JP4305857B2 - Pharmaceutical compositions containing decoys and methods of use thereof - Google Patents

Description

  The present invention relates to a composition comprising a compound (eg, a nucleic acid and analogs thereof) that specifically binds to a site on a chromosome to which a transcriptional regulatory factor binds, and a method of using the same. More particularly, the present invention relates to compositions comprising decoy compounds and methods of use thereof.

  Although various diseases such as asthma, cancer, heart disease, aneurysm, autoimmune disease and viral infection each show different symptoms, most of them have abnormal expression of one or several proteins ( It is suggested that it is caused by overexpression or underexpression). In general, the expression of these proteins is controlled by transcriptional regulatory factors such as various transcriptional activators and transcriptional repressor genes.

  NF-κB is a transcriptional regulator composed of p65 and p50 heterodimers. NF-κB is usually present in the cytoplasm in a form bound to its inhibitor IκB, and its nuclear translocation is prevented. However, when a stimulus such as cytokine, ischemia or reperfusion is applied for some reason, IκB is phosphorylated and decomposed, whereby NF-κB is activated and moves into the nucleus. The NF-κB transferred into the nucleus binds to the NF-κB binding site on the chromosome and promotes transcription of a gene downstream thereof. Examples of genes downstream of the NF-κB binding site include IL-1, IL-6, IL-8, inflammatory cytokines such as tumor necrosis factor α (TNFα), adhesion of VCAM-1, ICAM-1, etc. Factors are known.

NF-κB may be involved in the onset of progression of tumor malignancy (Rayet B et al., Oncogene 1999 Nov 22; 18 (49) 6938-47); NF-κB is responsible for tumor cell responses to hypoxic stress. Involved (Royds
JA et al., Mol Pathol 1998 Apr; 51 (2): 55-61); NF-κB decoy inhibits the expression of cytokines and adhesion molecules in synovial cells from patients with rheumatoid arthritis (Tomita T et al., Rheumatology ( (Oxford) 2000 Jul; 39 (7): 749-57); inhibition of synergy between multiple transcription factors including NF-κB alters the malignant phenotype of various cancers (Denhardt DT, Crit Rev Oncog 1996; 7 (3-4): 261-91); down-regulation of NF-κB activity by green tea polyphenols blocks the induction of nitric oxide synthase and suppresses A431 human epidermoid carcinoma cells (Lin JK et al., Biochem). Pharmacol 1999 Sep 15; 58 (6 : 911-5); amyloid β peptide found in the brain of Alzheimer's disease patients binds to the 75 kD neurotrophic factor receptor (p75NTR) in neuroblastoma cells, thereby making NF-κB time-dependent and dose-dependent. It is activated in a sexual manner (Kuper P et al., J
Neurosci Res 1998 Dec 15; 54 (6): 798-804); TNFα activated by NF-κB plays an important role in the development of glomerulonephritis (Ardalelou et al., Bull Acad Natl Med 1995 Jan; 179). (1) 103-15).

NFκB decoy inhibits cytokine and adhesion molecule expression in vivo in mouse nephritis induced by TNFα (Tomlta N et al., Gene Ther 2000).
Aug; 7 (15) 1326-32);

  It has been suggested that NF-κB suppresses MMP1 and MMP9, which are members of matrix metalloproteinases (MMPs), at the transcriptional level (Amplification of IL-1beta-induced metalloproteinase-9 expression by hypersensitivity oxidase sensitization. medium by activated activities of NF-kappaB and activating protein-1 and involved activation of the mitogen-activated protein kinase path. W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. J Immunol 2000 Nov 15,165 (10), 5788-97;. Nuclear factor kappaB activity is essential for matrix metalloproteinase-1 and -3 upregulation in rabbit dermal fibroblasts Biochem Biophys Res Commun. Bond M, Baker AH, Newby AC. 1999 Oct 22, 264 (2), 561-7; Synergistic up-regulation of metalloproteinase-9 by growt . Factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B.Bond M, Fabunmi RP, Baker AH, Newby AC FEBS Lett 1998 Sep 11,435 (1), 29-34; and Lipopolysaccharide activates matrix metalloproteinase-2 in endothelial cells through an NF-kappaB-dependent pathway. Kim H, Koh G. Biochem Biophys Res Commun. 2000 Mar 16, 269 (2), 401-5).

The role of NF-κB activity in the pathological condition of atopic dermatitis or in the animal model of atopic dermatitis induces increased expression of various cytokines accompanied by infiltration or activation of lymphocytes, and plays an important role in the onset and progression of the pathological condition (Role of B7-CD28 / CTLA-4 costulation and NF-kappa B in allergen-induced T cell chemotaxis by IL-16 and RANTES. Checkpoints for regulatory; Cruikshank WW, Center DM, Holgate ST, Djukanovic R. J Immunol 2000 Jan 1, 164 (1), 412-8; on of development and IFN-γ production by Th1cells in TCR-transgenic models.Anne O'Garra.Immunology 1999 65,41-44; Overproduction of Th2-specific chemokines in NC / Nga mice exhibiting atopic dermatitis-like lesions.Christian
Vestergaard et al. J Clin Invest 1999 1999, 1097-1105; Involvement of nuclear factor-kappa B activation in IgE synthesis in human B cells. Yanagihara Y, Basaki Y, Ikizawa
K, Kajiwara K, Kosio
T, Akiyama K. et al. J Allergy Clin Immunol 1996
Dec, 98 (6 Pt 2): S224-9).

In addition, it has been suggested that activation of NF-κB is one of important mechanisms in psoriasis vulgaris, contact dermatitis, and the like (Macrophage-derived cytokine and nuclear factor kappa B pp65 expression in syndrome) and skin of patents with positive arthritis.Danning CL, Illei GG, Hitchon C, Greer MR, Bumpas DT, McInnes IBArthrism Rheum 2000 Jun, 43 (6), 43 (6), n
endothelial cells by the common haptens
nickel and cobalt. Goebeler M, Roth J, Blocker EB, Sorg C, Schulze-Osthoff K. et al. J Immunol 1995 Sep 1; 155 (5): 2459-67).

GATA-3 is a transcription factor that plays an important role in the development and development of allergic diseases. , Cameron L, Wright E, Lavigne F, Toda M, Muro S, Ray A, Eidelman DH, Minshall E, Hamid Q. J
Allergy
Clin Immunol 2000 Jun 105 (6Pt1), 1146-52; Inhibition of allergy information in
a murine model of asthma by
expression of a dominant-negative mutant of GATA-3, Zhang DH, Yang L, Cohn
L, Parkyn L, Homer R, Ray P, Ray A, Immunity
1999 Oct 11 (4), 473-82).

  STAT-6 is a transcription factor that regulates IL-4 expression regulatory mechanism and IL-4 helper T cell reaction (Wang LH, Yang XY, Kirken RA, Resau JH, Farrar WL. Targeted disruption of stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4-drive T (H) 2 cell response. Blood 2000 Feb 15, 95 (4), 1249-57).

AP-1 is a transcription factor that plays an important role in the development and progression of allergic diseases (Transscriptive control of the IL-5 gene by human helper in cells). IL-4 synthesis, Mori A, Kaminuma O, Mikami T, Inoue
S, Okumura Y, Akiyama K, Okudaira H. et al. J
Allergy Clin Immunol 1999 May 103 (5Pt2), S429-36; The glucocorticoid receptor gene as a candidate for gene therapy in asthma, Mathieu M, Gougat C, Jaffuel D, Danielsen M, Godard P, Bousquet J, Demoly P, Gene Ther 1999 Feb, 6 (2), 245-52).

  Stat-1 and Ets are also transcription factors that are thought to play an important role in the development and development of allergic diseases.

  Thus, transcription factors including NF-κB have been suggested to be involved in various diseases through the expression of many genes under the transcriptional control. No method of treatment is provided.

  The present invention relates to a composition comprising a decoy compound suitable for treating skin diseases including atopic dermatitis, psoriasis vulgaris, contact dermatitis, keloids, ulcerative colitis, Crohn's disease, etc. Provide the product and its usage.

  The present invention provides a composition containing a decoy compound as a main component and treating (treating and preventing) a skin disease and a method for treating the disease. The present inventors have found that it is effective to administer a decoy compound in order to treat skin diseases, and have completed the present invention.

  The present invention relates to a pharmaceutical composition for treating and preventing skin diseases, the composition comprising at least one decoy and a pharmaceutically acceptable carrier.

  Preferably, the at least one decoy is selected from the group consisting of NF-κB decoy, STAT-1 decoy, GATA-3 decoy, STAT-6 decoy, AP-1 decoy, and Ets decoy. The

  Alternatively, the at least one decoy is selected from the group consisting of NF-κB decoy, STAT-1 decoy, GATA-3 decoy, STAT-6 decoy, AP-1 decoy, and Ets decoy. An oligonucleotide having one or more decoys attached thereto.

  The skin disease can be atopic dermatitis, psoriasis vulgaris, contact dermatitis, keloids, wounds, ulcerative colitis, or Crohn's disease.

  Preferably, the pharmaceutically acceptable carrier comprises petrolatum.

  Preferably, the pharmaceutically acceptable carrier is petrolatum, petrolatum containing 5% stearyl alcohol, or petrolatum containing liquid paraffin.

  Composition comprising decoy compound suitable for treating skin diseases including atopic dermatitis, psoriasis vulgaris, contact dermatitis, keloid, ulcerative colitis, Crohn's disease, etc. and use thereof Law is provided. A composition containing a decoy compound as a main component and treating (treating and preventing) a skin disease and a method for treating the disease are provided.

  The term “decoy” or “decoy compound” used in the present invention is a site on a chromosome to which NF-κB binds or a site on a chromosome to which another transcriptional regulatory factor of a gene controlled by NF-κB binds ( A compound that binds to a target binding site and antagonizes NF-κB or other transcription factors for binding to these target binding sites. Typically, a decoy or decoy compound is a nucleic acid and analogs thereof.

  If the decoy is present in the nucleus, the biological function provided by the decoy competing with the transcriptional regulator for binding to the target binding site of the transcriptional regulator, resulting in binding of the transcriptional regulator to the target binding site Is inhibited. The decoy includes at least one nucleic acid sequence that can bind to a target binding sequence. As long as it has binding activity to the target binding sequence, the decoy can be used in the preparation of the pharmaceutical composition of the present invention.

As an example of a preferable decoy, 5′-CCT-TGA-AGG-GAT-TTC-CCT-CC-3 ′ (SEQ ID NO: 1) (NF-κB decoy), 5′-GAT-CTA-GGG-ATT-TCC- GGG-AAA-TGA-AGC-T-3 ′ (SEQ ID NO: 2) (STAT-1 decoy), 5′-AGC-TTG-AGA-TAG-AGC-T-3 ′ (SEQ ID NO: 3) (GATA- 3 ') 5'-GAT-CAA-GAC-CTT-TTC-CCA-AGA-AAT-CTA-T-3' (SEQ ID NO: 4) (STAT-6 decoy), 5'-AGC-TTG- TGA-GTC-AGA-AGC-T-3 ′ (SEQ ID NO: 5) (AP-1 decoy) and 5′-AAT-TCA-CCG-GAA-GTA-TTC-GA-3 ′ (SEQ ID NO: 6) (Ets decoy) or this Oligonucleotides containing al complements, variants thereof or compounds containing them therein molecule, and the like. The oligonucleotide may be DNA or RNA, or may contain a nucleic acid modification and / or pseudo-nucleic acid within the oligonucleotide. Further, these oligonucleotides may be mutants thereof, or compounds containing them therein molecule, may be a single-stranded or double-stranded, may be either a linear or cyclic. A variant is a part of the above sequence that is mutated, substituted, inserted, or deleted, and has a nucleic acid binding site to which NF-κB or another transcriptional regulatory factor of a gene controlled by NF-κB binds. It means a nucleic acid that has the ability to specifically antagonize.

  More preferred decoys include double-stranded oligonucleotides containing one or more of the nucleic acid sequences or variants thereof. In order to indicate the number of nucleic acid sequences contained, a nucleic acid containing one or a plurality of the nucleic acid sequences is referred to as a chimera (table) decoy when it is two, and as a triple decoy when it is three. obtain.

  The oligonucleotide used in the present invention includes an oligonucleotide (S-oligo) having a thiophosphate diester bond in which the oxygen atom of the phosphodiester bond is substituted with a sulfur atom, or a non-charged methyl phosphodiester bond. Oligonucleotides modified to make the oligonucleotides less susceptible to degradation in vivo, such as oligonucleotides substituted with phosphate groups.

  As a method for producing the decoy used in the present invention, a chemical synthesis method or a biochemical synthesis method known in the art can be used. For example, when a nucleic acid is used as a decoy compound, a nucleic acid synthesis method generally used in genetic engineering can be used. For example, a target decoy nucleic acid may be directly synthesized using a DNA synthesizer, or these Or a part thereof may be synthesized and then amplified using a PCR method or a cloning vector. Furthermore, the nucleic acid obtained by these methods may be cleaved using a restriction enzyme or the like, and bound using DNA ligase or the like to produce the target nucleic acid. Furthermore, in order to obtain a more stable decoy nucleic acid in the cell, the base, sugar, and phosphate portion of the nucleic acid may be subjected to chemical modification such as alkylation or acylation.

  The present invention provides a pharmaceutical composition comprising a decoy compound as described above alone or in combination with a stabilizing compound, diluent, carrier, or other agent such as another ingredient or agent. The pharmaceutical composition of the present invention is used in such a form that the decoy is taken into the cells of the affected area or the cells of the target tissue.

  The pharmaceutical compositions of the invention can be administered in any sterile biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. The decoy compounds described above can be administered to a patient alone or in combination with other agents in a pharmaceutical composition mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. . In an embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

  Administration of the pharmaceutical composition of the present invention is accomplished orally or parenterally. Parenteral delivery methods include topical, dermal application, intraarterial (eg directly to tumor, aneurysm), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or Intranasal administration is mentioned.

  The pharmaceutical compositions of the present invention are suitable pharmaceutically, including excipients or other compounds that facilitate processing of the decoy compound, in addition to the decoy compound, in order to prepare a pharmaceutically usable formulation. An acceptable carrier can be included. Further details of techniques for formulation and administration are described, for example, in the final edition of “REMINGTON'S PHARAMEUTICAL SCIENCES” (Maack Publishing Co., Easton, Pa.).

  Hereinafter, the pharmaceutical composition of the present invention will be described in detail by exemplifying pharmaceutically acceptable carriers to be used for each administration form. In addition, the composition of this invention shown below is an illustration of this invention and does not restrict | limit this invention.

  Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art as dosage forms suitable for administration. Such carriers allow the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, ointments, syrups, slurries, suspensions, etc. suitable for consumption by the patient. .

  A pharmaceutical composition for oral use combines a decoy compound with a solid excipient as a carrier, grinds the resulting mixture, if desired, to obtain a tablet or dragee core, if desired After adding the appropriate further compound, it can be obtained via processing the mixture of granules. Suitable excipients are carbohydrate or protein fillers, including but not limited to: sugars including lactose, sucrose, mannitol, or sorbitol; derived from corn, wheat, rice, potato, or other plants Starches; celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including gum arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

  Dragee cores are provided with suitable coatings, such as concentrated sugar solutions. It may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragees for product identification or to characterize the quantity of active compound (ie, dose).

  Pharmaceutical preparations that can be used orally can include, for example, gelatin capsules, soft sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Gelatin capsules may contain the active ingredient in admixture with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the decoy compound can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin or liquid polyethylene glycol, with or without stabilizers.

  Pharmaceutical formulations for parenteral administration include aqueous solutions of decoy compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. If desired, the suspension may contain stabilizers or suitable agents or reagents that increase the solubility of the compounds that allow for the formulation of highly concentrated solutions.

  For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  The pharmaceutical compositions of the invention can be prepared in a manner similar to that known in the art (eg, conventional mixing, dissolving, granulating, dragee making, starch syrup, emulsifying, encapsulating, entrapping, or lyophilizing). By means).

Preferably, when administered topically into the affected cell or the tissue of interest, the pharmaceutical composition of the present invention comprises a synthetic or natural hydrophilic polymer as a carrier.
Examples of such hydrophilic polymers include hydroxypropyl cellulose and polyethylene glycol. The decoy compound of the present invention is mixed with such a hydrophilic polymer in a suitable solvent, the solvent is removed by a method such as air drying, and after molding into a desired form, for example, a sheet shape, Can be granted. Since the preparation containing such a hydrophilic polymer has a low water content, it is excellent in storage stability, and in use, it absorbs water and becomes a gel, so that the storage property of the decoy compound is excellent. In addition to the above-mentioned composition, a hydrophilic sheet formed by mixing polyhydric alcohol with cellulose, starch and derivatives thereof, or a synthetic polymer compound and adjusting the hardness can also be used.

  Such a sheet can be applied to the target site under a laparoscope, for example, using laparoscopic techniques. At present, laparoscopic surgery has developed remarkably as a non-invasive technique, and can be combined with the pharmaceutical composition of the present invention to provide a treatment method for non-invasive and repeatedly treatable diseases.

  Alternatively, when a nucleic acid or a modified product thereof is used as a decoy, the pharmaceutical composition of the present invention is in a form used in a commonly used gene transfer method, for example, a membrane-fused liposome preparation using Sendai virus or the like. , Using liposome preparations such as liposome preparations utilizing endocytosis, preparations containing cationic lipids such as lipofectamine (manufactured by Lifetech Oriental), or virus preparations using retrovirus vectors, adenovirus vectors, etc. Are particularly preferred, and membrane-fused liposome preparations are preferred.

  The liposome preparation may be a large unilamellar liposome (LUV), multilamellar liposome (MLV), or small unilamellar liposome (SUV). The size can also be a particle system of 200 to 1000 nm for LUV, 400 to 3500 nm for MLV, and 20 to 50 nm for SUV. In the case of a membrane fusion liposome preparation using Sendai virus or the like, an MLV with a particle system of 200 to 1000 nm is used. It is preferable to use it.

  The method for producing the liposome is not particularly limited as long as the decoy is retained, and a conventional method such as reverse phase evaporation (Szoka, F et al., Biochim. Biophys. Acta, Vol. 601 559 ( 1980)), ether injection method (Deamer, DW: Ann. NY Acad. Sci., Vol. 308 250 (1978)), surfactant method (Brunner, J et al .: Biochim. Biophys. Acta). , Vol.455 322 (1976)) or the like.

  As lipids for forming the liposome structure, phospholipids, cholesterols, nitrogen lipids and the like are used. Generally, phospholipids are preferable, and phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidine. Acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, lysolecithin and other natural phospholipids, or those hydrogenated according to conventional methods, dicetyl phosphate, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, di Palmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphati Ethanolamine, can be employed including synthetic phospholipids such Jer osteoprotegerin aroyl phosphatidylserine.

  Lipids containing these phospholipids can be used alone, but two or more can be used in combination. At this time, the binding rate of an electrically negative decoy nucleic acid can be increased by using a substance having an atomic group having a positive group such as ethanolamine or choline in the molecule. In addition to the main phospholipid at the time of liposome formation, additives such as cholesterols, stearylamine, α-tocopherol and the like that are generally known as additives for liposome formation can also be used.

  The liposomes thus obtained are purified from membrane fusion promoters such as Sendai virus, inactivated Sendai virus and Senda virus in order to promote uptake of affected cells or cells of the target tissue into cells. Membrane fusion promoting protein, polyethylene glycol and the like can be added.

An example of a method for producing a liposome preparation will be specifically described. For example, the above-mentioned liposome-forming substance is dissolved in an organic solvent such as tetrahydrofuran, chloroform, ethanol and the like together with cholesterol, and this is placed in a suitable container and the solvent is removed under reduced pressure. To form a liposome-forming substance film on the inner surface of the container. A buffer solution containing decoy is added and stirred, and the membrane fusion promoting substance is further added to the obtained liposome as desired, and then the liposome is isolated. Or liposomes containing decoy obtained as this is suspended in a suitable solvent, or once a material obtained by freeze drying can be used to treat redispersed in a suitable solvent. The membrane fusion promoting substance may be added after liposome isolation and before use.

  The pharmaceutical compositions of the present invention include compositions in which the decoy compound is contained in an amount effective to achieve the intended purpose. “Therapeutically effective amount” or “pharmacologically effective amount” is a term well recognized by those skilled in the art and refers to the amount of an agent effective to produce the intended pharmacological result. Thus, a therapeutically effective amount is an amount sufficient to reduce symptoms of the disease to be treated. One useful assay to ascertain an effective amount (eg, a therapeutically effective amount) for a given application is to measure the extent of recovery of the target disease. The amount actually administered will depend on the individual to whom the treatment is to be applied, and is preferably an amount optimized to achieve the desired effect without significant side effects. Determination of a therapeutically effective dose is well within the ability of those skilled in the art.

  For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in any suitable animal model. The animal model is also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

  A therapeutically effective amount refers to the amount of decoy compound that reduces the sign or condition of the disease. The therapeutic efficacy and toxicity of such compounds is comparable to standard pharmaceutical procedures in cell cultures or laboratory animals (eg ED50, therapeutically effective dose in 50% of the population; and LD50, 50% of the population). The dose which is lethal). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED50 / LD50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal experiments are used to formulate a range of quantities for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration. As an example, the dosage of the decoy compound is appropriately selected depending on the age and other patient conditions, the type of disease, the type of decoy used, etc. For example, intra-blood administration, intramuscular administration, intra-articular administration, skin application In general, 1 μg to 100 mg can be administered once to several times a day.

  The exact dose is chosen by the individual clinician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state (eg, tumor size and location; patient age, weight, and gender; food restriction time and frequency of administration, drug combination, response sensitivity, and resistance to therapy. / Response). Depending on the half-life and clearance rate of the particular formulation, sustained action pharmaceutical compositions can be administered every 3-4 days, weekly, or once every two weeks. Guidance on specific doses and methods of delivery is provided in literature known in the art.

The thus obtained pharmaceutical containing the decoy as a main component can be administered by various methods depending on the type of disease, the type of decoy used, etc., for example, ischemic disease, inflammatory disease, autoimmunity In the case of disease and cancer metastasis / invasion, and the epidemic, intravascular administration, application to the diseased site, administration into the diseased site, or intravascular administration to the diseased site, etc.
As a more specific example, for example, when PTCA is performed for organ infarction or the like, it can be administered to the affected blood vessel at the same time or before and after, and in organ transplantation or the like, the organ to be transplanted is used in advance in the present application. It may be used after being treated. Further, for example, in rheumatoid arthritis, it can be directly injected into the joint.

  For skin diseases, the composition of the invention can be administered topically to the affected area of the skin, preferably in the form of an ointment. This ointment is a semi-solid topical preparation with an appropriate consistency that can be easily applied to the skin. Usually, fat, fatty oil, lanolin, petrolatum, paraffin, wax, plaster, resin, plastic are used as carriers. , Glycols, higher alcohols, glycerin, water or emulsifiers, suspending agents, and these are used as a base and the decoy compound is evenly mixed. Depending on the base, it may be in the form of an oily ointment, an emulsion ointment, a water-soluble ointment. The oily ointment is based on animal and vegetable oils and waxes, or petrolatum, fluidized paraffin, etc. The emulsion ointment emulsifies an oily substance and water with an emulsifier, and is an oil-in-water type (O / W) or It can be either water-in-oil (W / O). The oil-in-water type (O / W) may be a hydrophilic ointment, and the water-in-oil type (W / O) may lack a water phase from the beginning and may contain hydrophilic petrolatum, purified lanolin, or water absorption that includes an aqueous phase. May contain ointment, hydrolanolin. The water-soluble ointment may contain as a main component a macrogol base that is completely soluble in water.

  The present invention will be described below with reference to examples, but these examples are illustrative of the present invention and are not intended to be limiting.

  The effect of the composition of the present invention was confirmed by the following procedure.

  1. First, by using NF-κB decoy ointment (the composition of which is shown in the following 2), it is confirmed that NF-κB decoy is surely introduced into epidermal stroma and intradermal cells. Confirmed by applying ointment to mice.

  FIG. 1 is a fluorescence micrograph (× 400) of a skin sample of the upper half of NC / Nga mice 4 days after NF-κB decoy ointment labeled with a fluorescent dye FITC was applied to the upper half of the skin. . As shown in FIG. 1, uniform fluorescence is observed throughout the stroma of the epidermis, and green fluorescence is observed in lymphocytes and hair follicle constituent cells in the dermis, and NF-κB decoy is expressed in the epidermis. It was confirmed that it was introduced into the dermal cells.

2. Next, NC / Nga mice (natural atopy model mice) were divided into two groups, a NF-κB decoy ointment local administration group (test group) and a control decoy ointment local administration group (control group). The local effects on atopic dermatitis were compared. Here, in order to increase the spontaneous incidence of atopic over dermatitis in the test mice to 100% from 80%, purchased 4-week-old male of the strain mice bred by adding Hitodani only, atopic Used as a dermatitis-induced mouse.

  To summarize this study, the purchased NC / Nga mice were divided into 2 groups, and each group of mice received NF-κB decoy ointment having the composition shown below in the protocol shown below (protocols 1 and 2). In accordance with the above, topical administration (applied to the skin) was applied to both ears, upper back half and head (NF-κB decoy ointment topical administration group). To other groups of mice, a control decoy ointment having the composition shown below was locally administered in the same manner as in the NF-κB decoy ointment topical administration group (control decoy ointment topical administration group), and atopic dermatitis in each group of mice. We compared the local effects on the effect.

Composition of NF-κB decoy ointment :
NF-κB decoy 10 mg, stearyl alcohol 30 mg,
Petrolatum 0.6g.

Control decoy ointment composition :
Control decoy 10 mg, stearyl alcohol 30 mg,
Petrolatum 0.6g
Protocol 1 :
Approximately 1 mg of NF-kB decoy or control decoy per mouse is administered by applying the ointment; applied 4 times a week from 2 weeks to 2 weeks in total, evaluated at 12 weeks of age .

Protocol 2 :
Approximately 2 mg of NF-kB decoy or control decoy per mouse is administered by applying the ointment; applied only once at 29 weeks of age and evaluated at 30 weeks of age.

  The local effect of the administered ointment on atopic dermatitis was evaluated macroscopically using a clinical skin symptom score. Furthermore, this local effect was evaluated microscopically using HE staining according to a conventional method.

  FIG. 2A shows the results of clinical skin symptom scores in the evaluation test of the local effect on atopic dermatitis according to the protocol 1 as an average value of each group. As shown in the figure, in the NF-κB decoy ointment topical administration group at the age of 12 weeks, the average clinical symptom score of 6 animals tended to be lower than in the control decoy ointment topical administration group, and the long-term administration of NF-κB decoy ointment The effect of was confirmed.

  FIG. 2B and FIG. 2C are photographs of the skin samples (12 weeks: HE staining) of the upper half of the mice in the control decoy ointment topical administration group and the NF-κB decoy ointment topical administration group, respectively. As is clear from FIG. 2B and FIG. 2C, in the NF-κB decoy ointment topical administration group mice, an improvement in epidermal thickening, an improvement in spinous layer thickening, and a decrease in granule layer were observed compared to the control decoy ointment topical administration group. It was shown that the skin tissue was pathologically improved by infiltration of the NF-κB decoy into the skin.

  FIG. 2D and FIG. 2E are photographs taken from the back of the control decoy ointment local administration group and the NF-κB decoy ointment local administration group, respectively. As seen in FIG. 2D and FIG. 2E, in the NF-κB decoy ointment topical administration group of mice, eczema accompanied by redness was improved as compared with the control decoy ointment topical administration group, and a reduction in pruritus was also observed. It shows that the symptoms of atopic dermatitis were improved by infiltration of NF-κB decoy into the skin.

  FIG. 2F shows the measurement results of the number of mast cells per unit area of skin in the NF-κB decoy ointment topical administration group and the control decoy ointment topical administration group as average values. As is clear from FIG. 2F, the number of mast cells was significantly smaller in the mice in the NF-κB decoy ointment topical administration group than in the control decoy ointment topical administration group. Thus, it was shown that NF-κB decoy suppressed the accumulation of mast cells that produce cytokines related to the pathology of atopic dermatitis.

  FIG. 3A shows the results of clinical skin symptom scores in the evaluation test of the local effect on atopic dermatitis according to the protocol 2 as an average value of each group. As shown in the figure, in the NF-κB decoy ointment topical administration group at 30 weeks after birth, the average clinical symptom score of 6 animals tended to be lower than that in the control decoy ointment topical administration group, and the NF-κB decoy ointment short term The effect was confirmed.

  FIG. 3B and FIG. 3C are photographs of the skin samples (12 weeks: HE staining) of the upper half of the mice with the local administration group of the control decoy ointment and the NF-κB decoy ointment topical administration group in Procol 2, respectively. As is clear from FIGS. 3B and 3C, in the NF-κB decoy ointment topical administration group, compared to the control decoy ointment topical administration group, the epidermis thickening was improved, the spinal layer thickening was improved, and the granular layer was decreased. It was shown that the skin tissue was pathologically improved by infiltration of the NF-κB decoy into the skin.

  FIG. 3D is a photograph taken from the back, and FIG. 3E is a photograph taken from the front of the control decoy ointment topical administration group of mice. As can be seen in FIG. 3D and FIG. 3E, each mouse in this group has redness-related eczema and pruritus, which are characteristic symptoms of atopic dermatitis.

  FIG. 3F is a photograph taken from the back of the NF-κB decoy ointment topical administration group of mice, and FIG. 3G is a photograph taken from the front. As seen in FIG. 3F and FIG. 3G, each mouse in this group had improved eczema with redness, and a reduction in pruritus, and the symptoms of atopic dermatitis were NF-κB decoy. It shows improvement by infiltration into the skin.

  Composition comprising decoy compound suitable for treating skin diseases including atopic dermatitis, psoriasis vulgaris, contact dermatitis, keloid, ulcerative colitis, Crohn's disease, etc. and use thereof Law is provided. A composition containing a decoy compound as a main component and treating (treating and preventing) a skin disease and a method for treating the disease are provided.

FIG. 1 is a fluorescence micrograph (× 400) of a skin sample of the upper half of a mouse administered with the composition of the present invention. FIG. 2A is a graph showing the results of clinical skin symptom scores (protocol 1) showing the effects of the present invention over time. FIG. 2B is a photograph of a skin sample of the upper half of a control group of mice stained with HE. FIG. 2C is a photograph of the skin specimen of the upper half of the test group of mice stained with HE. FIG. 2D is a photograph of the control group of mice taken from the back. FIG. 2E is a photograph of the mice in the test group taken from the back. FIG. 2F is a diagram showing measurement results of the number of mast cells per unit skin area in the control group and the test group. FIG. 3A is a graph showing the results of clinical skin symptom scores (protocol 2) showing the effects of the present invention over time. FIG. 3B is a photograph of a skin specimen of the upper half of a control group of mice stained with HE. FIG. 3C is a photograph of the skin specimen of the upper half of the test group of mice stained with HE. FIG. 3D is a photograph of the control group of mice taken from the back. FIG. 3E is a photograph of the control group of mice taken from the front. FIG. 3F is a photograph of the test group of mice taken from the back. FIG. 3G is a photograph of mice in the test group taken from the front.

Claims (7)

A pharmaceutical composition for the treatment of psoriasis vulgaris, a decoy of at least one NF-[kappa] B, and a pharmaceutically acceptable carrier including composition, the decoy double-stranded oligonucleotide Or a composition that is an S-oligo . The composition according to claim 1, wherein the NF-κB decoy is a double-stranded oligonucleotide or an S-oligo thereof containing at least one nucleic acid sequence to which NF-κB can bind. The NF-κB decoy contains a sequence in which a part of the sequence shown in SEQ ID NO: 1 is substituted, inserted or deleted, and specifically antagonizes an NF-κB nucleic acid binding site Or the composition of Claim 1 which is the S-oligo. The composition of claim 1, wherein the NF-κB decoy comprises a double-stranded nucleic acid comprising the sequence set forth in SEQ ID NO: 1 and its complement. The composition of claim 1, wherein the at least one NF-κB decoy is an oligonucleotide comprising one or more nucleic acid sequences capable of binding to NF-κB. The composition according to any one of claims 1 to 5 , wherein the pharmaceutically acceptable carrier comprises petrolatum. The composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable carrier is petrolatum containing 5% stearyl alcohol or petrolatum containing liquid paraffin.

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