KR20170051399A - Composition for skin permeation comprising cationic molecular transporters and anionic bioactive substance - Google Patents

Abstract

The present invention relates to a skin-permeating composition for delivering biologically active substances into skin, by containing a cationic compound and an ion assembly to which biologically active anionic substances are bonded via an ionic bond. According to the present invention, it is possible to deliver, to dermal layers or skin layers, various types of biologically active anionic substances such as diagnosis reagents, drugs, and fluorescent substances by allowing the biologically active anionic substances to easily pass through cellular membranes and skin membranes.

Description

TECHNICAL FIELD [0001] The present invention relates to a skin permeable composition comprising a cationic molecular transporter and an anionic physiologically active substance,

The present invention relates to a composition for permeating skin comprising a cationic molecular transporter and an anionic physiologically active substance and delivering the physiologically active substance into the skin, specifically, a part of the epidermal layer and the dermal layer.

The basic building block of all living organisms is a cell, which consists of the cytoplasm where the intracellular organelles are present, and the cell membrane that protects it and at the same time forms the boundary with the external environment. Cell membranes with selective mass permeability have a mosaic pattern of flow proteins in the bilayer of phospholipids, and therefore there are many limitations in the passage of substances with useful therapeutic activity through cell membranes. In particular, hydrophilic molecules, macromolecules such as highly charged molecules, peptides and oligonucleotides (eg, nucleic acids and genes) that are low in molecular weight are difficult to pass through the cell membrane, and therefore, a separate method for transporting these molecules into cells (See, for example, S. Futaki, Adv. Drug Delivery Rev. 2005, 57, 547-558 and PA Wender, Adv. Drug Delivery Rev. 2008, 60, 452-472) , And actually cause cytotoxicity.

Meanwhile, various molecular transporters capable of transferring physiologically active substances have been developed (SK Chung, et al. Int . J. Pharmaceutics , 2008, 354, 16-22); [KK Maiti, et al. Angew . Chem ... Int Ed 2007, 46 , 5880-5884];..... [KK Maiti, et al Angew Chem Int Ed 2006, 45, 2907-2912]; Republic of Korea Patent No. 10-0578732 call; claim 10-0699279 10-0849033, 10-1021078, etc.).

However, attempts have not been made to transfer water-soluble anionic physiologically active substances into the skin using such a molecular transporter. Accordingly, the present inventors have found that, in order to deliver a physiologically active substance having a phosphate group or a carboxylate group, such as salicylic acid, well known as ascorbic acid phosphate or a nonsteroidal antiinflammatory agent, The ion conjugate was prepared by ion-binding with the reported molecular transporter at a specific ratio. The present inventors completed the present invention by confirming that the ion conjugate thus prepared can improve the skin permeability.

[Patent Document 1] Korean Patent No. 10-0578732

[Patent Document 2] Korean Patent No. 10-0699279

[Patent Document 3] Korean Patent No. 10-0849033

[Patent Document 4] Korean Patent No. 10-1021078

[Non-Patent Document 1] S. Futaki, Adv . Drug Delivery Rev. 2005, 57, 547-558

[Non-Patent Document 2] PA Wender, et al . Adv . Drug Delivery Rev. 2008, 60, 452-472

[Non-Patent Document 3] SK Chung, et al . Int . J. Pharmaceutics , 2008, 354, 16-22

[Non-Patent Document 4] KK Maiti, et al . Angew . Chem . Int . Ed . 2007, 46, 5880-5884

[Non-Patent Document 5] KK Maiti, et al . Angew . Chem . Int . Ed . 2006, 45, 2907-2912

Accordingly, an object of the present invention is to provide a skin permeable composition for delivering a physiologically active substance into skin.

In order to accomplish the above object, the present invention provides a cationic compound represented by any of the following formulas (1) to (4): And an ionic bond in which an anionic physiologically active substance is ionically bound, and for delivering the physiologically active substance into the skin,

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above equations,

R ₁ and R ₂ are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heteroalkyl, - (CH ₂ ) mNHR ', - (CH ₂ ) _l CO ₂ R'',-COR''', -SO ₂ R '''' or a physiologically active substance to be transported, 'And R''''are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl or C ₃ -C ₈ heteroalkyl M is an integer from 2 to 5, and l is an integer from 1 to 5;

또는

이고;R ₃ is

or

ego;

이고;R ₄ is

ego;

이고;R ₅ is

ego;

n is an integer of 1 to 12;

The composition according to the present invention exhibits an excellent effect of penetrating molecules that are difficult to permeate to cell membranes or skin membranes, such as ascorbic acid phosphate, aspirin, an anionic water-soluble physiologically active substance into the skin, This can be useful for

1 to 5 show the cell membrane permeability of the ionic conjugate of the present invention prepared using biotin-FITC, IPF, UDPF and 4-ABF as physiologically active substances, respectively, by using fluorescence image (A), cell morphology image (B) (C) of a combined form of a fluorescence image and a cell type image.
FIGS. 6 and 7 show fluorescence images of the ionic conjugates prepared in various forms using biotin-FITC and 4-ABF as physiologically active substances, respectively, in the mouse skin.

The present invention relates to a cationic compound of any one of the following formulas (1) to (4): And an ionic bond in which an anionic physiologically active substance is ionically bound, and for delivering the physiologically active substance into the skin,

In the above equations,

R ₁ and R ₂ are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heteroalkyl, - (CH ₂ ) mNHR ', - (CH ₂ ) _l CO ₂ R'',-COR''', -SO ₂ R '''' or a physiologically active substance to be transported, 'And R''''are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl or C ₃ -C ₈ heteroalkyl M is an integer from 2 to 5, and l is an integer from 1 to 5;

또는

이고;R ₃ is

or

ego;

이고;R ₄ is

ego;

이고;R ₅ is

ego;

n is an integer of 1 to 12;

Hereinafter, the present invention will be described in more detail.

In the present invention, the ionic conjugate includes a cationic compound. The cationic compound may be any one of the above chemical formulas 1 to 4 and is used as a molecular transporter (refer to Korean Patent No. 10-0578732, No. 10-0699279, No. 10-0849033, No. 10-1021078, etc. ). The cationic compound has a structure in which a guanidine group or an arginine group having various side chain lengths is introduced into a sugar or sugar analog skeleton structure as shown in formulas (1) to (4), and thus has a water-soluble and excellent biological membrane permeability, It can easily pass through cell membranes and skin membranes in the form of ion-binding with various kinds of anionic physiologically active substances such as reagents, medicines, and fluorescent substances.

According to formula (1) The compound is a form in which 1 to 8 guanidine groups are introduced into a sugar alcohol derivative capable of introducing a desired functional group at a high concentration using a side chain structure of a dendrimer type in the skeleton structure. According to one embodiment of the present invention, the compound of formula (I) may be 1 to 12, for example, an alditol derivative having a stereostructure of sorbitol, mannitol or galactitol, and a salt thereof.

The compound according to formula (2) may be n-1 to 12, more preferably 8-guanidine group-introduced sorbitol derivatives and salts thereof.

The compound according to formula (3) may be n-1 to 12, more preferably sorbitol derivatives having 6 guanidine groups introduced thereto and salts thereof.

The compound according to formula (IV) may be arginine (when n is 1) or a polymer thereof (when n is any of 2 to 12), preferably n is 6 or 8.

In the present invention, the ionic conjugate includes an anionic physiologically active substance. The anionic physiologically active substance may be a water-soluble molecule containing a carboxylic acid (-CO ₂ H) group, a phosphoric acid (-OP (O) (OH) ₂ ) group or a sulfonic acid (-SO ₃ H) group. Preferably selected from ascorbic acid phosphate, vitagen, inositol phosphate (IP), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), acetylsalicylic acid, 4-aminobenzoic acid 4-aminobenzoic acid, 4-ABF), hyaluronic acid, and dextran sulfate.

The anionic physiologically active substance may be connected to a fluorescent material selected from the group consisting of fluorescein (FITC), dansyl (5-dimethylamino-1-naphthalene sulfonyl), and rhodamine.

The anionic bioactive materials according to one embodiment of the present invention are as follows:

In the present invention, the ionic conjugate is characterized in that the cationic compound: anionic physiologically active substance is ionically bonded at a ratio of 1: 1 to 4: 1 based on the charge. The charge ratio of the cationic molecular transporter compound and the anionic physiologically active substance is very important in the production of ionic conjugates for molecular transfer. When the binding ratio of the cationic molecular carrier: anionic physiologically active substance is 1: 1 or more, the number of guanidine groups per molecule is appropriate due to the formation of an ionic bond with the substrate, and the cell permeability is improved and the binding ratio is 4: , It is possible to increase the rate of permeation of the ionic conjugate through the cell membrane because the excess free free molecular form of the molecular carrier can be prevented from permeating through the cell membrane by forming an ionic bond with the substrate, ) Can be improved.

Accordingly, in the present invention, an ionic compound in which a cationic compound: an anionic physiologically active substance is ionically bonded at a ratio of 2: 1 based on the charge is more preferable (Fig. 1).

The skin permeable composition according to the present invention is characterized in that it penetrates into the skin, preferably to a depth of 125 to 200 μm, for example, 150 to 200 μm. This depth corresponds to the penetration of the entire stratum corneum and the viable epidermis layer and the dermis layer. Therefore, the composition for penetrating the skin according to the present invention can transmit the physiologically active substance through intracellular or subcutaneous [for example, between the skin epidermis and the dermis) (Test Example 2, and Figs. 6 and 7 ), And can significantly improve delivery of functional cosmetics or skin disease treatment agents. In addition, subcutaneous administration can be useful for transporting macromolecules such as proteins and genes as well as various kinds of therapeutic or diagnostic drugs required.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Manufacturing example  One: Fluorescently labeled Anionic  Preparation of Substrate

<1-1> Vitagen - FITC's  Produce

Was dissolved in a mixture of dimethylformamide and water (2.5: 1 (v / v)) (2 ml), to which was added fluorosine-5-isocyanate (160 mg, 0.418 mmol) Amine (300 L, 2.158 mmol), and the mixture was stirred at room temperature for 1 day. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (chloroform: ethanol: acetic acid = 16: 3: 1 -> dichloromethane: methanol = 1: 1). DOWEX 50WX8-400 ion exchange resin , And the pH was adjusted to 8-10 with 1N NaOH and lyophilized to obtain a yellow powdery compound (240 mg).

<1-2> FITC -4-aminobenzoic acid (4- ABF )

4-Aminobenzoic acid (50 mg, 0.3308 mmol) was dissolved in tetrahydrofuran and ethanol mixture (3: 2 v / v) (5 ml), and fluorocaine-5-isocyanate (154 mg, 0.397 mmol) Triethylamine (69 L, 0.4962 mmol) was added and stirred at room temperature for 1 day. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and then purified by column chromatography (dichloromethane: methanol = 10: 1) to obtain a yellow powdery compound (145 mg).

Example 1: Preparation of ionic conjugates containing cationic compounds of sorbitol skeleton having 8 guanidine groups

&Lt; 1-1 & gt ; Preparation of an ionic conjugate containing a cationic compound of sorbitol skeleton having 8 guanidine groups and non - GFP - FITC

(Hereinafter referred to as "sorbitol-based G8 molecular transporter" or "SG8") having sorbitol skeleton and 8 guanidine groups (+8; one positive charge per guanidine group) ) Was prepared according to the method described in Example 1 of Korean Patent No. 10-0699279. Next, 52 mg (30 μmol) of SG8 prepared above was dissolved in 500 μL of tertiary distilled water to prepare a cationic stock solution.

On the other hand, 84 mg (120 μmol) of Vitagen-FITC (-1; one phosphate is one negative charge) corresponding to the physiologically active substance of the preparation example <1-1> was dissolved in 2 mL of tertiary distilled water, .

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<1-2> SG8 and myo - Inositol-1P- FITC  &Lt; / RTI &gt;

A cation stock solution containing SG8 was prepared in the same manner as described in <1-1> above.

On the other hand, 92 mg (120 μmol) of myo -inositol-1P-FITC (hereinafter referred to as "IPF") (-1; one negative charge for one phosphate group) corresponding to the physiologically active substance was dissolved in 2 mL of tertiary distilled water (KC Seo et al . Journal of Carbohydrate Chemistry 2007 , 26 , 305-327)

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<1-3> SG8 and UDP - carba - GlcNAc - FITC  ( UDPF ) &Lt; / RTI &gt;

A cation stock solution containing SG8 was prepared in the same manner as described in <1-1> above.

On the other hand, 66 mg (60 μmol) of UDP-carba-GlcNAc-FITC (hereinafter referred to as "UDPF") corresponding to physiologically active substance (-2; one negative charge for two phosphate groups) was dissolved in 1 mL of tertiary distilled water (KC Seo et al . Chemical Communications , 2009 , 1733-1735)

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<1-4> SG8 and UDPF  &Lt; / RTI &gt;

Cationic compound: The anionic physiologically active substance was carried out in the same manner as in Example <1-3>, except that the anionic physiologically active substance was ion-bonded at a charge ratio of 1: 1, Ion-bonded ionic conjugate was obtained at a charge ratio of 1: 1.

&Lt; 1-5 > UDPF  &Lt; / RTI &gt;

Cationic compound: The anionic physiologically active substance was carried out in the same manner as in Example <1-3> except that the anionic physiologically active substance was ion-bonded at a charge ratio of 4: 1, Ion-bonded ionic bonds were obtained at a charge ratio of 4: 1.

Example 2: Preparation of ionic conjugates containing cationic compounds of sorbitol skeleton having 6 guanidine groups

&Lt; 2-1 & gt ; Preparation of an ionic conjugate containing a cationic compound of sorbitol skeleton having six guanidine groups and non - GFP - FITC

(Hereinafter referred to as "sorbitol-based G6 molecular transporter" or "SG6") having 6 guanidine groups (+6; one positive charge per guanidine group) Quot;) was prepared according to the method described in Example 8 of Korean Patent No. 10-0699279. Subsequently, 40 mg (30 μmol) of SG6 prepared above was dissolved in 500 μL of tertiary distilled water to prepare a cationic stock solution.

Meanwhile, 63 mg (90 μmol) of Vitagen-FITC prepared in the same manner as described in <1-1> was dissolved in 1.5 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

&Lt; 2-2 > IPF  &Lt; / RTI &gt;

A cation stock solution containing SG6 was prepared in the same manner as described in <2-1> above.

Meanwhile, 69 mg (90 μmol) of IPF prepared in the same manner as described in <1-2> was dissolved in 1.5 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<2-3> SG6 and UDPF  &Lt; / RTI &gt;

A cation stock solution containing SG6 was prepared in the same manner as described in <2-1> above.

Meanwhile, 50 mg (45 μmol) of UDPF prepared in the same manner as described in <1-3> was dissolved in 750 μL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

Example  3: Cationic  Arginine Octamer  Preparation of ionic conjugates containing compounds

<3-1> Arginine- Octamer  And Vitagen - FITC  &Lt; / RTI &gt;

The arginine-octamer (n = 8; +8) cationic compound (hereinafter referred to as "ARG8") according to Formula 4 of the present invention was obtained from Peptron. Then, 61 mg (30 μmol) of ARG8 was dissolved in 500 μL of tertiary distilled water to prepare a cation stock solution.

On the other hand, 84 mg (120 μmol) of Vitagen-FITC prepared in the same manner as described in <1-1> was dissolved in 2 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<3-2> ARG8  And IPF  &Lt; / RTI &gt;

A cation stock solution containing ARG8 was prepared in the same manner as described in <3-1> above.

On the other hand, 92 mg (120 μmol) of IPF prepared in the same manner as described in <1-2> was dissolved in 2 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<3-3> ARG8  And UDPF  &Lt; / RTI &gt;

A cation stock solution containing ARG8 was prepared in the same manner as described in <3-1> above.

Meanwhile, 66 mg (60 μmol) of UDPF prepared in the same manner as described in <1-3> was dissolved in 1 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<3-4> ARG8  And FITC -4-aminobenzoic acid &lt; / RTI &gt;

A cation stock solution containing ARG8 was prepared in the same manner as described in <3-1> above.

On the other hand, 65 mg (120 μmol) of FITC-4-aminobenzoic acid (hereinafter referred to as "4-ABF") -1 (one carboxylate) corresponding to the physiologically active substance of Production Example <1-2> Was dissolved in 2 mL of tertiary distilled water containing 10% DMSO to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

Example  4: Cationic  Arginine Hexamer  Preparation of ionic conjugates containing compounds

<4-1> arginine Hexamer  And Vitagen - FITC  &Lt; / RTI &gt;

An arginine hexamer (n = 6, +6) cationic compound (hereinafter referred to as "ARG6") according to Formula 4 of the present invention was obtained from Peptron. 46 mg (30 μmol) of the prepared ARG6 was dissolved in 500 μL of tertiary distilled water to prepare a cation stock solution.

Meanwhile, 63 mg (90 μmol) of Vitagen-FITC prepared in the same manner as described in <1-1> was dissolved in 1.5 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<4-2> ARG6  And IPF  &Lt; / RTI &gt;

A cation stock solution containing ARG6 was prepared in the same manner as described in <4-1> above.

Meanwhile, 69 mg (90 μmol) of IPF prepared in the same manner as described in <1-2> was dissolved in 1.5 mL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

<4-3> ARG6  And UDPF  &Lt; / RTI &gt;

A cation stock solution containing ARG6 was prepared in the same manner as described in <4-1> above.

Meanwhile, 50 mg (45 μmol) of UDPF prepared in the same manner as described in <1-3> was dissolved in 750 μL of tertiary distilled water to prepare an anion stock solution.

The anion solution prepared above was slowly added dropwise to the cationic solution, and the mixture was stirred for about 2-3 hours until the turbid solution became clear. Then, the mixture was lyophilized to obtain an ionic compound in which cationic compound: anionic physiologically active substance was ion-bonded at a charge ratio of 2: 1.

Test Example  1: Measurement of cell membrane permeability

In order to confirm the cell membrane permeability of the ionic binders prepared in the above examples, the fluorescence of FITC labeled with a physiologically active substance (substrate) was measured with a Confocal Laser Scanning Microscope (Olympus FV1000).

First, HeLa cells (ATCC ^® CCL-2 ™) were cultured on a dish plate. At this time, the cells were stabilized in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS for 24 hours, and cultured for 24 hours in serum-free medium for cell starvation Respectively. Then, the ionic conjugates prepared in Examples 1-1 to 4-3 were treated at a concentration of 48 μM (substrate concentration) for 1 hour at 37 ° C., washed five times with PBS (phosphate buffer solution) Membrane permeability was measured with a confocal microscope. At this time, Vitagen-FITC, IPF, UDPF and 4-ABF were used as control groups, respectively.

An Ar laser (wavelength: 488 nm) was used to excite the fluorescent material, and the magnification was observed after magnification of 60 times in total, and the results are shown in FIGS. 1 to 5. In FIGS. 1 to 5, the column A is the fluorescence of the cell after the sample treatment, the column B is the morphology image (DIC) of the cell after the sample treatment, and the column C is the image superimposed on the fluorescence image and the cell morphology image merge).

1 (1) to (4) in FIG. 1 are respectively a graph showing the results of the comparison between the control group (UDPF), the ionic coupler (1: 1) of Example <1-4>, the ionic coupler (4: 1) of Example < 1-5 >.

1, the fluorescence signal of the ionic conjugate according to the present invention (A- (2) to A- (4) in FIG. 1) (A- (1) of FIG. 1), and the fluorescent signal observed in the cells of the ionic bond (A- (3) in FIG. 1) having a binding ratio of cation: anion of 2: . Therefore, in the present invention, the optimal charge ratio for formation of an ionic bond is 4: 1, preferably 2: 1 for the cation: anion. From the above results, if the ratio of the molecular transporter (cation) is too low, the number of guanidine groups per molecule is relatively lowered due to the formation of ionic bonds with the substrate, so that the cell permeability is lowered. On the contrary, If the molecular weight is high, the excess free - form molecular transporter that competes with the substrate to form an ionic bond penetrates the cell membrane, so that the cell membrane permeation rate of the ionic conjugate is lowered.

(1) to (5) in FIG. 2 are respectively the same as the control group (butagen-FITC), the ionic conjugate of Example <4-1>, the ionic conjugate of Example <3-1> Ion conjugate and the ionic conjugate of Example < 1-1 > were treated on the cells.

As shown in FIG. 2, in the case of the ionic conjugate (A- (2) to A- (5) in FIG. 1), only intracellular fluorescence signals A stronger fluorescence signal than that of A- (1) in Fig. 1 was observed. This is due to the formation of ionic bonds between the anion of the phosphate group of the butazene corresponding to the substrate and a part of the guanidine group cation of the cationic compound, which is a molecular transport, and the enhancement of cell membrane permeability by the residual guanidine group of the molecular transporter.

(1) to (5) in FIG. 3 are respectively the same as the control group (IPF), the ionic conjugate of Example <4-2>, the ionic conjugate of Example <3-2>, the ionic conjugate of Example < The image of the case where the ion-binding substance of Example <1-2> was treated on the cells.

As shown in FIG. 3, in the case of the ionic conjugate (A- (2) to A- (5) in FIG. 2), intracellular fluorescence signals A- (1)). This is due to the formation of ionic bonds between the anion of the phosphate group of the IPF corresponding to the substrate and a part of the guanidine group cation of the cationic compound, which is a molecular transport, and the enhancement of cell membrane permeability by the remaining guanidine group of the molecular transporter.

(1) to (5) of FIG. 4 are respectively a graph showing the results of measurement of the ionic conductivity of the ionic compound of Example <4-3>, the ionic compound of Example <3-3>, the ionic compound of Example <2-3> The image of the case where the ion-binding substance of Example < 1-3 > is treated on the cell.

As shown in FIG. 4, in the case of the ionic conjugate according to the present invention (A- (2) to A- (5) in FIG. 3), only the UDPF substrate was treated with cells, A- (1)). This is due to the formation of ionic bonds between the anion of the phosphate group of UDPF corresponding to the substrate and a part of the guanidine group cation of the cationic compound, which is a molecular transport, and the enhancement of cell membrane permeability by the residual guanidine group of the molecular transporter.

FIGS. 5 (1) and (2) show images obtained by treating the cells with the ionic conjugates of the control group (4-ABF) and the example <3-4>, respectively.

As shown in FIG. 5, in the case of the ionic conjugate according to the present invention (A- (2) in FIG. 4), only intracellular fluorescence signals (A- )) Was observed. This is due to the formation of an ionic bond between the anion of the carboxyl group of 4-ABF corresponding to the substrate and a part of the guanidine group cation of the cationic compound, which is a molecular transport, and the enhancement of cell membrane permeability by the residual guanidine group of the molecular transporter.

Test Example  2: Measurement of permeation and distribution into the skin of rats

In 26 mg of the ionic conjugate of ARG8 and non-FITC prepared in Example <3-1>, lettuce-FITC was taken as 16 mg based on the substrate and dissolved in 84 μL of water. Then, 300 mg of PEG 400 was mixed with 4% A sample solution was prepared. In addition, 4% sample solutions were prepared in the same manner as above using the ionic conjugates prepared in Examples <1-1> and <3-4>, respectively.

After 4-week-old BALB / c nude mice were anesthetized with gas, 50 μL of the sample solution prepared above was applied to the skin of the hind paw to 1 cm × 1 cm area, and left in the dark room for 3 hours. After 3 hours, the legs were washed with distilled water and 70% ethanol and euthanized with carbon dioxide. Then, the thigh skin tissue was peeled off, placed on a glass slide, fixed with a cover slip, and then percutaneous permeability of each slide sample was observed with a two-photon laser scanning microscope (Leica). Using a femtosecond laser (wavelength 960 nm) to excite the fluorescent material, the subcutaneous layer was continuously photographed at a depth of 2 μm from the skin surface and images of representative specific depths (45 μm and 90 μm) The results are shown in Figs. 6 and 7, respectively.

(B) is an ionic conjugate of 4% ARG8 and Vitagen-FITC, (C) is 4% SG8, and Vitagen-FITC (B) is an ionic bond of 4% ARG8 and 4-ABF, (C) is 4% of 4-ABF, SG8 &lt; / RTI &gt; and 4-ABF.

As commonly seen in FIGS. 6 and 7, when compared with the control group, non-FITC or 4-ABF, the ionic conjugate of the present invention in which each substrate and the molecular transporter compound are ion- It was well permeated. In addition, it can be observed that the intensity of fluorescence is weakened by gradually decreasing the amount of physiologically active substance as the imaging depth is deepened. According to the results of percutaneous permeation measured with a two-photon laser scanning microscope (Leica), it is observed that the physiologically active substance having fluorescence penetrates to a depth of at least 125 to 200 μm, and this depth corresponds to the stratum corneum ) And the entire surface of the viable epidermis layer and the dermis layer.

From the above series results, it can be seen that the ionic conjugates prepared according to the present invention exhibit a high cell membrane and subcutaneous permeability. Accordingly, the present invention can be usefully used to deliver a water-soluble physiologically active substance having anion to a cell or subcutaneously (skin layer or dermal layer).

Claims (5)

A cationic compound of any of formulas (2) to (4); And
An anionic physiologically active substance containing a carboxylic acid group (-CO ₂ H) or a phosphate group (-OP (O) (OH) ₂ ) is directly ion-bonded at a ratio of 2: 1 to 4: A composition for transdermal administration for applying the composition to skin to deliver the physiologically active substance into the skin,
(2)

(3)

[Chemical Formula 4]

In the above equations,
R ₄ is

ego;
R ₅ is

;
n is an integer of 1 to 8; The method according to claim 1,
Wherein the anionic physiologically active substance is selected from the group consisting of ascorbic acid phosphate, vitagen, inositol phosphate (IP), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), acetylsalicylic acid, Wherein the composition is selected from the group consisting of 4-aminobenzoic acid (4-ABF), hyaluronic acid, and dextran sulfate. The method according to claim 1,
Wherein the anionic physiologically active substance is connected to a fluorescent substance selected from the group consisting of fluorescein (FITC), dansyl (5-dimethylamino-1-naphthalene sulfonyl) and rhodamine , A composition for transdermal administration. The method according to claim 1,
Wherein the composition for percutaneous administration is permeated between the skin epidermal layer and the dermal layer. 5. The method of claim 4,
Wherein the composition for percutaneous administration is permeated through the skin to a depth of 125 to 200 mu m.

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