suppository. The pharmaceutical composition of the present invention can be formulated by a known method, and can be appropriately formulated depending on the administration method and patient. For example, the pharmaceutical composition of the present invention contains, as an active ingredient, a complex for co-delivering the antioxidant of the present invention and a gene to a subject, and a pharmaceutically acceptable carrier such as an excipient, It can be formulated by a known method together with a binder, a disintegrant, a lubricant, a flavoring agent, a coloring agent, a solubilizing agent, a suspending agent, a coating agent and the like.

  The effective dose of the pharmaceutical composition of the present invention varies depending on the disease state and patient, but in general, it is administered so that the antioxidant is 1 μg to 1 g and the plasmid DNA is 0.6 mg to 600 mg per day. be able to. The pharmaceutical composition of the present invention is administered once, divided into several times a day. Alternatively, it is administered continuously. Doctors and other medical personnel appropriately determine the dosage or administration interval, administration time, administration procedure, administration site, etc. of the pharmaceutical composition of the present invention according to the patient's attributes and / or pathological conditions. Can be determined. Also within the scope of the invention are pharmaceutical compositions of the invention that are administered in such a manner or dosage determined accordingly.

  Diseases suitable for prevention and treatment with a pharmaceutical composition comprising a complex for co-delivery of an antioxidant of the present invention and a gene to a subject as an active ingredient include diseases caused by abnormal epigenetics. Examples of such diseases include, but are not limited to, inflammatory diseases such as inflammatory lung disease (COPD), colorectal cancer, Alzheimer type dementia and the like.

  Specific examples of the present invention are shown below. These examples are intended to provide an illustration for understanding the invention and are not intended to limit the scope of the invention.

Example 1 Production of Dimer-type Mn Porphyrin Complex (MnPD) (1) Synthesis of H ₂ 4PyP ₃ P (5- (4-pyridyl) -10,15,20-triphenyl-21H, 23H-porphyrin) To the three-necked flask, 500 ml of propionic acid was added, and the mixture was stirred and refluxed at 173 ° C. for 30 minutes. After the temperature was kept constant, 14 ml of benzaldehyde and 6 ml of 4-pyridinecarboxaldehyde were added, followed by 12.5 ml of purified pyrrole.

  After heating and stirring for 1.5 hours, the mixture was allowed to cool to 90 ° C., and 350 ml of ethylene glycol was added dropwise. After cooling to room temperature, it was left overnight in a refrigerator.

  The solution after cooling was filtered with suction, washed with a cooled ethanol / methanol = 1: 1 solution, and then a mixture of six kinds of purple powder of porphyrin was recovered.

The obtained porphyrin mixture was eluted with 100% CHCl ₃ by silica gel chromatography to elute TPP (tetraphenylporphyrin). Next, H ₂ 4PyP ₃ P, which is the target compound, was eluted. The solvent of the fraction was distilled off to obtain a purple target porphyrin. The synthesis was confirmed by UV spectrum measurement and ¹ H-NMR. The results are shown below.

Maximum absorption wavelength of H ₂ 4PyP ₃ P : λmax 421 nm (soret band)
¹ H-NMR of H ₂ 4PyP ₃ P : 9.44 (2H) (hydrogen atoms at positions 2 and 6 of the pyridine ring), 9.02 (2H) (hydrogen atoms at positions 3 and 5 of the pyridine ring), 8 .98 (2H) (hydrogen atom at β position of pyrrole), 8.95 (2H) (hydrogen atom at β position of pyrrole), 8.87 (4H) (hydrogen atom at β position of pyrrole), 8.24 (6H) (hydrogen atom of phenyl group), 7.91-7.84 (9H) (hydrogen atom of phenyl group), 4.70 (3H) (hydrogen atom of methyl group), -2.91 (2H) (Hydrogen atom in the ring)
Structural formula of H ₂ 4PyP ₃ P:

(2) dimeric porphyrin complex _(H 2 PD: 1,3-di [5-(N-methylene - pyridinium-4-yl) -10,15,20 triphenyl-21H, 23H-porphyrin] benzene dibromide ) Synthesis 100 ml of porphyrin H ₂ 4PyP ₃ P obtained in (1) above and 19 mg of metaxylene dibromide were added to a 100 ml three-necked flask, 5 ml of dry DMF was added, and the mixture was heated to reflux at 90 ° C. under nitrogen flow for 24 hours. did.

The solvent of the reaction mixture was distilled off by a rotary evaporator. Methanol was added and insoluble H ₂ 4PyP ₃ P was removed by filtration, followed by evaporation and evaporation of the solvent.

The obtained porphyrin mixture was eluted with a mixed solvent of chloroform / methanol / DMF = 4: 1: 0.02 by silica gel chromatography to elute H ₂ 4PyP ₃ P and a mono-substituted product. Next, H ₂ PD, which was the target compound eluted third, was eluted. The solvent of the fraction was distilled off to obtain the desired porphyrin. The synthesis was confirmed by UV spectrum measurement and ¹ H-NMR. The results are shown below.

Maximum absorption wavelength of H ₂ PD: λmax 415 nm (soret band)
¹ H-NMR of H ₂ PD: 9.72 (4H) (hydrogen atom at positions 2 and 6 of the pyridine ring) 9.13 (4H) (hydrogen atom at positions 3 and 5 of the pyridine ring) 8.94 (4H) (Β-position hydrogen atom of pyrrole) 8.79 (4H) (hydrogen atom of β-position of pyrrole) 8.54 (8H) (hydrogen atom of β-position of pyrrole) 8.33 (1H) (position of 2-position of xylyl) (Hydrogen atom) 8.22 (4H) (hydrogen atom at positions 2 and 6 of the phenyl group) 8.09 (2H) (hydrogen atom at positions 4 and 6 of xylyl) 7.91-7.85 (7H) (xylyl) And hydrogen atoms at positions 3, 4, and 5 of the phenyl group) 7.62-7.56 (12H) (hydrogen atoms at positions 2, 4, and 6 of the phenyl group) 7.37-7.35 (8H ) (Hydrogen atom of phenyl group 3, 5 position) 6.40 (4H) (hydrogen atom of benzyl) -2.96 (4H) (water inside ring Atom)
Structural formula of H ₂ PD:

₍₃₎ the three-necked flask Mn introduction 100ml into _H 2 PD added resulting _H 2 PD (2), was dissolved in methanol, 10 equivalents of manganese acetate tetrahydrate was added The mixture was heated to reflux at 50 ° C., and the reaction was carried out using a UV-visible spectrum until the introduction of the metal was confirmed from the shift of the porphyrin Sore band and the change in absorbance.

  After completion of the reaction, the solvent was distilled off by evaporation. The obtained solid was redissolved in water, an appropriate amount of ammonium hexafluorophosphate was added, and suction filtration was performed to collect a green solid on the filter paper. The synthesis was confirmed by UV spectrum measurement and elemental analysis. The results are shown below.

Maximum absorption wavelength of MnPD: 467 nm (soret band)
Elemental analysis of MnPD: Theoretical value (%) C: 65.48, H: 4.42, N: 7.31 Measured value (%) C: 65.43, H: 4.59, N: 7.32
Structural formula of MnPD:

Example 2: Preparation of DNA-complexed MnPD-containing liposome (1) Preparation of MnPD-containing liposome In a 20 ml eggplant flask, 0.12 mg of MnPD, 0.0124 g of dioleoylphosphatidylethanolamine (DOPE), didodecyldimethylammonium bromide (DDAB) 3 4 mg was added and dissolved in 5 ml of a mixed solvent of chloroform / methanol = 1: 1. By using a rotary evaporator, the solvent was distilled off to produce a dry thin film.

PBS (-) 5 ml was added to the dried thin film, and ultrasonic stirring was performed in ice water for 40 minutes to obtain a MnPD liposome dispersion. The particle diameter was 71.9 ± 17.6 nm, and the zeta potential was +25.8 mV.
(2) Liposome complexed with DNA In the Eppendorf tube, the liposome solution of (1) and the plasmid DNA (pDNA) solution are mixed at an arbitrary molar ratio and allowed to stand at room temperature for 1 hour, which is a constituent component of the liposome. The cationic lipid DDAB and the anionic pDNA were combined so that the charge ratio (+/−) was 1. The particle diameter was 558.8 ± 136.1 nm, and the zeta potential was +4.51 mV.

Example 3 Preparation of MnPD / pDNA Complex-Containing PLA Particles 50 μL of 0.4 mM MnPD and 75 μL of 0.0856 mg / mL pGL3 solution were added to an Eppendorf tube and allowed to stand at room temperature for 1 hour to be complexed. Here, pGL3 is a plasmid DNA containing DNA encoding luciferase. The complex solution was transferred to a 10 mL vial, and 150 μL of DMSO (dimethyl sulfoxide) was added and dissolved. The mixture was stirred at a speed of 400 rpm for 1 hour, 0.0025 g of polylactic acid (PLA; weight average molecular weight 10,000) was added, and the mixture was stirred again under the same conditions. After 1 hour, 2 mL of distilled water was added, transferred to a dialysis membrane with a molecular weight cut off of 100-500, and stirred for 2 days. Thereafter, the solution in the dialysis membrane was transferred to a 50 mL centrifuge tube, centrifuged at 1000 rpm for 15 minutes, and collected.

  The particle size was 365.8 ± 433.9 nm.

Example 4: Evaluation of cell accumulation ability of MnPD-containing liposomes and DNA-complexed MnPD-containing liposomes Incorporation of MnPD-containing liposomes and DNA-complexed MnPD-containing liposomes into the cell membrane was evaluated using A549 cells (human alveolar basal epithelial adenocarcinoma cells). ).

A549 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics under conditions of 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours. MnPD, MnPD-containing liposomes, and DNA-complexed MnPD liposomes were added so as to have various concentrations of 0 to 100 μM. After culturing for 4 hours, the medium was removed, and porphyrin adhering to the cell membrane was removed by washing with PBS. After washing, the cells were lysed with 100 μl / well cell lysis and the cell lysate was collected in a sample tube. Finally, nitric acid (0.5N HNO ₃ ) was added to the collected cell lysate, the cells were destroyed and allowed to stand. The supernatant of the cell-containing nitric acid solution was used as a sample, and the central metal of the metal porphyrin incorporated into the cells was quantified by atomic absorption spectrometry.

  When the addition concentration of the DNA-complexed MnPD-containing liposome was 20 μM, about 5% of the addition amount was taken up into the cells.

Example 5: In vitro antioxidant activity evaluation of DNA-conjugated MnPD-containing liposomes A549 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics under conditions of 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours. MnPD and DNA-complexed MnPD liposomes were added so as to have various concentrations of 0 to 100 μM. After culturing for 4 hours, the medium was removed, and porphyrin adhering to the cell membrane was removed by washing with PBS. After washing, the cells were lysed with 100 μl / well cell lysis and the Mn porphyrin complex cell lysate was collected in a sample tube. The sample was allowed to stand in hot water for 5 minutes to inactivate endogenous catalase. Add 100 μl of 10 mM hydrogen peroxide solution, 100 μl of recovered Mn porphyrin complex-containing cell lysate and 800 μl of 50 mM phosphate buffer (pH 7.4) into the cell of the Clark-type dissolved oxygen electrode, and hydrogen peroxide is decomposed by the porphyrin complex. The oxygen generated by the decomposition of hydrogen peroxide was electrochemically detected and traced.

  Oxygen production was also observed when DNA-complexed MnPD liposomes were added, indicating that DNA-complexed MnPD liposomes exhibited antioxidant activity even in the intracellular environment.

Example 6: In vitro gene expression evaluation of DNA-conjugated MnPD-containing liposomes A549 cells were cultured in 10% FBS, 1% antibiotic-containing DMEM medium under conditions of 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 96-well plate at a cell density of 1 × 10 ⁴ cells / well, and cultured in an incubator for 24 hours.

  Using pGL3 encoding luciferase, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio in PBS (−) so that the pDNA concentration was 200 ng / well, and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, the medium was removed, and the complex adhering to the cell membrane was removed by washing with PBS. After washing, the cells were lysed with 20 μl / well cell lysis, 100 μl of luciferin solution was added, and the well-mixed one was collected in an Eppendorf tube, and the chemiluminescence amount of luciferin was measured with a luminometer.

  The gene expression efficiency was about 1000 times that of Naked DNA, indicating that the prepared complex had sufficient gene expression ability.

Example 7: Evaluation of HDAC2 expression level by Western blotting A549 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics under conditions of 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours.

  Using pDNA encoding HDAC2, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio in PBS (−) so that the pDNA concentration was 1000 ng / well, and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, the medium was removed, and the complex adhering to the cell membrane was removed by washing with PBS. After washing, cells were lysed with 100 μl / well cell lysis, 90 μl of which was collected in an Eppendorf tube, and the remaining 10 μl was collected for protein quantification. From the protein quantification results, the dilution factor was determined and diluted with sample buffer. In order to cleave the disulfide bond of the protein, heat treatment was performed at 95 ° C. for 5 minutes. The prepared sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) (concentrated gel: 30% acrylamide, Tris pH 6.8, 10% SDS, 10% APS, TEMED, DDW; separation gel: 30% acrylamide, Tris pH 8.8. 10% SDS, 10% APS, TEMED, DDW), and the protein was transferred from the gel to the PVDF membrane using a tank type blotting apparatus. The primary antibody treatment (anti-HDAC2 mouse antibody and anti-β-actin rabbit antibody): wash buffer = 1: 1000 solution is performed overnight on the protein-transferred membrane, followed by secondary antibody (HRP-conjugated anti-antibody). Mouse antibody, HRP-conjugated anti-rabbit antibody): The secondary antibody treatment was performed with a wash buffer = 1: 1000 solution. The expression level of HDAC2 was semi-quantitatively evaluated by detecting luminescence using Prime Western Blotting Detection System.

  The expression level of HDAC2 increased about 3-fold compared to untreated cells, and it was shown that a sufficient amount of HDAC2 was expressed by using the prepared complex.

Example 8 Evaluation of HDAC2 Activity of Complex by ELISA Culture of A549 cells was carried out in 10% FBS, 1% antibiotic-containing DMEM medium under conditions of 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours.

  Using pDNA encoding HDAC2, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio in PBS (−) so that the pDNA concentration was 1000 ng / well, and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, the medium was removed, and the complex adhering to the cell membrane was removed by washing with PBS. After washing, the cells were lysed with 100 μl / well cell lysis and collected in an Eppendorf tube.

  The collected samples were evaluated for HDAC2 activity by detecting changes in fluorescence intensity using ELISA. When the HDAC2 activity of untreated cells was taken as 100%, it was shown that the HDAC2 activity increased to 103% when the prepared complex was added.

Example 9: Examination of HDAC2 protective effect of DNA-conjugated MnPD-containing liposome against reduction of HDAC2 expression level by hydrogen peroxide A549 cells were cultured in 10% FBS, 1% antibiotic-containing DMEM medium, 5% CO ₂ , 37 ° C. It went on condition of. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours.

  Using pDNA encoding HDAC2, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio in PBS (−) so that the pDNA concentration was 1000 ng / well, and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, hydrogen peroxide was added to a concentration of 100 μM / well, followed by culturing for 4 hours. After culturing for 4 hours, the medium was removed and the cells were lysed with 100 μl / well cell lysis, 90 μl of which was collected in an Eppendorf tube, and the remaining 10 μl was collected for protein quantification. From the protein quantification results, the dilution factor was determined and diluted with sample buffer. In order to cleave the disulfide bond of the protein, heat treatment was performed at 95 ° C. for 5 minutes. The prepared sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) (concentrated gel: 30% acrylamide, Tris pH 6.8, 10% SDS, 10% APS, TEMED, DDW; separation gel: 30% acrylamide, Tris pH 8.8. 10% SDS, 10% APS, TEMED, DDW), and the protein was transferred from the gel to the PVDF membrane using a tank type blotting apparatus. The primary antibody treatment (anti-HDAC2 mouse antibody and anti-β-actin rabbit antibody): wash buffer = 1: 1000 solution is performed overnight on the protein-transferred membrane, followed by secondary antibody (HRP-conjugated anti-antibody). Mouse antibody and HRP-conjugated anti-rabbit antibody): Wash buffer = 1: 1000 was treated with secondary antibody. HDAC2 expression level was quantified semi-quantitatively by detecting luminescence using Prime Western Blotting Detection System. The HDAC2 protective effect of the complex was evaluated by quantifying HDAC2 whose expression level was reduced by hydrogen peroxide and examining whether each complex could suppress the decrease in HDAC2 expression level.

  When the HDAC2 expression level of untreated cells was 100%, the HDAC2 expression level was reduced to about 60% by hydrogen peroxide treatment. On the other hand, when the prepared complex was added, the expression level increased to about 200%.

Example 10: Examination of HDAC2 activity improvement effect of liposomes containing DNA-complexed MnPD against reduction of HDAC2 expression level by hydrogen peroxide A549 cells were cultured in 10% FBS, 1% antibiotic-containing DMEM medium, 5% CO ₂ , 37 It carried out on the conditions of ℃. Confluent cells were counted, seeded on a 12-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours.

  Using pDNA encoding HDAC2, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio in PBS (−) so that the pDNA concentration was 1000 ng / well, and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, hydrogen peroxide was added to a concentration of 100 μM / well, followed by culturing for 4 hours. After culturing for 4 hours, the medium was removed, the cells were lysed with 100 μl / well cell lysis, and collected in an Eppendorf tube. An ELISA was used for the collected samples, and the change in chemiluminescence amount was detected to examine the effect of improving the HDAC2 activity of each complex.

  When HDAC2 activity of untreated cells was taken as 100%, HDAC2 activity was reduced to 50% by hydrogen peroxide treatment. In contrast, the addition of the prepared complex restored HDAC2 activity to 60%.

Example 11: Examination of steroid resistance inhibitory effect of DNA-conjugated MnPD-containing liposomes in COPD model by hydrogen peroxide and LPS A549 cells were cultured in 10% FBS, 1% antibiotic-containing DMEM medium, 5% CO ₂ , 37 It carried out on the conditions of ℃. Confluent cells were counted, seeded on a 48-well plate at a cell density of 1 × 10 ⁵ cells / well, and cultured in an incubator for 24 hours.

  Using pDNA encoding HDAC2, pDNA was mixed with MnPD-containing liposomes at an arbitrary charge ratio so that the pDNA concentration would be 200 ng / well in PBS (−), and allowed to stand at room temperature for 1 hour. Formed.

A PBS (−) solution of each complex was added to the culture medium of the above A549 cells, and the medium was changed after culturing in an incubator under conditions of 5% CO ₂ and 37 ° C. for 4 hours. Thereafter, after culturing for 48 hours under the same conditions, hydrogen peroxide was added to a concentration of 100 μM / well, followed by culturing for 4 hours. After culturing for 4 hours, the medium was changed, LPS was added to a concentration of 0.2 ng / well, dexamethasone was added to a concentration of 100 μM / well, and the cells were cultured for 16 hours. Thereafter, the medium was removed, the cells were lysed with 50 μl / well cell lysis, and the cell lysate was collected in an Eppendorf tube. In the COPD model by co-treatment with hydrogen peroxide and LPS, administration of steroid dexamethasone cannot suppress IL-8 production (steroid resistance). Quantifying IL-8 production in a COPD model, and evaluating whether the administration of the conjugate normalizes the action of steroids and decreases the production of IL-8 did. Moreover, evaluation was performed by ELISA and the change of IL-8 production amount was computed from the change of fluorescence intensity.

  In untreated cells, IL-8 production was about 400 pg / mL, and treatment with hydrogen peroxide and LPS increased IL-8 production to about 1200 pg / mL. In contrast, when dexamethasone was added, IL-8 production could not be suppressed (steroid resistance). However, when DNA-conjugated MnPD-containing liposomes were co-administered, IL-8 production decreased to about 500 pg / mL, indicating that the prepared complex succeeded in suppressing steroid resistance in the COPD model. It was.

Example 12: Preparation of DNA complexed lipid-coated MnPD-containing PLA nanoparticles Manganese porphyrin dimer (MnPD) -containing polylactic acid (PLA) nanoparticles were prepared by the following procedure. First, 1 mg of MnPD and 100 mg of PLA were dissolved in a methanol / acetone mixed solvent. The resulting solution was added to 30 mL of polyvinyl alcohol solution and stirred. After stirring, the particles were settled by centrifugation. Next, the supernatant was removed, 30 mL of distilled water was added, and centrifugation was performed. This operation was repeated twice. Finally, the supernatant was removed, 10 mL of distilled water was added, and the particles were resuspended. The obtained suspension was freeze-dried to obtain MnPD-containing PLA nanoparticles. The particle diameter was 265 ± 78 nm, and the zeta potential was −6.7 mV.

The resulting particles were lipid coated by the following procedure. First, 2 mg cationic lipid (1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP)) was dissolved in chloroform, and the solvent was evaporated using a rotary evaporator to obtain a lipid thin film. To the obtained lipid thin film, 4 mg of MnPD-containing PLA particles suspended in PBS was added and subjected to ultrasonic treatment. After sonication, MnPD-containing PLA nanoparticles that were allowed to stand and were lipid-coated were obtained.

  Finally, 690 ng of lipid-coated MnPD-containing PLA nanoparticles were added with 200 ng of plasmid DNA encoding HDAC2 in PBS and allowed to stand at room temperature for 1 hour to obtain DNA-complexed lipid-coated PLA nanoparticles containing MnPD. . The particle diameter was 414 ± 17 nm, and the zeta potential was +21 mV.

Example 13: Examination of anti-inflammatory effect and steroid resistance inhibitory effect of DNA complexed lipid-coated MnPD-containing PLA nanoparticles in COPD model with hydrogen peroxide and LPS A549 cells were cultured in 10% FBS, 1% antibiotic-containing DMEM medium At 5% CO ₂ and 37 ° C. Confluent cells were counted, seeded on a 96-well plate at a cell density of 5 × 10 ⁴ cells / well, and cultured in an incubator for 24 hours.

A PBS (−) solution of the DNA complexed lipid-coated MnPD-containing PLA nanoparticles prepared in Example 12 was added to the above-described culture medium of A549 cells. After culturing for 48 hours under conditions of 5% CO ₂ and 37 ° C. in an incubator, hydrogen peroxide was added to 100 μM / well and incubated for 4 hours. Thereafter, LPS was added to a concentration of 0.2 ng / well, dexamethasone (Dex.) Was added to a concentration of 100 μM / well, and the mixture was incubated for 16 hours. Thereafter, the medium was removed, and the collected cells were lysed with 50 μl / well of cell lysis, and the expression level of inflammatory cytokine (IL-8) was evaluated by ELISA.

  The results are shown in FIG. Cells treated with hydrogen peroxide and LPS increased IL-8 production. In cells to which DNA complexed lipid-coated MnPD-containing PLA nanoparticles were added, IL-8 production was significantly suppressed compared to cells treated with hydrogen peroxide and LPS (p <0.05). Furthermore, the addition of carrier and dexamethasone led to a further decrease in the amount of IL-8 compared to the carrier alone (p <0.05).

  The complex of the present invention can simultaneously achieve elimination of reactive oxygen species by an antioxidant and improvement of epigenetic abnormality by plasmid DNA by co-delivery of an antioxidant and plasmid DNA. Therefore, epigenetic abnormalities and reactive oxygen species, which conventionally had only symptomatic treatment, are expected to be applied to the radical treatment of diseases related to pathology.

Claims (13)

  A complex for co-delivery of an antioxidant and a gene to a subject, the complex comprising a carrier capable of co-delivery of the antioxidant and the gene, an antioxidant, and plasmid DNA.   The complex according to claim 1, wherein the carrier capable of co-delivery of an antioxidant and a gene is a liposome, a biodegradable polymer or a lipid-coated biodegradable polymer. The liposome is a cationic liposome, and the cationic liposome is:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
At least one cationic lipid selected from the group consisting of:
Diacylphosphatidylcholine;
Lysophosphatidylcholine;
Diacylphosphatidylglycerol;
Diacylphosphatidic acid;
Diacylphosphatidylserine;
Diacylphosphatidylethanolamine;
Sphingomyelin;
Ceramide;
Diacylglycerol; and cholesterol;
At least one non-cationic lipid selected from the group consisting of (wherein the acyl group contained in the non-cationic lipid is a saturated or unsaturated acyl group having 12 to 20 carbon atoms), The composite according to claim 2.   The complex according to claim 2, wherein the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan. The lipid-coated biodegradable polymer is a biodegradable polymer coated with a cationic lipid, where the cationic lipid is:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
Selected from the group consisting of
And here, the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan.
The composite according to claim 2.   The antioxidant is selected from the group consisting of porphyrin antioxidants, phthalocyanine antioxidants, ascorbic acid, glutathione, uric acid, melatonin, urobilinogen, tocopherols, tocotrienols, carotenoids, xanthophylls, polyphenols, flavonoids The complex according to any one of claims 1 to 5. The antioxidant is a porphyrin antioxidant, and the porphyrin antioxidant is
Cationic metalloporphyrin complex represented by the following formula (I):
(Where
M represents a metal atom for forming a complex;
Ar ₁ , Ar ₂ , Ar ₃ , and Ar ₄ each independently represent an unsubstituted or optionally substituted carbocyclic or heterocyclic aromatic group, and Ar ₁ , Ar ₂ , At least one of Ar ₃ and Ar ₄ is an aromatic group having a cationic group); and a cationic metalloporphyrin dimer represented by the following formula (II):
(Where
M represents a metal atom for forming a complex;
Ar ₁ and Ar ₁ ′ represent pyridine, wherein 1-position of pyridine is bonded to X and 4-position is bonded to porphyrin,
Ar ₂ , Ar ₃ , and Ar ₄ , and Ar ₂ ′, Ar ₃ ′, and Ar ₄ ′ are each independently an unsubstituted or optionally substituted carbocyclic or heterocyclic fragrance Indicates a group,
X is —CH ₂ -phenyl-CH ₂ — or represented by any of the following formulas (III) to (V):
_{-C 2 H 4 - (NH-} CH 2 CH 2) a - ···· (III)
(Wherein, a represents an integer of 1 to 10)
_{-C 2 H 4 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 - ··· (IV)
_{-C 3 H 6 - (NH-} CH 2 CH 2 CH 2) b-NH-CH 2 CH 2 CH 2 - ··· (V)
(In the formula, b represents an integer of 3 to 5))
The complex according to any one of claims 1 to 5, which is selected from the group consisting of: Plasmid DNA is as follows:
DNA encoding an enzyme or protein involved in the control of epigenetics;
DNA encoding an antisense nucleic acid or double-stranded RNA that suppresses the expression of an enzyme or protein involved in the control of epigenetics;
DNA encoding an antibody or fragment thereof that binds to an enzyme or protein involved in the control of epigenetics;
The complex according to any one of claims 1 to 7, comprising DNA selected from the group consisting of:   A pharmaceutical composition comprising the complex according to any one of claims 1 to 8.   A pharmaceutical composition for treating COPD, comprising the complex according to any one of claims 1 to 8, wherein the plasmid DNA is a plasmid DNA encoding histone deacetylase 2 (HDAC2) Said pharmaceutical composition. A pharmaceutical composition for treating COPD, comprising a complex composed of a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA,
Here, the carrier capable of co-delivery of the antioxidant and the gene is a liposome composed of didodecyldimethylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE),
The antioxidant is represented by the following formula (VI):
A plasmid Mn porphyrin dimer, and the plasmid DNA is a plasmid DNA encoding HDAC2.
Said pharmaceutical composition. A pharmaceutical composition for treating COPD, comprising a complex composed of a carrier capable of co-delivery of an antioxidant and a gene, an antioxidant, and plasmid DNA,
Here, the carrier capable of co-delivery of the antioxidant and the gene is polylactic acid particles coated with 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP),
The antioxidant is represented by the following formula (VI):
A cationic Mn porphyrin dimer represented by: the antioxidant is present in the polylactic acid particle injection,
The plasmid DNA is a plasmid DNA encoding HDAC2, and the plasmid DNA is attached to the surface of polylactic acid particles coated with lipids.
Said pharmaceutical composition. A complexing agent for complexing an antioxidant and plasmid DNA, selected from the group consisting of cationic liposomes and biodegradable polymers;
Here cationic liposomes are:
Didodecyldimethylammonium bromide (DDAB);
N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA);
1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP);
1,2-distearoyl-3-trimethylammoniumpropane (DSTAP);
Dioleoyl-3-dimethylammoniumpropane (DODAP);
Dioctadecyl-dimethyl-ammonium chloride (DODAC);
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE);
2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanium trifluoroacetate (DOSPA);
3β-N- (N ′, N′-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride;
At least one cationic lipid selected from the group consisting of:
Diacylphosphatidylcholine;
Lysophosphatidylcholine;
Diacylphosphatidylglycerol;
Diacylphosphatidic acid;
Diacylphosphatidylserine;
Diacylphosphatidylethanolamine;
Sphingomyelin;
Ceramide;
Diacylglycerol; and cholesterol;
Composed of at least one non-cationic lipid selected from the group consisting of (wherein the acyl group contained in the non-cationic lipid is a saturated or unsaturated acyl group having 12 to 20 carbon atoms) And the biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactide-co-glycolide) copolymer, polycaprolactone, polydioxanone, and chitosan,
The complexing agent.