JP4971609B2 - Nucleic acid skin preparation - Google Patents

Description

The present invention relates to pharmaceutical preparations containing nucleic acids. More specifically, the present invention relates to a skin external preparation containing a nucleic acid oligonucleotide.

The therapeutic effect of an applied preparation such as an ointment or lotion requires appropriate permeability control of the active ingredient (main agent). For example, in the case of many skin diseases, it is necessary to allow the main agent to penetrate to the epithelium and dermis of the skin tissue. As a barrier at that time, a so-called skin barrier composed of keratinous and epithelial tissue can be mentioned. In general, high fat solubility and low molecular weight (up to about 800 molecular weight) are required as the physicochemical properties of the main agent that easily passes through the skin barrier. For example, steroids, vitamin D, and the like are known to efficiently penetrate the skin barrier, but they are relatively low-molecular and highly fat-soluble. On the other hand, it is known that highly water-soluble and high molecular weight molecules are quite difficult to pass through the skin barrier. Although pharmaceutical studies have been carried out, few formulations with sufficient permeability have been known.

In recent years, with the advance of biotechnology, new drugs for treating diseases by controlling gene expression have been actively developed. One of them is a nucleic acid medicine using an oligonucleotide or the like, many of which are polymers having a molecular weight of 5,000 or more, and have extremely high water solubility. For example, when developing a coating formulation based on a highly water-soluble, high-molecular-weight nucleic acid drug for skin diseases such as atopic dermatitis, a formulation with excellent skin permeability was reported as described above. The development was desired.

A skin disease is a disease that is easily damaged not only physically but also physically because the affected part is exposed to the outside. For example, atopic dermatitis, which has been increasing in recent years, often involves a wide area of the body, is refractory, and patients are troubled for a long time. This is an allergic disease found in a wide range of ages from children to adults. Skin has a role of a barrier that protects the body from external substances and irritation, but in atopic dermatitis, when the barrier function of this skin is reduced, environmental antigens such as mites are more likely to enter the skin, resulting in allergies. A reaction occurs. However, steroid ointment often used for the treatment of atopic dermatitis suppresses allergic reactions, but also has a risk of lowering the barrier function of the skin, and is not suitable for long-term use.

Since the external preparation for skin exhibits a medicinal effect by permeating the skin, it is essential to break this barrier and penetrate into the dermis. However, when the active ingredient of the external preparation for skin is a macromolecule such as a nucleic acid, it is further difficult to penetrate the skin as it is. For these reasons, it has been difficult to develop an external preparation for skin containing a macromolecule such as a nucleic acid as an active ingredient.

In view of the above background, it has been desired to develop a drug that penetrates into the skin while maintaining its skin barrier function and exhibits its medicinal effects.

As a result of intensive studies to solve these problems, the present inventors have unexpectedly found that viscous polysaccharides, or urea-based compounds and phospholipids are excellent as penetration enhancers, and are further used as moisturizing agents in cosmetics. The inventors have found that alginic acid or a pharmacological salt thereof used can achieve the purpose most effectively, and the present invention has been completed.

Alginic acid is a polysaccharide derived from seaweed and is used in foods and cosmetics as a thickener, a coating agent, and a moisturizer, but its effect as a penetration enhancer was not known at all.

This patent document discloses a polysaccharide gum preparation such as alginate of a non-steroid anti-inflammatory drug (NSAID). However, this uses a low molecular weight compound as a mixed preparation with alginate (alginate), but has not examined the skin permeability of a high molecular weight compound such as a nucleic acid.

The present disclosure discloses a pulmonary sustained-release preparation comprising a combination of alginic acid and an oligonucleotide. Although this is a preparation of alginic acid and nucleic acid, it is a drug intended for the lung, and it did not mention skin permeability.

In recent years, gene / nucleic acid drugs have attracted attention, and a number of gene therapies have been attempted. However, there is no nucleic acid pharmaceutical established as a therapeutic agent for skin diseases, and if this is realized, it may be applicable to diseases for which conventional drugs have no effect.

The problem to be solved by the present invention is to provide an external preparation for skin containing nucleic acid as an active ingredient. In particular, it must have high skin permeability while protecting the skin barrier function.

The present inventors have found that an oligonucleotide exhibits high skin permeability by using a nucleic acid drug such as NF-κB decoy oligonucleotide and using it as a mixed preparation of sodium alginate or soybean phospholipid and petrolatum. It was. That is, the present invention provides a high skin permeability external preparation for skin containing nucleic acid as an active ingredient.

The gist is
(1) A pharmaceutical composition comprising a nucleic acid drug and a penetration enhancer for delivering the nucleic acid drug into cells of the dermis or epidermis tissue,
(2) The composition according to (1), wherein the nucleic acid drug is a gene or an analog thereof,
(3) The composition according to (1) or (2), wherein the gene or an analog thereof is a polynucleotide or an oligonucleotide,
(4) The composition according to (1) to (3), wherein the oligonucleotide is a decoy (decoy molecule), antisense, ribozyme, aptamer or siRNA
(5) The composition according to (1) to (4), wherein the decoy has a transcription factor inhibitory action,
(6) The composition according to (1) to (5), wherein the decoy is NF-κB, STAT-1, GATA-3, STAT-6, AP-1, Ets, E2F decoy oligonucleotide,
(7) The composition according to (1) to (6), wherein the decoy is an NF-κB decoy oligonucleotide represented by SEQ ID NO: 1.
(8) The composition according to (1) to (7), comprising 0.01 to 5% by weight of a nucleic acid drug,
(9) The composition according to (1) to (8), comprising 0.1 to 1% by weight of a nucleic acid drug,
(10) The composition according to (1) to (9), wherein the penetration enhancer is a viscous polysaccharide, or a urea compound and a phospholipid,
(11) The composition according to (1) to (10), wherein the viscous polysaccharide is alginic acid or a pharmacologically acceptable salt thereof,
(12) The composition according to (1) to (11), wherein the viscous polysaccharide is sodium alginate,
(13) The composition according to (1) to (12), comprising 0.01 to 10% by weight of a viscous polysaccharide,
(14) The composition according to (1) to (13), comprising 0.1 to 5% by weight of a viscous polysaccharide,
(15) The composition according to (1) to (14), further comprising a phosphate,
(16) The composition according to (1) to (15), wherein the phosphate is sodium dihydrogen phosphate,
(17) The composition according to (1) to (16), comprising 0.01 to 10% by weight of a phosphate,
(18) The composition according to (1) to (17), comprising 0.1 to 5% by weight of a phosphate,
(19) The composition according to (1) to (18), wherein the urea compound is urea or cyclic urea,
(20) The composition according to (1) to (19), wherein the urea compound is urea,
(21) The composition according to (1) to (20), which contains 0.01 to 10% by weight of a urea compound.
(22) The composition according to (1) to (21), comprising 0.1 to 5% by weight of a urea compound.
(23) The phospholipid is one or more selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) or sphingomyelin (SPM). , (1) to (22) composition,
(24) The composition according to (1) to (23), wherein the phospholipid is phosphatidylcholine,
(25) The composition according to (1) to (24), wherein the phospholipid is soybean-derived phosphatidylcholine,
(26) The composition according to (1) to (25), comprising 0.1 to 20% by weight of phospholipid,
(27) The composition according to (1) to (26), comprising 1 to 16% by weight of phospholipid,
(28) A preventive, ameliorating, or therapeutic agent for skin diseases comprising the composition according to (1) to (27),
(29) Skin diseases are atopic dermatitis, contact dermatitis, photosensitivity dermatitis, chronic dermatitis of hands and feet, seborrheic dermatitis, monetary dermatitis, generalized exfoliative dermatitis, congestive The preventive, ameliorating, therapeutic agent according to (28), which is dermatitis, topical abrasion dermatitis, drug dermatitis or psoriasis,
About.

Since the nucleic acid skin external preparation of the present invention has high skin permeability, it can efficiently deliver an active ingredient nucleic acid to the affected area. Since the skin permeability is high, the nucleic acid may be used at a low dose, and is excellent in safety and economy. In addition, since the mechanism of action is different from the low-molecular-weight drugs that are currently used anti-inflammatory agents, the effect can be expected even when the low-molecular-weight drugs are not effective.

The present invention provides a pharmaceutical composition comprising a nucleic acid drug and a penetration enhancer for delivering the nucleic acid drug into cells of the dermis or epidermal tissue.

The nucleic acid drugs used in the present invention are usually decoys, antisenses, ribozymes, aptamers, siRNAs and the like, but are preferably decoys. A decoy refers to a double-stranded oligonucleotide that competes with a nucleic acid binding sequence when a transcriptional regulator binds to the nucleic acid. Decoy, decoy oligonucleotide, and decoy ODN are used interchangeably. Nucleic acid drugs can be used alone or in admixture of two or more. The skin external preparation of the present invention contains 0.01 to 5% by weight (hereinafter referred to as "%"), particularly 0.1 to 1%. Preferably it is done.

Examples of the nucleic acid drug of the present invention include decoys such as NF-κB, STAT-1, GATA-3, STAT-6, AP-1, Ets, and E2F, but NF-κB is a suitable target. . Examples of preferred decoys include 5'-ccttgaagggatttccctcc-3 '(SEQ ID NO: 1) (NF-κB decoy), 5'-gatctagggatttccgggaaatgaagct-3' (SEQ ID NO: 2) (STAT-1 decoy), 5'-agcttgagatagagct- 3 '(SEQ ID NO: 3) (GATA-3 decoy), 5'-gatcaagaccttttcccaagaaatctat-3' (SEQ ID NO: 4) (STAT-6 decoy), 5'-agcttgtgagtcagaagct-3 '(SEQ ID NO: 5) (AP-1 decoy) ), 5'-aattcaccggaagtattcga-3 '(SEQ ID NO: 6) (Ets decoy), 5'-ctagatttcccgcg-3' (SEQ ID NO: 7) (E2F decoy), or an oligonucleotide containing these complements, variants thereof Or a compound containing these in the molecule. The oligonucleotide may be DNA or RNA, or may contain a nucleic acid modification and / or pseudo-nucleic acid within the oligonucleotide. These nucleic acid drugs include double-stranded oligonucleotides containing one or more of the nucleic acid sequences or variants thereof.

In addition, as the penetration enhancer in the present invention, viscous polysaccharides, phosphates, urea, phospholipids and the like are usually used. Preferably, viscous polysaccharides, phosphates, or phospholipids are used alone or in combination of two or more. Are mixed and used. More preferably, a mixture of viscous polysaccharide and phosphate is used.

As the viscous polysaccharide, alginic acid, lamiranan, carrageenan, flucefuran, porphyran, grateropyran, glucan, fucoidan, hyaluronic acid and the like can be used, but preferably alginic acid and carrageenan, more preferably alginic acid. Alginic acid or a pharmacologically acceptable salt thereof can be used as alginic acid, but sodium alginate is usually preferred. Commercially available alginic acid such as “Kimika Argin” (manufactured by Kimika) can be used.

These viscous polysaccharides can be used alone or in admixture of two or more, and are used in the external preparation for skin of the present invention in an amount of 0.01 to 10% by weight (hereinafter referred to as “%”), particularly 0.1 to 5%. % Is preferable.

Phosphate is usually sodium dihydrogen phosphate, calcium monohydrogen phosphate, tripotassium phosphate, tricalcium phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, triphosphate Sodium, calcium dihydrogen phosphate, and the like are used, and sodium dihydrogen phosphate is preferably used. These phosphates can be used alone or in admixture of two or more, and are preferably blended in the skin external preparation of the present invention in an amount of 0.01 to 10% by weight, particularly 0.1 to 5%.

As the phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and sphingomyelin are usually used, but phosphatidylcholine is preferably used, and soybean-derived phosphatidylcholine is more preferably used. These phospholipids can be used alone or in admixture of two or more, and are preferably blended in the skin external preparation of the present invention in an amount of 0.01 to 20% by weight, particularly 1 to 16%.

Preferably, 0.01% to 10% urea is used in addition to phospholipid. More preferably, 0.1% to 5% of urea is used. Urea may be urea or cyclic urea. The cyclic urea is usually preferably a 5-membered ring, and the nitrogen atom may be substituted with lower alkyl. Specifically, for example, ethylene urea, N, N′-dimethylethyleneurea, N, N′-diethylethyleneurea and the like can be mentioned.

The external preparation for skin of the present invention includes, in addition to the above essential components, water, oils, waxes, silicones, surfactants, alcohols, alcohols, and many other substances within the qualitative and quantitative ranges that do not affect the effects of the present invention. In addition to ingredients used in pharmaceuticals and cosmetics such as monohydric alcohols, water-soluble polymer thickeners, pH regulators, fragrances, antioxidants, chelating agents, dyes, pigments, preservatives, other medicinal ingredients, UV absorbers, etc. Inorganic or organic components can also be blended.

The target diseases of the external preparation for skin of the present invention are, for example, atopic dermatitis, contact dermatitis, photosensitivity dermatitis, chronic dermatitis of hands and feet, seborrheic dermatitis, monetary dermatitis, Systemic exfoliative dermatitis, congestive dermatitis, topical abrasion dermatitis, drug dermatitis or psoriasis.

The external preparation for skin of the present invention can be prepared according to a conventional method using the above essential components and optional components as necessary, and can be made into various dosage forms such as liquids, lotions, oils, creams, ointments and the like. .

The dose varies depending on age, body weight, symptom, therapeutic effect, administration method, treatment time, etc., but usually an amount that can cover the affected area is applied 1 to 3 times per day.

Subsequently, in order to specifically describe the present invention, examples are given below, but the present invention is not limited thereto.

1. Skin permeability test 1-1. To clarify the skin permeability of at NF-[kappa] B decoy oligonucleotide single transdermal administration in rats, ³⁵ S-labeled ³⁵ S- NF-κB decoy oligonucleotide (hereinafter ³⁵ S -NF (abbreviated as -κB decoy), the radioactivity concentration in blood and skin was measured.
The chemical structure and label position of ³⁵ S-NF-κB decoy are shown below.
³⁵ S-5'-CCTTGAAGGGATTTCCCTCC-3 '
³⁵ S-3'-GGAACTTCCCTAAAGGGAGG-5 '

³⁵ S-NF-κB decoy was prepared as follows according to the method of Agrawal et al. (Proc. Natl. Acad. Sci. USA, 88, 7595-7599, 1991). Create H-phosphonate-type oligonucleotide object of nucleotide sequence, oxidizing the H of H-phosphonate to ³⁵ S at ³⁵ S ₈ before deprotection (elementals sulfur), it performs the normal deprotection reaction, ³⁵ S purposes A labeled oligonucleotide was obtained.

(1) Preparation of dosage formulation After collecting a predetermined amount of ³⁵ S-NF-κB decoy stock solution and unlabeled NF-κB decoy in an agate mortar or in a plastic container, add a large excess of ethanol to remove NF-κB decoy. Precipitated. Next, after removing the supernatant, the solvent was completely removed under a nitrogen gas stream to prepare ³⁵ S-NF-κB decoy drug substance.

Formulation A (Vaseline alone)

³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Stearyl alcohol 5.0% 20.0 mg
White petrolatum 94.5% 378.0 mg
────────────────────────────
Total 100.0% 400.0 mg

A mixed base was prepared by melting and mixing a predetermined amount of stearyl alcohol and white petrolatum in a water bath at 70 to 80 ° C. and allowing to cool to room temperature. Add the specified amount of the mixed base to the ³⁵ S-NF-κB decoy base prepared in advance and mix well, adding 0.4 mg of NF-κB decoy (content of unlabeled NF-κB decoy: 78.88%) / 500 kBq / 100 mg of formulation A was prepared.

Formulation B (1% phospholipid formulation)
³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Distilled water for injection 30.0% 120.0 mg
Purified hydrogenated soybean phospholipid 1.0% 4.0 mg
White petrolatum 65.5% 262.0 mg
Isopropyl myristate 3.0% 12.0 mg
────────────────────────────
Total 100.0% 400.0 mg

^A predetermined amount of distilled water for injection was added to the S-NF-κB decoy drug substance and dissolved at room temperature, and then a specified amount of purified hydrogenated soybean phospholipid was added and stirred until evenly dispersed. Next, a predetermined amount of white petrolatum and isopropyl myristate are melt-mixed in a 60 ° C water bath, mixed with the mixture allowed to cool to room temperature, and stirred uniformly to obtain NF-κB decoy. A preparation B of 0.4 mg (content of unlabeled NF-κB decoy: 78.88%) / 500 kBq / 100 mg was prepared. Formulations C to E were prepared in the same manner.

Formulation C (2% phospholipid formulation)
³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Distilled water for injection 30.0% 120.0 mg
Purified hydrogenated soybean phospholipid 2.0% 8.0 mg
White petrolatum 64.5% 258.0 mg
Isopropyl myristate 3.0% 12.0 mg
────────────────────────────
Total 100.0% 400.0 mg

Formulation D (6% phospholipid formulation)
³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Distilled water for injection 30.0% 120.0 mg
Purified hydrogenated soybean phospholipid 6.0% 24.0 mg
White petrolatum 60.5% 242.0 mg
Isopropyl myristate 3.0% 12.0 mg
────────────────────────────
Total 100.0% 400.0 mg

Formulation E (16% phospholipid formulation)
³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Distilled water for injection 30.0% 120.0 mg
Purified hydrogenated soybean phospholipid 16.0% 64.0 mg
White petrolatum 50.5% 202.0 mg
Isopropyl myristate 3.0% 12.0 mg
────────────────────────────
Total 100.0% 400.0 mg

Water-soluble preparation A (1% alginate preparation)
³⁵ S-NF-κB Decoy 0.5% 2.0 mg
Alginic acid base (1% sodium alginate /
Sodium dihydrogen phosphate aqueous solution appropriate amount) 99.5% 398.0 mg
────────────────────────────────────
Total 100.0% 400.0 mg

Add a predetermined amount of alginate base to ³⁵ S-NF-κB decoy base, dissolve, thoroughly stir with a vortex mixer, and then pipet thoroughly with a micropipette to disperse uniformly. ³⁵ S-NF An aqueous preparation A of 0.4 mg (content of unlabeled NF-κB decoy: 78.88%) / 500 kBq / 100 mg was prepared as -κB decoy.

The following media were used as preparation preparation media. Stearyl alcohol (Lot No. 20714C, listed in Japanese Pharmacopoeia, Japanese fats and oils), white petrolatum (Lot No. 662275, listed in Japanese Pharmacopoeia, Nikko Pharmaceutical), purified hydrogenated soybean phospholipid (Lot No. 3228, NIKKOL Resinol) S-10E, Nikko Chemicals), isopropyl myristate (Lot No. WAE 5335, chemical, Wako Pure Chemical Industries), distilled water for injection (Lot No. 3B76N, Japanese Pharmacopoeia, Otsuka Pharmaceutical), alginate base (Appropriate amount of 1% sodium alginate / sodium dihydrogen phosphate aqueous solution).

(2) 7-week-old male Crj: CD (SD) rats were used as one animal per group. Rats with a body weight range of 220-330 g were used.

(3) Preparation of the application site The day before administration, the rat was shaved with an electric clipper and an electric shaver so as not to damage the skin under light ether anesthesia. The shaved area was confirmed on the day of administration, and it was confirmed that there was no inflammation or redness. This was designated as “normal skin”. Furthermore, on the day of administration, the shaved part was crimped and peeled with cellophane tape 10 times, and the skin layer was peeled off as “damaged skin”. Animals were used separately as shown in Table 1.

Table 1
────────────────────
Test animal number Administration formulation Skin condition

────────────────────
1 Formulation A Normal
2 損傷 Damage
3 Formulation B Normal
4 損傷 Damage
5 Formulation C Normal
6 〃 Damage
7 Formulation D Normal
8 損傷 Damage
9 Formulation E Normal
10 損傷 Damage
11 Water-soluble preparation A Normal
12 〃 Damage────────────────────

(4) Application and protection method
^{The 35} S-NF-κB decoy preparation was applied to the back skin so that the application area was 3 cm wide x 3 cm long. The dose of the preparation was 0.4 mg / head, the dose of the preparation was 100 mg / head, the dose of radioactivity was 500 kBq / head, and the number of doses was once.
After that, place a paper box form (4 cm wide x 4 cm long x 1 cm high) so as to surround the application part, and use adhesive elastic tape (elastopore, Nichiban) to surround the box form. The application part was protected by fixing. The coating time was 24 hours.

(5) Method of removing the dosage form After moistening with slightly warm water, use a total of 5 pieces, one for each piece of absorbent cotton (4 cm x 5 cm) that has been squeezed firmly. Wiping was performed.

(6) Measurement of skin radioactivity concentration
^The animals were treated with ³⁵ S-NF-κB decoy at a dose of 0.4 mg / head, and the animals were subjected to ether anesthesia.

The radioactivity concentration in the skin at the administration site was measured as follows. After removal of the drug product, the administration site skin was removed at a thickness of 2 mm and 2.5 x 2.5 cm from the center, and then frozen at around -20 ° C with light pressure applied between two slide glasses. Next, after punching ^two sections from the dermis side so that each section was 1 cm ² , the dermis side after freezing was fixed to the stage under liquid nitrogen with an OCT compound. After fixation, it was sliced from the epidermis side to a thickness of 10μm using a cryostat under freezing. After every 4 slices (40 μm equivalent), slice the sliced slices, add the tissue solubilizer SOLUENE-350 (2 mL), heat and dissolve, add 10 mL of scintillator, and leave at room temperature. After that, the radioactivity was measured using a liquid scintillation counter, and the radioactivity concentration of each layer was calculated from the obtained value. 24 hours after application, the remaining part of the applied part was wiped off.

Results) Skin permeability test (see Tables 2-6, Figures 1-4)
As for the radioactivity concentration in the skin, it was confirmed that the water-soluble preparation A (alginic acid preparation), which is a water-soluble gel, significantly increased the skin permeability as compared with the preparation A (peteline single preparation). In addition, it was found that preparations B to E (purified hydrogenated soybean phospholipid (Soybean phosphatidyl choline, SPC) preparation) also enhanced skin permeability compared to preparation A (pepperine alone preparation).

Table 2
Normal skin radioactivity (ng eq. Of NF-κB Decoy ODNs / cm ³ )
───────────────────────────────
Depth (μm) Formulation A Formulation B Formulation C Formulation D Formulation E
───────────────────────────────
40 127723 109155 60207 41009 129858
80 176569 260189 106498 71782 261867
120 195258 241277 119444 100643 331157
160 160645 236509 126704 132561 365296
200 122080 186778 126413 144684 367088
240 103637 152236 132612 138648 349545
280 67479 111365 135825 96451 316355
320 34559 93319 161536 67699 243472
360 14894 86309 122965 39964 148861
400 5278 74650 77955 25426 72110
440 2269 38523 31019 15169 45368
480 533 18588 10410 11974 24153
520 228 11102 4495 4411 12401
560 165 4944 2098 2069 7742
600 150 1178 1303 1304 4462
640 202 355 1032 969 3003
680 0 275 630 798 1457
720 0 294 586 667 1123
760 0 367 510 386 1212
800 0 382 706 371 1339
840 125 209 554 334 1353
880 220 245 341 336 1764
920 165 274 151 196 764
960 0 150 159 236 657
1000 0 0 157 199 581
1040 0 0 183 145 372
1080 0 0 260 333 149
1120 0 0 110 449 273
1160 0 0 0 580 385
1200 0 0 178 212 208
1240 0 0 120 179 101
1280 0 0 186 155 185
1320 0 136 183 188 110
1360 0 0 0 0 172
1400 0 0 0 182 187
1440 0 0 153 390 120
1480 160 0 145 0 193
1520 0 0 227 140 177
1560 119 0 206 0 105
1600 0 0 125 0 0
1640 0 0 105 0 0
1680 0 0 144--
1720 0 0 0--
1760 0----
1800 0----
───────────────────────────────
(Formulation A: Vaseline alone, Formulation B: Phospholipid 1%, Formulation C: Phospholipid 2%, Formulation D: Phospholipid 6%, Formulation E: Phospholipid 16%, 0: Below detection limit,-: Not measured )

Table 3
Damaged skin radioactivity (ng eq. Of NF-κB Decoy ODNs / cm ³ )
───────────────────────────────
Depth (μm) Formulation A Formulation B Formulation C Formulation D Formulation E
───────────────────────────────
40 151237 480991 522326 635059 160513
80 328816 834599 1151740 1033450 645599
120 496468 766617 1370503 2164656 1169895
160 482113 394444 756279 1947898 1549725
200 300 313 135 651 339 598 987 221 994 644
240 200318 42853 143062 523925 508349
280 126664 18898 40206 281962 153527
320 69 206 12445 17463 189409 50979
360 32528 7118 11278 112898 17764
400 14366 5194 9775 68852 6992
440 6132 3667 6440 40039 3406
480 3148 3523 4018 24236 2014
520 1360 3213 3056 15703 2114
560 731 2460 2247 8663 1534
600 753 1495 1404 6344 1245
640 422 1276 1236 4234 1547
680 546 834 1079 2860 1541
720 929 746 1456 2152 1321
760 779 366 1021 1962 1030
800 2136 206 611 1898 582
840 923 158 625 2432 402
880 465 190 234 2118 125
920 257 216 564 1741 552
960 0 414 347 1388 331
1000 150 419 435 1061 934
1040 131 432 326 517 1649
1080 0 255 817 2815 1004
1120 0 116 965 755 645
1160 0 0 459 341 478
1200 174 0 0 228 281
1240 0 240 221 353 256
1280 0 0 0 819 1068
1320 0 446 212 591 783
1360 0 265 230 834 802
1400 144 0 0 509 560
1440 0 284 0 527 249
1480 0 285 0 722 664
1520 122 232 0 558 133
1560 144 0 319 483 0
1600 0 467 267 360 149
1640 0 0 1386 471 172
1680 148 0 259 267 0
1720-0 841 0 0
1760-0 137 0 219
1800-0 0 210 0
1840--0 105 0
1880---0 0
───────────────────────────────
(Formulation A: Vaseline alone, Formulation B: Phospholipid 1%, Formulation C: Phospholipid 2%, Formulation D: Phospholipid 6%, Formulation E: Phospholipid 16%, 0: Below detection limit,-: Not measured )

Table 4
Normal skin radioactivity (ng eq. Of NF-κB Decoy ODNs / cm ³ )
───────────────────
Depth (μm) Formulation A Water-soluble formulation A
───────────────────
40 127723 305858
80 176569 456038
120 195258 449339
160 160645 356230
200 122080 245652
240 103637 135583
280 67479 75834
320 34559 42958
360 14894 21062
400 5278 8044
440 2269 2884
480 533 1413
520 228 1130
560 165 379
600 150 492
640 202 285
680 0 399
720 0 163
760 0 163
800 0 143
840 125 143
880 220 0
920 165 0
960 0 118
1000 0 0
1040 0 0
1080 0 0
1120 0 0
1160 0 0
1200 0 0
1240 0 0
1280 0 0
1320 0 0
1360 0 0
1400 0 0
1440 0 0
1480 160 0
1520 0 0
1560 119 0
1600 0 0
1640 0 0
1680 0 0
1720 0 0
1760 0-
1800 0-
───────────────────
(Formulation A: Vaseline alone, water-soluble preparation A: sodium alginate, 0: below detection limit,-: not measured)

Table 5
Damaged skin radioactivity (ng eq. Of NF-κB Decoy ODNs / cm ³ )
───────────────────
Depth (μm) Formulation A Water-soluble formulation A
───────────────────
40 151237 332272
80 328816 599862
120 496468 744654
160 482113 905723
200 300 313 1227719
240 200318 958660
280 126664 602954
320 69206 214862
360 32528 38100
400 14366 10253
440 6132 5402
480 3148 3394
520 1360 6428
560 731 2011
600 753 3392
640 422 3124
680 546 5698
720 929 2787
760 779 2580
800 2136 4018
840 923 2534
880 465 2108
920 257 651
960 0 517
1000 150 381
1040 131 263
1080 0 121
1120 0 0
1160 0 132
1200 174 124
1240 0 0
1280 0 157
1320 0 0
1360 0 0
1400 144 991
1440 0 233
1480 0 879
1520 122 215
1560 144 1251
1600 0 609
1640 0 172
1680 148 0
1720 0 0
1760 0 0
1800 0 0
───────────────────
(Formulation A: Vaseline alone, water-soluble preparation A: sodium alginate, 0: below detection limit,-: not measured)

Table 6
Radioactivity concentration (% of dose)
─────────────────────────────
Type of preparation Normal skin Damaged skin─────────────────────────────
Formulation A 9.18 (0-640 μm) 20.36 (0-920 μm)
4.65 (121-640 μm) 11.39 (121-800 μm)
Formulation B 14.97 (0-960 μm) 24.74 (0-1120 μm)
9.35 (121-800 μm) 5.77 (121-800 μm)
Formulation C 11.07 (0-1120 μm) 39.68 (0-1160 μm)
8.47 (121-800 μm) 12.11 (121-800 μm)
Formulation D 8.95 (0-1320 μm) 73.54 (0-1680 μm)
6.23 (121-800 μm) 38.44 (121-800 μm)
Formulation E 24.51 (0-1560 μm) 48.53 (0-1520 μm)
17.86 (121-800 μm) 30.29 (121-800 μm)
Aqueous preparation A 19.30 (0-840 μm) 52.62 (0-1080 μm)
8.19 (121-800 μm) 37.03 (121-800 μm)
─────────────────────────────
(The upper part of each formulation represents the total radioactivity penetrating the skin. The lower part represents the radioactivity penetrating into the dermis.)

Moreover, when the same test was conducted on pig skin, the following results were obtained.

Table 7
Radioactivity concentration (% of dose)
─────────────────────────────
Type of preparation Normal skin Damaged skin─────────────────────────────
Formulation A 4.95 (0-880 μm) 3.35 (0-640 μm)
2.83 (121-880 μm) 2.73 (121-640 μm)
Formulation C 3.49 (0-2040 μm) 10.18 (0-960 μm)
3.08 (121-2040 μm) 4.58 (121-960 μm)
Formulation E 3.22 (0-1080 μm) 19.75 (0-1560 μm)
2.69 (121-1080 μm) 12.59 (121-1560 μm)
Aqueous preparation A 33.78 (0-1440 μm) 41.59 (0-2440 μm)
22.57 (121-1440 μm) 34.76 (121-2440 μm)
─────────────────────────────
(The upper part of each formulation represents the total radioactivity penetrating the skin. The lower part represents the radioactivity penetrating into the dermis.)

From the above, from the viewpoint of skin permeability, water-soluble preparation A (alginic acid preparation) and preparation E (Vaseline + SPC 16% preparation) are more preferable.

By using these preparations, the required amount of NF-κB decoy drug substance can be reduced to about 1/5 (0.4% concentration) of preparation E, and about 1/13 (0.15% concentration) of aqueous preparation A.

2. Cumulative skin irritation test
Cumulative skin irritation was examined when a water-soluble preparation A containing ³⁵ S-NF-κB decoy (alginic acid preparation) and an alginate base alone were administered to the back skin of rabbits for 14 days.

(1) A Japanese white female rabbit (Kbl: JW, SPF) with hair removed from the back of the animal was purchased from Minowa Production Center of Kitayama Labes Co., Ltd. The age at the time of drug administration was 17 weeks and the body weight was 3.10 to 3.74 kg.

(2) Preparation of application site and application For each animal, the normal site was provided with two damaged sites, and the administration site was set so that the number of each administered substance was six.

On the day before the start of administration, the fine hair on the back is removed with an electric clipper, and four administration sites are used according to the Draize method (Draize, JH, et al, J. Pharmacol. Exp. Ther., 82, 377-390 (1944)). Set (see FIG. 5). Based on the method of Fukawa et al. (Fukawa Kazunaga et al., Pharmaceutical Journal 102, 89-98 (1982)), two points that are point-symmetric are treated with normal shaving. And stripped with cellophane tape to give damaged skin. As for the test preparations A to D, 0.2 g was weighed and spread evenly on a lint cloth cut to 2.5 × 2.5 cm and applied to the skin, and a 5 × 5 cm taping tape was applied and fixed thereon. Further, the covering was fixed using a cloth cover (stocknet, ALCARE) and a tube-type net bandage (pressnet, ALCARE). With regard to the aqueous preparation base and aqueous preparation A, 0.2 mL was administered using a pipette and a spatula while confirming penetration into the skin, and then lint cloth and 5 × 5 cm taping tape cut to 2.5 × 2.5 cm from above. Was affixed and fixed. Covering and fixing with a cloth cover and a tube-type net bandage were similarly performed. After the administration was completed, the lint cloth and taping tape were removed, and the administration site was wiped with slightly warm water.

On the first day of administration, administration was started by setting the dosage of preparations A to D to 0.5 g, and the dosage of alginic acid base material and aqueous preparation A to 0.5 mL. However, administration should be performed in a 2.5 × 2.5 cm section. It was difficult. Therefore, the dosages were changed to 0.2 g and 0.2 mL, which were the maximum doses, from the second day of administration for the preparations A to D and from the middle of the first administration day for the alginate base material and the aqueous preparation A, respectively. For animal No. 13, 0.5 mL of the alginate base material was administered to normal skin and damaged skin on the first day of administration, 0.5 mL of water-soluble preparation A was applied to normal skin, and 0.2 mL was applied to damaged skin. Other animals received 0.2 mL of both alginic acid base and water-soluble preparation A in both normal and damaged skin from the first day of administration.

(3) Determination of irritation Macroscopic determination was performed before daily administration and at the end of final administration (1 hour after removal of administered substance). Judgment was carried out in accordance with Draize's criteria for erythema (scab formation) and edema at the administration site. Judgment results were recorded as scores based on the Draize criteria. Furthermore, it was divided into normal skin and damaged skin, and the average value and standard deviation were calculated for erythema (scab formation), edema and their total score at each observation time point. The lower the score, the less irritating.

Results) Alginic acid base alone (water-soluble preparation A only) and water-soluble preparation A (alginic acid preparation) did not show irritation to normal rabbit skin. Erythema and edema developed in the damaged skin, but the extent was mild, and it was a transient change that recovered even after continued administration. (See Table 8, Figs. 6-7)

Provided is a nucleic acid skin external preparation having high skin permeability and low irritation.

It is the graph showing the result of the skin permeability test of NF-κB decoy oligonucleotide using rat normal skin. Formulations A, B, C, D, E are compared. It is the graph showing the result of the skin permeability test of NF-κB decoy oligonucleotide using rat damaged skin. Formulations A, B, C, D, E are compared. It is the graph showing the result of the skin permeability test of NF-κB decoy oligonucleotide using rat normal skin. Formulation A and water-soluble formulation A are compared. It is the graph showing the result of the skin permeability test of NF-κB decoy oligonucleotide using rat damaged skin. Formulation A and water-soluble formulation A are compared. The preparation part of the application | coating part by the Draize method used in the skin irritation test of a rabbit is shown. It is the figure which looked at the rabbit back from the top. It is the graph which represented the average value of the erythema and edema score of the administration site | part of NF- (kappa) B decoy oligonucleotide using a rabbit over time. Comparison is made when water-based preparation A alone (no nucleic acid) is administered to normal and damaged skin. Error bars in the figure represent standard deviation. It is the graph which represented the average value of the erythema and edema score of the administration site | part of NF- (kappa) B decoy oligonucleotide using a rabbit over time. The case where water-soluble preparation A is administered to normal skin and damaged skin is compared. Error bars in the figure represent standard deviation.

Claims (10)

Into cells of the dermis or epidermis tissue, decoy (decoy molecules), antisense, ribozyme, for delivering a nucleic acid based agents selected from an aptamer or siRNA, pharmaceutical compositions comprising said nucleic acid-based drugs and hydrogenated soybean phospholipid object. Decoy has transcriptional factor inhibitory action, The composition of claim 1. The composition according to claim 1 or 2 , wherein the decoy is NF-κB, STAT-1, GATA-3, STAT-6, AP-1, Ets, E2F decoy oligonucleotide. The composition according to claims 1 to 3 , wherein the decoy is an NF-κB decoy oligonucleotide represented by SEQ ID NO: 1. A nucleic acid-based drug containing 0.01 to 5 wt%, claims 1 to 4 composition. Comprising a nucleic acid-based agent 0.1-1 wt%, claims 1 to 5 composition. The composition according to any one of claims 1 to 6 , comprising 0.1 to 20% by weight of hydrogenated soybean phospholipid. The composition according to any one of claims 1 to 7 , comprising 1 to 16% by weight of hydrogenated soybean phospholipid. Prevention of claims 1 to skin disorders comprising a composition of 8, wherein, improving the therapeutic agent. Skin diseases are atopic dermatitis, contact dermatitis, photosensitivity dermatitis, chronic dermatitis of hands and feet, seborrheic dermatitis, monetary dermatitis, generalized exfoliative dermatitis, congestive dermatitis, The preventive, ameliorating, or therapeutic agent according to claim 9 , which is local abrasion dermatitis, drug dermatitis or psoriasis.

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