WO2005088299A1 - Method of assaying dermal permeability of transdermal drug mediated by dermal transporter - Google Patents

Abstract

A method of assaying the dermal permeability of a transdermal drug in which the degree of dermal permeability of transdermal drug mediated by a dermal transporter is measured and assessed; and a method of screening a substance capable of enhancing or suppressing the dermal permeability of transdermal drug mediated by a transporter. A chamber in which an epidermatic side and subcutaneous tissue side of skin segment are partitioned from each other is provided. A propylene glycol containing solution in which a transdermal drug, such as indomethacin, is dissolved is injected to the epidermatic side, while a solution containing any of glucose and other energy sources, such as Hanks solution, is injected to the subcutaneous tissue side. After the passage of given period of time under conditions suitable for living of the skin segment, preferably with the solution of subcutaneous tissue side maintained at body temperature while the solution of epidermatic side maintained at room temperature, the degree of dermal permeability of transdermal drug mediated by a dermal transporter is measured and assessed.

Description

 Specification

 Method for assaying skin permeability of transdermal drug via skin transporter

 [0001] The present invention relates to a method for assaying skin permeability of a transdermal drug or a candidate transdermal drug, and more particularly, to a method for transdermal drug or transdermal drug delivery using a chamber sectioned on the skin section into a subcutaneous tissue side and an epidermal side. The present invention relates to a method for measuring skin permeability of a transdermal drug or a transdermal candidate drug, which measures and evaluates the degree of skin permeability of a skin candidate drug through a skin transporter (a transporter provided in skin tissue).

 Background art

 [0002] Transdermal drug delivery is often used as a route of administration for potent low molecular weight therapeutic agents, and avoids first-pass metabolism, reduces pain, reduces drug delivery, and reduces the likelihood of conventional dosage forms such as tablets and injections. It has excellent effects, including the possibility of sustained release. For example, transdermal administration of nonsteroidal anti-inflammatory drugs (NSAIDs) has been introduced to avoid disadvantages of the oral route, such as gastrointestinal inflammation and ulceration (e.g., J. Pharm.Sci.

 86: 503-508, 1997). However, transdermal delivery has limited applications due to low skin penetration.

 [0003] The skin is a physical and biochemical, both-sided barrier. The outermost layer of skin, the stratum corneum (SC), has been described as the largest barrier to the penetration of various substances due to its physical structure. In addition, xenobiotic metabolizing enzymes in the skin are the second biochemical noria. For example, the expression and activity of daltathione S-transferase (GST) P1-1 has been detected in human epidermal cells (see, for example, J. Dermatol. Sci. 30: 205-214, 2002). Expression of xenobiotic metabolizing enzymes has also been detected in normal human keratinocytes and constitutive activity has been observed (eg, J. Invest. Dermatol. 102: 970-975, 1994; J. Biol. Chem. 273: 32071). -32079, 1998, J. Invest. Dermatol. 112: 337-342, 1994).

[0004] In recent years, the expression of several transporters in the skin has been reported. Neuronal glutamate transporter EAAC1 and glialglutamate transporter (GLT-1 ) Is detected in keratinocytes (see, for example, J. Invest. Dermatol. 112: 337-342, 1994), and sodium-dependent multivitamin transporter (SMVT) has been found to be expressed in skin (eg, J. Invest. Dermatol. 120: 428-433, 2003). Since glutamate and biotin are essential for normal cell function, the presence of such transporters has been shown to support a role for substrates in the skin. On the other hand, the expression of a xenobiotics transporter has recently been reported. They transport a number of organic ion drugs and are present in normal human keratinocytes, multidrug metabolism-related proteins (MRPs) (see, for example, J. Invest. Dermatol. 116: 541-548, 2001) and Includes organic-one transport polypeptide (OATP) family members (see, for example, J. Invest. Dermatol. 120: 285-291, 2003). Expression of such transporters in the skin may also be an active factor in the transdermal penetration of drugs. However, the involvement of transporters in the transdermal penetration of drugs has not yet been clarified.

[0005] On the other hand, indomethacin, which is an NSAID, is transdermally administered and used as a hydrophobic model conjugate for investigating a novel enzyme in transdermal delivery (for example, see J. Pharm. Sci. 84: 482-488, 1995; J. Control Release 90: 335-343, 2003; J. Control Release 88: 243-252, 2003). Indeed, several approaches have been taken to promote its efficient transdermal penetration (J. Control Release 75: 155-166, 2001; Biol. Pharm. Bull. 25: 779-782, 2002) ₀ Manthol has also been used as a paracellular marker.

 [0006] An object of the present invention is to clarify that carrier-mediated transport is involved in transdermal permeation of a therapeutic agent, and then through a skin transporter of a transdermal drug or a transdermal candidate drug. An object of the present invention is to provide a method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug, which measures and evaluates the degree of skin permeability in all cases.

[0007] The present inventors used indomethacin, an NSAID transdermally delivered, as a hydrophobic model drug in order to investigate the characteristics of the mechanism of transdermal delivery. ^[14 C] Indometa syn and [] Man - transdermal penetration of the tall, both forces of absorption and secretion were also measured with time, transdermal transport of ^[14 C] Indomethacin is a paracellular marker [] Man - Thor Ri, found that high Nari force found that ^[14 c] Transdermal transport capacity of indomethacin, a name tool transcellular transport in paracellular transport. Also, ^[14 c] indomethacin and [¾] Man - comparing the preparative Lumpur on permeate unlabeled indomethacin effect was investigated whether transdermal penetration saturation of ^[14 c] indomethacin. When unlabeled indomethacin (500 M) is added to calories, [ ¹⁴ c] the power to increase the permeability of indomethacin in the secretion direction [¾] It is clear that it does not affect the permeability of mannitol, It was found that the effect was specific to [ ¹⁴ c] indamecin, and that the saturable process was involved in the transdermal permeability of [ ¹⁴ c] indomethacin. Furthermore, the effect of NaN and NaF was observed to determine whether the saturation of [ ¹⁴ C] indomethacin is energy-dependent.

 Three

. As a result, the penetration of [ ¹⁴ C] indomethacin was also increased by NaN and NaF,

 Three

We found that the osmotic step in the direction was ATP-dependent. These results on unidirectional, ATP-dependent and saturable penetration of [ ¹⁴ C] indomethacin indicated that transporter (s) may be involved in the transdermal penetration of indomethacin . We also compared the expression of various foreign transporters in hairless mouse skin and normal human skin. The present invention has been completed based on the above findings.

 Disclosure of the invention

 [0008] That is, the present invention provides (1) a solution in which a transdermal drug or a candidate transdermal drug is dissolved is injected into one of the chambers divided into a subcutaneous tissue side and an epidermis side by a skin section, On the other hand, a predetermined solution is injected, and the degree of skin permeability of the transdermal drug or the candidate transdermal drug through the skin transporter is measured and evaluated after a predetermined time under the survival condition of the skin section. And (2) maintaining the solution on the subcutaneous tissue side at body temperature and the solution on the epidermis side at room temperature, and after a predetermined period of time, Measuring the degree of skin permeability of the transdermal drug candidate through the skin transporter; evaluating the skin permeability of the transdermal drug or the transdermal drug candidate according to (1) above, About.

[0009] Further, in the present invention, (3) the measurement of the degree of skin permeability "evaluation is one or more measurements of saturation, inhibition effect, directionality and energy dependence of skin penetration". Especially The skin permeation test method of the transdermal drug or transdermal candidate drug described in (1) or (2) above, or (4) radioactive isotope or fluorescent substance as the transdermal drug or transdermal candidate drug The method for assaying skin permeability of a transdermal drug or a transdermal candidate drug described in any of (1) to (3) above, wherein a labeled transdermal drug or a transdermal candidate drug is used, (5) Transdermal drug As a solution in which a drug or a transdermal candidate drug is dissolved, a solution containing an energy source in which the subcutaneous tissue or a transdermal drug candidate is dissolved in the subcutaneous tissue side, and a transdermal drug or a transdermal drug candidate in the epidermis side (1)-(4), wherein the method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug is described in (1)-(4) above, ) Polyhydric alcohol power The transdermal drug according to the above (5), which is propylene glycol. Is the method of assaying the skin permeability of a candidate transdermal drug, and (7) the energy of a solution in which a transdermal drug or a transdermal candidate drug is dissolved dissolves a transdermal drug or a candidate transdermal drug on both the subcutaneous tissue side and the epidermis side. (1) a method for assaying the skin permeability of the transdermal drug or the candidate transdermal drug described in (1)-(4), wherein the solution contains an energy source. (5) The method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug according to any one of the above (5) to (7), which is a Hanks solution; The drug or transdermal candidate drug is used for both the subcutaneous tissue side and the epidermis side, and the transcutaneous drug labeled with a radioisotope or a fluorescent substance or the transdermal drug candidate is examined for saturated transdermal penetration. The method for assaying the skin permeability of a transdermal drug or a transdermal drug as described in any of (1) to (8) above, (10) As described in (1)-(8) above, which is characterized by changing the pH of the solution on the epidermis side, the method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug described in ) NaN and NaF were used for both the subcutaneous tissue side and the epidermis side,

 Three

The penetration of the transdermal drug or the transdermal candidate drug according to any one of the above (1)-(8), characterized in that it is examined whether the penetration of the transdermal drug or the transdermal candidate drug is energy-dependent. It relates to a method for testing sex.

Further, the present invention relates to (12) a solution in which a transdermal drug or a transdermal candidate drug is dissolved, and a solution in which a transdermal drug or a transdermal candidate drug is dissolved in a test substance, and a subcutaneous tissue side of the skin slice. The percutaneous drug or the percutaneous candidate drug is injected into the subcutaneous tissue side in one chamber partitioned into the epidermis side and a predetermined solution is injected into the other, and after a predetermined time under the conditions for survival of the skin section, Skin permeability of a transdermal drug or a transdermal candidate drug characterized by measuring the degree of skin permeability of the agent via the skin transporter and comparing and evaluating the degree of skin permeability (13) maintaining the solution on the subcutaneous tissue side at body temperature and the solution on the epidermis side at room temperature, and after a predetermined time, permeation of the transdermal drug or the candidate transdermal drug through the skin transporter The present invention relates to the method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or a transdermal candidate drug according to the above (12), wherein the degree of sex is measured.

The present invention is also characterized in that (14) the measurement of the degree of skin permeability 'evaluation is one or more measurements of the saturation, inhibition effect, directionality and energy dependence of skin penetration' evaluation. And (15) a radioactive isotope as a transdermal drug or a transdermal candidate drug as described in (12) or (13) above, or a (15) transdermal drug or a transdermal candidate drug. Skin permeability of transdermal drug or transdermal candidate drug as described in (12)-(14) above, characterized in that a transdermal drug or transdermal candidate drug labeled with a body or a fluorescent substance is used. (16) As a solution in which a transdermal drug or a transdermal candidate drug is dissolved, a solution containing an energy source in which the subcutaneous tissue or the transdermal candidate drug is dissolved, Contains polyvalent alcohol in which epidermis dissolved transdermal drug or transdermal candidate drug (12)-(15), wherein the method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a candidate transdermal drug is described in (12)-(15), 17) Polyhydric alcohol power A method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug according to the above (16), which is propylene glycol; and (18) a transdermal drug or a candidate transdermal drug. Wherein the solution obtained by dissolving is a solution containing an energy source obtained by dissolving a transdermal drug or a candidate transdermal drug on both the subcutaneous tissue side and the epidermis side. (19) The method according to (16), wherein the solution containing an energy source is a Hanks solution. Skin penetration of the transdermal drug or transdermal candidate drug according to any of 18) (20) Transdermal drugs labeled with radioactive isotopes or fluorescent substances using unlabeled transdermal drugs or transdermal candidate drugs on both the subcutaneous tissue side and epidermis side Saturated transdermal penetration of transdermal candidate drugs A method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a candidate transdermal drug according to any one of the above (12) to (19), (12)-(19), wherein the method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a candidate transdermal drug according to (12)-(19), NaF is used for both the subcutaneous tissue side and the epidermis side, transdermal drug or transdermal

 Three

 Measuring or evaluating whether the penetration of the candidate drug is energy-dependent, and promoting skin permeability of the transdermal drug or the transdermal candidate drug as described in any of (12) to (19) above. Alternatively, the present invention relates to a method for screening an inhibitor.

 Brief Description of Drawings

 FIG. 1 is a diagram showing an outline of a transdermal permeation test of indomethacin by a Ussing-type Chamber method using hairless mouse skin.

FIG. 2 is a diagram showing the results of percutaneous penetration of [ ¹⁴ C] indomethacin (A) and [] mantol (B) into hairless mouse skin.

FIG. 3 is a graph showing the effect of unlabeled indomethacin on the transdermal penetration of [ ¹⁴ C] indomethacin (A) and [及 び] mantol (B).

FIG. 4 is a graph showing the effects of NaN and NaF on percutaneous penetration of [ ¹⁴ C] indomethacin (A) and [¾] mantol (B) in the secretion direction.

 Three

^[5] Fig. ^{5, [14} C] Indomethacin (white) and ^[3 H] Man - for Torr (black) shows the effect of pH value.

[Fig. 6] Fig. 6 is a view showing non-linear percutaneous permeability of [ ¹⁴ C] indomethacin in the absorption direction.

 FIG. 7 shows the results of transdermal penetration of Fluo-3 into hairless mouse skin.

 FIG. 8 shows the results of expression of transporter mRNA in hairless mouse skin and normal human skin.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] As a method for assaying the skin permeability of the transdermal drug or the transdermal candidate drug of the present invention, a solution in which the transdermal drug or the transdermal candidate drug is dissolved is applied to the skin section on the subcutaneous tissue side and the epidermal side. Parcel Inject into one of the chambers, and inject the prescribed solution into the other, and after a predetermined period of time under the conditions for survival of the skin section, through the skin transporter of the transdermal drug or the candidate transdermal drug. The method is not particularly limited as long as it is a method for measuring and evaluating the degree of skin permeability in all cases.The temperature (solution temperature) of the solution on the subcutaneous tissue side or the epidermis side is not particularly limited. Maintain the solution on the subcutaneous tissue side at body temperature and the solution on the epidermal side at room temperature, or maintain the temperature of the solution on both the subcutaneous tissue side and the epidermal side at body temperature. Although the degree of skin permeability of the candidate drug through the skin transporter can be measured, it is preferable to maintain the solution on the subcutaneous tissue side at body temperature and the solution on the epidermis side at room temperature. Further, as the above-mentioned measurement of the degree of skin permeability 'evaluation, one or two or more measurements of skin saturation, inhibition effect, directional directivity and energy dependency can be preferably mentioned. However, a method for measuring and evaluating a transdermal drug or a transdermal candidate drug at a low concentration using another high-sensitivity detector is also included in the present invention.

 [0014] The above-mentioned transdermal drug or transdermal candidate drug is not limited to the existing transdermal drug, but may be used as an oral drug or a candidate substance to be developed as a transdermal drug in the future. The transdermal drug or the transdermal candidate drug may be a chemical substance or a composition such as an extract of animals, plants and microorganisms. For example, centrally acting drugs (sedatives 抗 antiepileptic drugs Bronchodilators, antibiotics and corticosteroids, antifungals, antivirals, cardiovascular drugs, drugs for parasitic skin diseases, antineoplastic drugs, local anesthetics, eye drops, nasal drops, peripheral vessels Dilators (such as whiskers), germicidal disinfectants for skin, wounds Dermatological drugs, hormonal drugs, antihistamine drugs, drugs for purulent skin diseases, topical enzyme drugs, skin ulcer drugs, cosmetics, hair restorer, hair restorer, animals and plants Extracts such as strong extracts, crude drugs, nucleic acids, polypeptides and the like can be mentioned. Hereinafter, examples of these transdermal drugs or transdermal candidate drugs will be described.

[0015] Analgesics, antipruritics, astringents, and anti-inflammatory drugs (NSAIDs, steroids, etc.) include amcinod, prednisolone acetate valerate, diflucortron valerate, dexamethasone valerate, betamethasone valerate, diflorazone acetate, acetate Hydrocortisone, difluprednate, betamethasone dipropionate, dexamethasone, triamcinolone acetonide, halucino-d, full methasone pivalate, mometasone furoate, fluocinod, fluocinolone acetonide, Rudroxycortide, Prednisolone, Alclomethasone Propionate, Clobetasol Propionate, Dexamethasone Propionate, Deprodone Propionate, Metamethasone Propionate, Clobetasone Butyrate, Hydrocortisone Butyrate, Hydrocortisone Butyrate, Hydrometizone Propionate, Betamethazolone Propionate, , Zinc white starch, zinc white, ibuprofen piconol, indomethacin, difenamate, ketoprofen, glycyrrhetinic acid, dumbbell oxide, diclofenac sodium, suprofen, piroxicam, felbinac, bufexamac, flurbiprofen, bendazac, heparin The substance genole, hydrocortisone 'crotamiton and the like can be mentioned.

 [0016] As antibiotics and corticosteroid mixed preparations, oxytetracycline hydrochlortisone, tetracycline hydrochloride. Hydrocortisone acetate, betamethasone valerate. Gentamicin, betamethasone valerate. Fradiomycin, triamcinolone. Fradiomycin combination preparation, Fradiomycin 'fluocinolone acetonide, fradiomycin sulfate' prednisolone, erythromycin, pimaricin, acyclovir, bleomycin sulfate, hydrocortisone 'Fradiomycin' combination, oxytetracycline hydrochloride 'Polymyxin B sulfate, chloramue-col. And compounding agents.

 [0017] Drugs for parasitic dermatological diseases' Antifungal agents * Antiviral agents include salicylic acid, croconazole hydrochloride, neticonazole hydrochloride, clotrimazole, ketoconazole, isoconazole nitrate, econazole nitrate, oxyconazole nitrate, sulconazole nitrate, miconazole nitrate , Bifonazole, lanconazole, siccanin, ofloxacin, minosacrine hydrochloride, terbinafine hydrochloride, butenafine hydrochloride, tolnaftate, nadifloxacin, acyclovir, vidarabine and the like.

 [0018] In addition, local anesthetics and ophthalmic agents include lidocaine, aminoethyl benzoate, atopine sulfate and naphazoline sulfate, vasodilators include isosorbide dinitrate and nitroglycerin, and bronchodilators include Sulphaziazine, kanamycin sulfate, erythromycin, tetracycline hydrochloride, chloramphenicol, gentamicin sulfate, fradiomacin sulfate, colistin 'fradiomycin, nocitracin' fradiomycin sulfate, respectively. Can be mentioned.

[0019] Furthermore, antihistamines include diphenhydramine, diphenhydramine lauryl sulfate, Rotamiton, estradiol as a hormonal drug, popidone and iodine as germicidal disinfectants for skin, lysozyme chloride and bromelain as topical enzyme drugs, fluorouracil as an anti-neoplastic agent, skin ulcer drug As a transdermal drug or a candidate transdermal drug, chlorhexidine hydrochloride, diphenhydramine combination drug, diflucortron valerate, lidoin, Compounds containing sicon extract, tribenoside 'lid force-in, urea, lactolimus hydrate, gelatin, hydrocortisone acetate' hinokitiol, etretinate, calcipotriol, and tacalcitol can be exemplified.

 [0020] Examples of the dosage form of the transdermal drug or the transdermal candidate drug include haptics, tapes, plasters, ointments, creams, liquids, lotions, dusting agents, mousse types, aerosol types, and the like. be able to.

 [0021] The skin section to be used is preferably a section of hairless skin, and its origin is not particularly limited. However, skin sections of mammals such as mice, rats, dogs, dogs, and humans, and particularly those which are easy to prepare. Skin sections of hairless mice are preferred. The skin section can be prepared by a conventional method such as lightly separating the subcutaneous fat of the excised skin. Further, as the skin slice, a skin slice in which the expression of a specific skin transporter is suppressed or a skin slice in which the expression of a specific skin transporter is amplified can be used.

 A preferred example of the chamber sectioned into the skin section and the subcutaneous tissue side and the epidermis side is a Ussing-type Chamber to which the skin section can be attached in the vertical direction. A solution in which a transdermal drug or a candidate transdermal drug is dissolved is stored in one of the subcutaneous tissue side and the epidermis side of the partitioned chamber, and a predetermined solution is stored in the other. The solution is maintained at body temperature (approximately 36-37 ° C), and the solution on the epidermis side is maintained at room temperature. Also, the skin on both sides of the chamber is supplied with 95% 0/5% CO gas, for example.

 twenty two

 The degree of skin permeability is measured and evaluated under the conditions of existence.

In order to facilitate the measurement of skin permeability, a transdermal drug or a transdermal candidate drug labeled with a radioisotope or a fluorescent substance can be advantageously used. For example, a transdermal drug or a transdermal drug can be used. As the skin candidate drug, a dermal drug substance or a dermal drug substance labeled with a radioactive isotope such as ³ H, "C, ¹²⁵ 1 or ¹³¹ 1 can be advantageously used. To measure skin permeability by confocal microscopy, use it as a cell membrane marker.

A fluorescent dye such as FM4-64 can coexist.

 [0024] The solution on the subcutaneous tissue side in which the transdermal drug or the transdermal candidate drug is dissolved is a solution containing an energy source, that is, a solution containing glucose or other nutrients serving as an energy source. Specific examples thereof include Hanks' solution, Ringer's solution, and Kleps-Henseleit solution, which are preferably similar in composition, and a solution on the epidermis side in which a transdermal drug or a candidate transdermal drug is dissolved. The liquid is not particularly limited as long as it can dissolve the transdermal drug or the transdermal candidate drug. In addition to the solution containing the above energy source, polyhydric alcohols such as propylene glycol, glycerol, ethylene glycol, and sorbitol Liquid and ethanol-containing liquid.Examples include the use of a propylene glycol-containing liquid and a Nountus liquid. Can do. Therefore, in a preferred embodiment, the subcutaneous tissue side contains a solution containing an energy source in which the transdermal drug or transdermal candidate drug is dissolved, and the epidermis side contains the transdermal drug or transdermal drug as a solution in which the transdermal drug or transdermal candidate drug is dissolved. The solution containing the polyhydric alcohol in which the candidate skin agent was dissolved was used as a test method for the percutaneous drug or the skin permeability of the candidate candidate skin agent. A method for assaying the skin permeability of a transdermal drug or a candidate transdermal drug, which is a solution containing an energy source in which a transdermal drug or a transdermal candidate drug is dissolved on both the side and the epidermis, can be mentioned.

 When assaying the skin permeability of a transdermal drug or a candidate transdermal drug, an unlabeled transdermal drug or a candidate transdermal drug was applied to the subcutaneous tissue as described in the Examples below. To determine the saturable transdermal penetration of transdermal drugs or transdermal candidate drugs labeled with radioisotopes or fluorescent substances, and to change the pH of the solution on the epidermis side. Investigate the effect of skin permeability on the difference and use NaN and NaF on the subcutaneous tissue side and epidermis side.

 Three

 It is preferable to use it together to determine whether the penetration of a transdermal drug or a transdermal candidate drug is energy-dependent.

Next, as a method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or a transdermal candidate drug of the present invention, a solution in which a transdermal drug or a transdermal candidate drug is dissolved, and a transdermal drug Alternatively, a solution prepared by dissolving a transdermal candidate drug and a test substance is A predetermined solution is injected into the subcutaneous tissue side in the chamber partitioned into the epidermis side and the other side, and the skin of the transdermal drug or the transdermal candidate drug after a predetermined time under the survival condition of the skin section The method is not particularly limited as long as it is a method of measuring the degree of skin permeability through a transporter and comparing and evaluating the degree of skin permeability, and the test substance may be a chemical substance. It can also be a composition of extracts of animals, plants and microorganisms, etc. For example, the oxidation of probenecid, a mitochondria that is an inhibitor of the organic acid (a-one) transport carrier present on the basal side of epithelial cells. (Carboxycyanide p-trifluoromethoxyphenylhydrazone), which uncouples selective phosphorylation, TEA (tetraethylammonium), an adrenergic and cholinergic blocker, etc. The The temperature (solution temperature) of the solution on the subcutaneous tissue side and the epidermis side is not particularly limited. For example, the solution on the subcutaneous tissue side is maintained at the body temperature, the solution on the epidermis side is maintained at room temperature, or the subcutaneous tissue side and the epidermis side are maintained. While maintaining the temperature of both solutions at the body temperature, the degree of skin permeability of the transdermal drug or the candidate transdermal drug through the skin transporter can be measured after a predetermined time. It is preferred to maintain the solution at body temperature and the epidermal solution at room temperature. Further, as the above-mentioned measurement of the degree of skin permeability 'evaluation, one or two or more measurements of skin saturation, inhibition effect, directionality and energy dependency can be preferably mentioned. The method of evaluating a transdermal drug or a candidate transdermal drug at a low concentration using another high-sensitivity detector is also included in the present invention.In the evaluation, control and comparative evaluation in the absence of a test substance are included in the evaluation. It is preferable to ヽ.

In the method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or a candidate transdermal drug of the present invention, a transdermal drug or a transdermal drug labeled with a radioisotope, a fluorescent substance, or the like is used as the transdermal drug or the candidate transdermal drug. Preferably, a transdermal candidate agent is used. Further, as a solution in which the transdermal drug or the transdermal candidate drug is dissolved, as in the above-described assay method of the present invention, the subcutaneous tissue has an energy such as a liquid or a Nx solution in which the transdermal drug or the transdermal candidate drug is dissolved. A solution containing a single source can be advantageously used, and a solution containing a single source of energy, such as a solution containing a polyhydric alcohol such as propylene glycol or a nontus solution in which the transdermal drug or a candidate transdermal drug is dissolved on the epidermis side Can be advantageously used. Further, at the time of screening, as in the above-described assay method of the present invention, an unlabeled transdermal drug or transdermal candidate is used. Use the drug on both the subcutaneous tissue side and the epidermis side to measure and evaluate the saturable transdermal penetration of transdermal drugs or transdermal candidate drugs labeled with radioisotopes or fluorescent substances, and solutions on the epidermal side Of the skin permeability due to the difference in pH, and the use of NaN and NaF for both the subcutaneous tissue side and the epidermis side.

 Three

 It is preferred to measure 'evaluate' whether the penetration of the drug is energy dependent.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these Examples. In the examples, all data were expressed as the standard error of the mean, and statistical analysis was performed by Student's test. Differences between the means were considered significant when the p-value was less than 0.05.

 Example 1

[0029] [Materials and animals]

[ ¹⁴ C] Indomethacin (740 MBqZmol) and [ ³ H] mantol (740 GBqZmol) were purchased from PerkinElmer Life Sciences, Inc. and American Radiolabeled Chemicals Inc. X, respectively. Fluo-3-AM and FM4-64 were purchased from Dojindani Laboratory and Molecular Probe, respectively. SUPERSCRIPT ™ II RNase H— was purchased from Invitrogen Corp. Normal human adult skin cDNA was also purchased from Invitrogen Corp. and BioChain Institute Inc. In addition, 5- to 7-week-old male hairless mice (HR-1) were purchased from Japan SLC, Inc., and 5- to 7-week-old male hairless mice (FVB) were purchased from CLEA Japan. Purchased from the company. FVB / Mrpl (1-Z—) mice were prepared according to the method described in the literature (Nature Med. 3: 1275-1279, 1997). Animal experiments were conducted in accordance with the guidelines on animal experiments at Kanazawa University Takaramachi Campus.

 [Reverse transcription polymerase chain reaction (RT-PCR)]

 The skin of the hairless mouse was collected, RLT buffer containing β-mercaptoethanol was added, and total RNA was extracted using the RNeasy Mini Kit (QIAGEN). One microgram of total RNA was combined with oligo (dT) (Life technologies) and SUPERSCRIPT ™ II RNase H—

 12-18

Reverse transcription was performed using 200 U of Reverse Transcriptase to prepare cDNA. The mixture after the reaction was subjected to PCR for 30 cycles using an appropriate set of mouse primers (Table 1) and human primers (Table 2). The PCR product is electrophoresed on a 2% agarose gel and brominated. Stained with jam. The amount of each PCR product was measured with an AE-6955 Light Capture instrument (ATTO).

[Table 1]

Gene-specific primers for mouse in Table

Gene * One Forward primer Reverse pnmer ¹ Product size

,

 One

 One

[0031] [Table 2]

Table II. Gene-specific primers for human

Gene 'Forward primer ^1, Reverse primer ⁰ Product size (bp) OR 1 (ABCB 1) 5 * -GCTCCTGACTATGCCAAAGC-3' 5-ATTAGGCCTTCCGTGCTGTA -3 98

 MRP1 (ABCC 1) 5'-CATGAAGGCCATCGGACTCT-3 "5.CAGGTCCACGTGCAGACAG-3 '259

 M P2 (ABCC2) 5'-CTCATTCAGACGACCATCCA-3 '5'-GGCTGCCGCACTCTATAATC-3' 133

 M P3 (ABCC3) S'-GACTTCCAGTGCTCAGAGGG-S '5'-TGTCAGTCTCCAGGTCGATG-3' 139

 P4 (ABCC4) 5'-CTGAATTAGCAGAATCAGGATCC-3 '5'-TTCAATCTGTGTGCAATGGTTAGC-3' 205

 M P5 (ABCC5) 5 -3 '5 -3' 379

 MRPG (ABCC6) 5'-CCCATTGGTCACCTGCTAAACC-3 '5-CAGCTGCAAACACCAGGCCATT-3' 442

 RP7 (ABCC7) 5'.GGGTAGACACACATGAAGTCCAA-3 '5'-C3AAGGCAGTGTACCCTTGATG-3' 534

 G then UT5 (SLC2A5J 5'-AGCGTCTGTAGGGCTTTCTTG-3 '5'-CTCCTTGCAAACGTAGATGGC-3' 158

 AE2 (SLC A2) 5-TTACGTCAAGAAGGTCCGGAC-3 '5'-GGTCGGTGAAGATACGGGTGA-3' 249

 NHE3 (SLC9A3J 5'-GAGTCCTTCAAGTCGACCAAG-3 '5'-CAGGG GTCAATTCCTGCAG-3' 28S

 PEPT1 (SLC15A1) 5'-ACCGCCATCTACCATACGTT-3 '5-GAGCGACACAATGGTCTTGA-3' 105

 PEPT2 (SLC15A2) 5'-GCCATTOCTGACTCGT £ 3 (3TT-3 '5'-TGTGTACCAC GTCCTCCC-3, 124

 MCT1 <SLC16A1) 5'-AGCCCTCATGCGACCAATCG.3 '5' "CCATACATGTCATTGAGCCGAC-3 '675

 ff-CAGTACTTCTTCAGTTTTGCAA 5tB

 MCT3 (SLC16A3) 5'-ACCATCCTGGGCTTCATTGA-3 '5'-CAGAAGAAGTTGCCCAGCAG.3' 425

 MCT4 (SLC16A4) S'-CCTCTTACCTTGTTTCTGTAGC-y 5 * -TATAACCAGCCTGCTATAGGTG-3 '340

 CT5 (SLC16A5) 5'-AGCTTCTACGCCCTGCAGAA-3 'S'-TTGCCCAACTCACATGGCAG-S' 321

 NPT1 (SLC17A1) 5'-AACGAG6CCGACTTACTTCTATGA-3 '5'-ACCAGGGAGGATGTGATGTATT-3' 232

 NPT2 (SLC3 A1) 5 -CCAGAAGGTCATCAATACGGACTTC-3 '5'-ACAGAGGGCAATCTGGAAAGCGCT-3 "274

 OATP-A (SLC21A3) ff-GTGATACAGTTCAATGCATTCG S'-CTCATCGCTGACAAGATTAG-S '584

 OATP-B (SLC21A9) 5 * -CAGAAG 3TGGTATCAGCCTGA-3 'S'-CTGCTAAGACCTTTCGCCGA-S' 174

 OATP-C (SLC21A6) S'-ATCAGTTGCCGGACTAACCAT.B '5' «CATGTGAGGTGCCTCCAAGT ~ 3 '368

 OATP-D (SLC21A11) S'-CTGTGAATGCCAAACCGATTC-S '5'-AGGGATGAAGCCCAACAAAC-3' 343

 OATP-E (SLC21A12) 5, -T ACCGCCGTTCCCATCCTT-3 '5'-CTGAGCTTGTTCACAAAGAAGCC-3' 376

 OATP8 (SLC21A8) 5 'TCAG GCC 3GCCTAACCTT-3' 5 -3 '427

 OCT1 (SLC22A1) 5-GATTTAAAGATGCTTTCCCTCGAA-3 '5'-TCCCTCAGCC GAAGACTATGAA-3' 521

 OCT2 (SLC22A2) 5-TTGCTGGAGGTCTGGTGCTGTT.3 '5'-GGTTGAGTTGTATGGGCTTTGTGATGAG-3' 250

 OCT3 (SLC22A3) 5'-TGATCATCTTTGGTATCCTGGCATC-3 '5.-MCTTTCTCAAATCCTTGGTCGGCA-3' 562

 OCTN1 (SLC22A) 5'-TCATTCAACTGGTACCTGTGG-3 '5'-GACTACCCATGACGATGTAG-3'246

 OCTN2 (SLC22AS) 5'-CCATAATGCTGTGGATGACC-3 '5'-CCAAGGTAAACGAAGTAGGG-3' 412

 OAT1 (SLC22A6) 5'-T <3TCCGAACCTCTCTTGCTGTGC-3 '5'-TTCCTCCTCCTTGTGTGGGTGG-3' 510

 OAT2 (SLC22A7} 5 -3 'S'-CAACCCAAGGGGCACCTTTAT ^ -473

 OAT3 (SLC22A6) 5 '^ CATACOCTGGTGGTCTTGT-3' 5'-GAACTTGGCTGGGACATCGAr-3 '332

 OAT4 (SLC22A9) 5'-TTCTTCCTCCCGGAGACCCA-3 '5'-GGCTGAGTGAAGGTGTAAAGACA-3' 451

 URAT1 (SLC22A12) 5'-CAGCTTTACCCAGAAGCCCT-3 '5 "-GTAGGAGTTTCACGGGCATC-3' 197

 ENT1 (SLC29A1) 5'-CTGAGCGGAACTCTCTCAGT-3 '5'-CTGGCAATAGCGCAGATCATG-3' 379

Beta-acln 5'-AAGAGATGGCCACGGCTGCT-3 '5 * -TCCTTCTGCATCCTGTCGGCA-3' 275 a. Sequences were taken from GenBank; the accession numbers were as fotk> ws: N .000927 (MDR1), NM01989d (RP1), N _000392 (MRP2), MM—020038 (MRP3), N _005845 (iiH afi.uk/ b. MRP4), ΝΜ_005βθθ (MRP5) .NM.001171 (RP6), N _000492 (WRP7) .M55S31 (GLUT5) _t N _O03O4O (AE2), N _004174 (NHE3), NM.005073 (PEPT1) .S78203 (PEPT2), MM— 003051 (MCT1). NM_004731 (CT2), NM_00 207 (MCT3). N _00 696 (MCT4), NM— 004695 (CT5), NM_005074 (NPT1), NM_003052 (NPT2), NM_ 134431 (OATP-A). NM_0O7256 (OATP-B), N _006 46 (OATP-C), NM.013272 (OATP-D) .N .01635 (OATP-E), NM_019B44 (OATP-8), NM_003O57 (OCT ", N _003058 (OCT2 ), NM.021977 (OCT3).

 AB007446 (OCTN1), AB015050 (OCTN2), NM.00 790 (OAT1), NM.006672 (OAT2), N.00 254 (OAT3), NM.018484 (OAT4).

 NM.153378 (U AT1), U81375 (ENT1). NM_001101 (β-actin). [Infiltration experiment using Ussing-type Chamber Method]

The permeation experiment was performed by the method described in the literature (J. Pharm. Pharmacol. 49: 108-112, 1997, J. Pharm. Sci. 92: 1502-1508, 2003). Briefly, the hairless mice were euthanized, immediately skin excised by a lightly separated skin sections subcutaneous fat, longitudinally within Ussing- type Chamber with exposed areas of 0. 766cm ² (exposed area) Attached to. As shown in FIG. 1, Hank's solution (HBSS, 136.7 mM NaCl, 5.36 mM KC1, 0.952 mM CaCl ゝ 0.1812 mM, was placed in one chamber on the side of the subcutaneous tissue sectioned by the skin section. MgSO,

0.4441 mM KH PO, 0.385 mM Na PO, 25 mM D-glucose and 10 mM

M HEPES, pH 7.4) 1.2 mL of propylene glycol into the chamber on the epidermis side : 1.2 mL of a mixed solution of water (1: 1) was added, and the temperature of the solution was maintained at 37 ° C. on the subcutaneous tissue side and room temperature on the epidermis side. To maintain the viability of the skin tissue, the solution on both sides of the chamber was supplied with 95% 0/5% CO gas during the transport experiments. O /

 2 2 2

CO bubbles mix the solution around the tissue, not just supplying oxygen to the tissue

2 To determine the transdermal permeability of [ ¹⁴ C] indomethacin and [¾] mantol, use the test compound on the subcutaneous tissue side or epidermis side and invert 0.4 mL of the solution at a given time. Taken from the side and replaced with the same amount of fresh solution. The collected sample was measured with a liquid scintillation counter. In order to examine the effect of pH on the transdermal penetration of [ ¹⁴ C] indomethacin, Hanks solution (pH 7.4) was used on the subcutaneous tissue side, whereas the test solution on the epidermis side was made of propylene glycol: 20 mM. It consisted of a phosphate buffer (1: 1). The value of the permeability coefficient (μL / cmVhr, P and P) is the sec abs of the permeability plot against time (hr).

It was determined from the inclination force of the linear part (μL / cm 2).

[Thin layer chromatography (TLC) method]

Using TLC, it was examined whether indometha single chloride occurs during the experiment according to the method described in the literature (Pharm. Res. 17: 432-438, 2000). Briefly, after transdermal permeation experiments with [ ¹⁴ C] indmetacin, the medium opposite to the test compound was extracted with getyl ether (6 mL). The extract was evaporated to dryness, the residue was dissolved in ethanol and the solution was plotted on a TLC plate. Metabolites were separated with a mixture of black form: acetic acid (95: 5v Zv) and quantified using a radioisotope scanner. The obtained intensities were compared with those of standard [ ¹⁴ c] indomethacin.

[Confocal microscopy]

The skin of FVB and FVBZMrpl (—Z—) mice, which had been depilated with a hairless mouse and depilation cream for 24 hours, were each sliced on ice with a microslicer (DTK-2000, Dosaka EM Co., LTD.) On ice. : 100 μm). After skin sections that were-tight immersed 2 hours Fluo- 3-AM and _{(10 i u M) FM4-64 (} l i u M) Hanks solution of 4 ° C was added (pH7. 4), the skin sections Place on a LabTek chambered micro cover glass (24X60mm) and attach to a confocal microscope (Zeiss Axiovert 100M LSM510). At room temperature (20-22 ° C), The fluorescence was observed with a water immersion lens (× 630) using 88 nm and 543 nm lasers.

[Directional transdermal penetration of indomethacin]

The percutaneous permeability of [ ¹⁴ C] indomethacin (47 M) and [ ³ H] mantol (0.25 M) was measured from subcutaneous to epidermal side using a Ussing-type chamber method (square). The hairless mouse skin was examined for 4 hours from the epidermis to the subcutaneous tissue side (circle) for 4 hours. The results are shown in Figure 2 (each point represents the standard error of the mean in three to four experiments.) ₀ [ ¹⁴ C] Indomethacin percutaneous penetration was linear and secretory. (Subcutaneous tissue side to epidermis side; P

 sec

) Was 4 times stronger than the direction of absorption (from the epidermis to the subcutaneous tissue; P) (FIG. 2—A). this

 abs

In contrast, transdermal penetration of [ ³ H] mantol did not show any unidirectional transport (Figure 2-B). The permeability of [ ¹⁴ C] indomethacin was significantly higher (more than 5 times) than that of [ ³ H] mantol.

 [The radioactivity that appears on the opposite side is mainly due to the parent compound. ]

 Indomethacin chloride is a substrate of CMOATZMRP2 (Pharm. Res. 17: 432-438, 2000), and indomethacin is OAT1 (J. Pharmacol. Exp. Ther. 303: 534-539, 2002), OAT2 (J Pharmacol. Exp. Ther. 298: 1179-1184, 2001), OAT3 (J.

Pharmacol. Exp. Ther. 303: 534-539, 2002) and NPT1 (Biochem. Biophys. Res. Commun. 270: 254-259, 2000), but cMOATZMRP2 (Pharm. Res. 17: 432-438). , 2000). Indomethacin Force To determine whether or not metabolites were metabolized during percutaneous penetration experiments, [ ¹⁴ c] indomethacin was added to the subcutaneous tissue side, and the radioactivity that appeared on the epidermis side was analyzed by thin-layer chromatography (TLC) for 6 hours. Separated. ⁴ C] recovery of indomethacin was 80% or more. More than 95% of the labeled compounds were [ ¹⁴ C] indomethacin. Therefore, the metabolism of indomethacin in this experiment was considered to be negligible.

[Saturated transdermal penetration of indomethacin]

One of the important features of active transport is saturability, so we investigated whether transdermal infiltration of [ ¹⁴ c] indomethacin was saturable. Percutaneous penetration of [ ¹⁴ C] indomethacin (47 M) and [¾] mantle (0.25 M) was performed in the presence of unlabeled indomethacin (500 μ500) added to both sides of a Ussing type chamber. Fig. 3 shows the results of a 4-hour study in the absence (black pillars) and without (black pillars). (Each column represents the standard error of the mean in 3-4 experiments.) ₀ As a result, unlabeled indomethacin (500 μΜ) increased the power of [ ¹⁴ C] indomethacin by increasing the P Second, sec abs had no effect (Fig. 3—A). In contrast, the effect of unlabeled indomethacin on the permeability of [II] mannitol was unobserved (Figure 3-B). Thus, the effect of unlabeled indomethacin is specific for the penetration of [ ¹⁴ c] indomethacin, and the transdermal penetration of [ ¹⁴ C] indomethacin is saturable in the direction of secretion.

[ATP-dependent transdermal penetration of indomethacin]

[ ¹⁴ C] Indomethacin Osmotic Force To investigate whether it is energy-dependent, the effect of ATP consumption (depletor) on [ ¹⁴ C] indomethacin infiltration was examined. 30 minutes after loading NaN and NaF (10 mM) on both sides of Ussing-type chamber, [ ¹⁴ C]

 Three

The percutaneous permeability of tasin (47 M) and [ ³ H] man-tol (0.25 M) was determined in the presence (open circles) and absence (black circles) of added NaN and NaF (10 mM). 4 hours later

Three

The results are shown in FIG. 4 (each point represents the standard error of the mean in four to five experiments.) ₀ As a result, the P force of [ ¹⁴ C] indomethacin increased in the presence of NaN and NaF ( sec 3

Figure 4-A) was found, but no effect was observed on the permeability of [] mannitol (Figure 4B). These results indicated that the absorption direction was dependent on the osmotic energy of [ ¹⁴ C] indomethacin.

[Effect of pH on transdermal penetration of indomethacin]

Since the skin environment is weakly acidic, the effect of pH on the transdermal permeability of [ ¹⁴ C] indomethacin was confirmed. The pH of hairless mouse skin remained relatively constant (pH 5.9) (J. Investig. Dermatol. Symp. Proc. 3: 110-113, 1998) and was similar to that of human skin ( ^{pH 4 - 6) (2 1} ). Epidermal side of the chamber one ^[14 C] Indomethacin ⁽⁴ 7 / ζ Μ) and ^[3 H] Man -. The coefficient of transdermal permeability Torr (0. 25 M), different pH values (pH 5 0, 6 The results observed in the absorption direction (A) and in the secretion direction (B) at 0, 7.4) are shown in FIG. 5 (each column represents the standard error of the mean in four to five experiments). _{0 Since} the pKa value of indomethacin is 4.5, changes in the pH of the epidermal medium (5.0, 6.0 and 7.4) changed the degree of indomethacin ionization. It was found that when the pH value on the epidermis side decreased, the S abs of [ ¹⁴ C] indomethacin increased. On the other hand, there is no change in P of [ ³ H] man-Toll. Not observed (Figure 5-A). Conversely, in the epidermis side, ^[14 C] indomethacin or [¾] Man - the p Thor, such effect of _P H is observed ChikaraTsuta (Figure 5-B). As a result, [ ¹⁴ C]

 sec

The unidirectional penetration of indomethacin disappeared as the pH decreased on the epidermis side

[Saturable penetration of indomethacin at pH 5.0 on the epidermis side]

On the epidermis side, P of [ ¹⁴ C] indomethacin at pH 5.0 was replaced with unlabeled indomethacin

 abs

Were examined in the absence or presence of The percutaneous permeability of [ ¹⁴ C] indomethacin (47 M) and [ ³ H] mantle (0.25 μΜ) (Β) was compared with unlabeled indomethacin (500 the presence of M) (white) and absence (the result of examination after 4 hours black) shown in FIG. 6 (each point represents the mean mechanic standard error of 3-5 experiments.) ₀ Note In both the presence and absence of unlabeled indomethacin, the pH value on the epidermis side of the chamber was kept at 5.0. As a result, on the epidermis side, at pH 5.0, P of [ ¹⁴ C] indomethacin was increased by unlabeled indomethacin (500 M) (FIG. 6-A). But [ ³ H] abs

No effect of unlabeled indomethacin (500 μΜ) on mannitol P was observed.

 abs

(Fig. 6—B). Penetration in the direction of absorption of [ ¹⁴ C] indomethacin was considered to be saturable.

 [Directional transdermal penetration of Fluo-3]

The directional transdermal permeation of Fluo-3 is converted to a lipid-soluble acetomethyl ester form by converting the carboxyl group of Fluo-3 to cell membrane permeability, and hydrolyzed by intracellular esterase to form Fluo-3. (1— [2— Amino— 5— (2,7—dichloro— 6—hydroxy— 3— oxo-9-xanthenyl) phenoxy]-2- (2-amino-5-methylphenoxy) ethane- Ν, Ν, Ν Ν, Ν tetraacetic acid, pentaacetoxymethyl ester; 10 μM) and ¾: Infiltrate Hanks' solution with the photopigment FM4-64 (1 μΜ) and place it in a Ussing-type chamber. Fluo-3 excreted from both sides was examined. Further, the directional transdermal permeation of Fluo 3 was similarly examined for a sample prepared by adding probenecid (5 mM), FCCP (5 μM), and TEA (5 mM) to Fluo-3-AM, respectively. Fig. 7 shows the results. As a result, the percutaneous penetration of Fluo-3 produced in the skin tissue was linear, and the direction of absorption (from the inside of the skin tissue to the side of the subcutaneous tissue; the control on the left side of Fig. 7) was the same as that of the secretion. Epidermis side from inside tissue; right of Fig. 7 50 times higher strength than control). Also, among probenecid, FCCP and TEA, when FCCP coexisted, the absorption was approximately twice as high as that of Fluo-3 alone.

 [Expression of transporter in skin]

 RT-PCR and PCR were performed on the total RNA of the hairless mouse skin (panel A) and the cDNA of normal human skin (panel B), respectively. Human cDNA was obtained from three 80-year-old Z females (panels B-I), 44-year-old Z males (panel B-Π) and 44-year-old Z males, 58-year-old Z females, and 65-year-old Z females. The mixture (panels B-III) was used. PCR products were analyzed by 2% agarose gel electrophoresis and stained with bromide tube. Fig. 8 shows the results.

[0034] In panel A, one to 32 lanes include 1, Mdrla; 2, Mdrlb; 3, Mrpl; 4, Mrp2; 5, Mrp3; 6, Mrp4; 7, Mrp5; 8, Mrp6; 9, Smvt; 10 , PepTl; ll, PepT2; 12, Mctl; 13, Mct2; 14, Mct3; 15, Mct4; 16, Nptl; 17, Oatpl; 18, Oapt2; 19, Oapt 3; 20, Oatp4; 21, Oatp5; 22, Oatpll; 23, Oatpl4; 24, mPGT; 25, Octl; 26, Oct2; 27, Oct3; 28, Octnl; 29, Octn2; 30, Octn3; 31, Oatl; 32 and Oat2, respectively.

 [0035] In addition, lane 1-38 of panel B contains 1, j8-actin; 2, Uratl; 3, Mrpl; 4, and Mrp2.

 ; 5, Mrp3; 6, Mrp4; 7, Mrp5; 8, Mrp6; 9, Mrp7; 10, Oatl; ll, Oat2; 12, O at3; 13, Oat4; 14, Oatp— A; 15, Oatp— B; 16, Oatp-C; 17, Oatp-D; 18, O atp—E; 19, Oatp8; 20, Mdrl; 21, Octl; 22, Oct2; 23, Oct3; 24, Octnl; 25, Octn2; 26, Nptl ; 27, Npt; 28, Peptl; 29, Pept2; 30, Ae2; 31, Nhe3; 32, Glut5; 33, Entl; 34, Mctl; 35, Mct2; 26, Mct3; 37, Mct4; 38, Mct5 Shown respectively.

 [0036] In hairless mouse skin, MRP1, MRP3, MRP4, MRP5, peptide transporter 1 (PEPT1), PEPT2, monocarboxylic acid transporter 1 (MCT1), MCT2, MCT4, ΟΑΤΡ3, ΟΑΤΡ11, prostaglandin The expression of transporter (PGT), organic cation transporter 3 (OCT3), organic cation Z-calcin transporter 1 (OC TNI), OCTN2 and OCTN3 was observed (FIG. 8A).

Further, the expression of the transporter was observed in the cDNA of normal human skin (manufactured by ResGen Invitrogen and Biochain Institute). MRP1, MRP3, MRP4 in normal human skin , MRP5, MRP6, GLUT5, AE2, MCT1, MCT4, MCT5, OATP-B, OATP-D, OATP-E, OCTNl, OCTN2 and ENT1 expression were observed (Fig. 8-B). The expression of MRP2, MDR1, PEPT1, PEPT2, MCT2, MCT3, OCT1, OCT3 and URAT1 was detected in some donors, suggesting that there is individual difference in expression.

 [Discussion]

Saturation with unlabeled indomethacin (500 M) was clearly observed in the direction of secretion, but saturated permeation of [ ¹⁴ C] indomethacin was found in the epidermal compartment at low pH and also in the direction of absorption. Was. These results can be explained if multiple transport systems are involved in indomethacin transport. Such nonlinear kinetics of the direction of absorption is important in terms of the efficiency of transdermal absorption. In order to saturate the percutaneous permeation of indomethacin in the absorption direction, the concentration on the skin surface when indomethacin was transdermally administered was generally higher than the concentration of unlabeled indomethacin (500 μΜ) used in the above example. very high. Thus, the results of the above examples suggest that the permeability of transdermally administered drugs depends on the concentration administered.

[0038] The decrease in the pH value in the epidermal compartment is due to the power of [ ¹⁴ C] indomethacin that increases Ρ

abs did not affect sec. As a result, unidirectional penetration of [ ¹⁴ C] indomethacin disappears in the epidermal compartment at pH 5.0. P-power with one possibility thus increased

 abs

There was non Ioni spoon in the stratum corneum (SC) is ^[14 c] indomethacin include that increased distributed. This causes a high concentration gradient of the stratum corneum (SC) force to the subcutaneous tissue side, leading to a non-linear P of [ ¹⁴ C] indomethacin (Figure 6-A). But proton dependent

 abs

That sexual transport system is involved in the P of ^[14 c] indomethacin, there is another hypothesis.

 abs

 In previous studies, we demonstrated that monocarboxylic acid transporter 1 (MCT1) was involved in the intestinal absorption of weak organic acids in a pH-dependent manner (Biochem.

 Biophys. Res. Commun. 214: 482-489, 1995, J. Pharm. Pharmacol. 51: 1131-1121, 1999). On the other hand, the expression of a transporter of the OATP family, which transports organic compounds, was also confirmed.

[0039] To identify the transporter (s) that may be involved in the transdermal penetration of [ ^14C ] indomethacin, expression of the transporters in hairless mouse skin was determined. RT—Checked by PCR. MRP1, MRP3, MRP4 and MRP5 expression confirmed, P

EPT1, PEPT2, MCT1, MCT2, MCT4, OATP3, OATPl l, mPGT, OCT3

, OCTNl, OCTN2 and OCTN3 expression were also observed in hairless mouse skin (Figure 8). Indomethacin has been approved by OATl (J. Pharmacol. Exp. Ther. 303: 534-539, 2002), OAT2 (J.

Pharmacol. Exp. Ther. 298: 1179-1184, 2001) and Oat3 (J. Pharmacol. Exp. Ther.

303: 534-539, 2002), and a low substrate of NPT1 (Biochem. Biophys. Res.

Commun. 270: 254-259, 2000) Power previously reported. However, in the results of this example, expression of ΟΑΤ1, OAT3 and NPT1 was not detected, suggesting that other transporter forces S may be involved in penetration of indomethacin. The transporter expression in normal human skin was also examined and compared to hairless mouse skin. Expression of MRP, MCT and OCTN family members in hairless mouse skin was similar to expression in normal human skin, suggesting that hairless mouse skin could be a good model to study the function of these transporters. As in recent studies, expression of MRP family members other than MRP2 was detected in all individuals (J. Invest. Dermatol. 116: 541-548, 2001). Even in hairless mouse skin, the force of observing the expression of Mrpl, Mrp3, Mrp4 and Mrp5 was not observed, indicating that the expression characteristics were similar to human skin. Expression of OATP-B, OATP-D and OATP-E in human skin was observed in all individuals. This result was consistent with a recent report (J. Invest. Dermatol. 120: 285-291, 2003). OATP-B, as a substrate, is a sulfated conjugate of steroids. Puru (J. Pharmacol. Exp. Ther. 306: 703-708, 2003). OATP-C was considered to transport both forms of the steroid conjugate (Pharm. Res. 18: 1262-1269, 2001), whereas OATP-D translocated prostaglandins to special tissues and cells. It plays an important role when locating (Am. J. Physiol. Renal Physiol 285: F1188-1197, 2003). Immunohistochemical staining revealed that OATP-B force was expressed in all layers of the epidermis but not subcutaneously. In addition, the OATP family substrate, taurocholate, reduced estrone sulfate uptake by 33% in normal human epidermal keratinocytes. (J. Invest. Dermatol. 120: 285-291, 2003). These findings suggest that the organo-on transport system may be involved in the uptake of its substrate by keratinocytes, and that such transporters play an important role in the transdermal delivery of drugs. This suggests the need for further analysis to determine if any. MCT1, MCT2 and MCT5 expression was detected in human skin. Expression of MCT2 and MCT3 could be detected in only a few cases (FIG. 8). MCT1 and MCT4 were detected in multiple skin-derived cell lines, suggesting that MCT is a major determinant of pH adjustment in melanoma (Mol. Cancer Ther. 1: 617-628, 2002). In previous studies, we found that MCT1 played an important role in the transport of monocarboxylic acids, including benzoic acid and exogenous and endogenous weak organic acids such as lactic acid in the small intestine and brain. (Biochem. Biophys. Res. Commun. 214: 482-489, 1995, J. Pharm. Pharmacol. 51: 1113-1121, 1999, Pharm. Res. 17: 55-62, 2000). Expression of MCTs in the skin may indicate a role in skin pH regulation and transport of weak organic acids. Expression of OCTN family members was observed in all individuals (FIG. 8). The OCTN family is involved in the transport of caltin, a cofactor essential for the long-chain fatty acid oxidation. An in vitro cal-tin transport system in human cultured skin fibroblasts has been characterized. The Km for the uptake of caltin was: and was close to that of human OCTN2 (N. Engl. J. Med. 319: 1331-1336, 1988, Pediatr. Res. 28: 247-255, 1990, Biochem. Pharmacol. 55: 1729-1732, 1998). OCTN1 functions as a multi-selective, pH-dependent organic cation transporter that can function in the apical membrane of kidney and other tissues as proton Z organic cation antiporter and Z or organic cation Z cation exchanger (J. Pharmacol. Exp. Ther. 289: 768-773, 1999). In contrast, OCTN2 is thought to be a multi-selective transporter that mediates both organic cation transport and carnitine transport (J. Biol. Chem. 275: 40064-40072, 2000). OCTN family members in the skin may be involved in the uptake of carnitine or organic cation conjugates. Thus, further study of the functions in the skin is needed, but the expression of these transporters in the skin indicates that they may be involved in substrate transport as active skin barrier systems . Industrial applicability

 According to the present invention, it has been clarified that a transporter (one or more) is involved in transdermal penetration of a transdermal drug such as indomethacin or a transdermal candidate drug. In addition, mRNA expression of multiple transporters of the MRP, OATP, MCT, and OCTN families was observed in both hairless mouse skin and normal human skin. As will be further elucidated, the presence of these diverse transporter types indicates a potential role for active noria in controlling transdermal penetration of xenobiotics. INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to develop an excellent transdermal drug delivery system for transdermal drugs and cosmetics that can be used without further clarification of the physiological role of transporters in the skin.

Claims

The scope of the claims  [1] A solution in which a transdermal drug or a candidate transdermal drug is dissolved is injected into one of the chambers divided into a subcutaneous tissue side and an epidermis side by a skin section, and a predetermined solution is injected into the other. A transdermal drug, wherein the degree of skin permeability of the transdermal drug or the transdermal candidate drug through a skin transporter is measured and evaluated after a predetermined time under the survival condition of the skin section. Test method for skin permeability of transdermal candidate drug.  [2] Maintain the solution on the subcutaneous tissue side at body temperature and the solution on the epidermis side at room temperature, and measure the degree of skin permeability of the transdermal drug or the candidate transdermal drug through the skin transporter after a predetermined time. The method for assaying skin permeability of a transdermal drug or a transdermal candidate drug according to claim 1, wherein the evaluation is performed.  [3] The method according to claim 1, wherein the measurement of the degree of skin permeability is an evaluation of one or more of saturation, inhibition effect, directionality and energy dependence of skin penetration. Or a method for assaying skin permeability of a transdermal drug or a candidate transdermal drug according to 2 above.  [4] The transdermal drug according to any one of [13] to [13], wherein a transdermal drug or a transdermal drug candidate labeled with a radioisotope or a fluorescent substance is used as the transdermal drug or the transdermal drug candidate. A method for assaying skin permeability of a drug or a transdermal candidate drug.  [5] As a solution in which the transdermal drug or the transdermal candidate drug is dissolved, a solution containing an energy source in which the subcutaneous drug or the transdermal candidate drug is dissolved in the subcutaneous tissue side, and a transdermal drug or a transdermal drug candidate in the epidermis side are used. The method for assaying skin permeability of a transdermal drug or a transdermal candidate drug according to any one of claims 14 to 14, wherein a dissolved polyhydric alcohol-containing solution is used, respectively.  6. The method for assaying skin permeability of a transdermal drug or a transdermal candidate drug according to claim 5, wherein the polyhydric alcohol power is propylene glycol. [7] The solution in which the transdermal drug or the transdermal candidate drug is dissolved is a solution containing an energy source in which the transdermal drug or the transdermal candidate drug is dissolved on both the subcutaneous tissue side and the epidermis side. 14. A method for detecting skin permeability of a transdermal drug or a transdermal candidate drug according to any one of 1 to 4. [8] The solution according to claim 5-7, wherein the solution is a Hanks solution containing an energy source. A method for assaying skin permeability of a transdermal drug or a transdermal candidate drug according to any one of the above.  [9] Unlabeled transdermal drug or transdermal candidate drug is used for both the subcutaneous tissue side and epidermis side, and the radioactive isotope or fluorescent substance is used to label the transdermal drug or the transdermal candidate drug for transdermal drug saturation. 19. The method for assaying skin permeability of a transdermal drug or a transdermal candidate drug according to claim 18, wherein the penetration is examined.  [10] The method for assaying skin permeability of a transdermal drug or a candidate transdermal drug according to claim 18, wherein the pH of the solution on the epidermis side is changed.  [11] Transdermal drug or transdermal candidate drug using NaN and NaF for both subcutaneous tissue and epidermis  Three  19. The method for assaying skin permeability of a transdermal drug or a candidate transdermal drug according to claim 18, wherein it is determined whether the penetration of the drug is energy-dependent. [12] A solution in which a transdermal drug or a candidate transdermal drug is dissolved, and a solution in which a transdermal drug or a candidate transdermal drug is dissolved in a test substance are partitioned into a subcutaneous tissue side and an epidermal side by a skin section. Injected into the subcutaneous tissue side of the chamber and into the other, a predetermined solution was injected into the other, and after a predetermined period of time under the survival conditions of the skin section, through the skin transporter of the transdermal drug or the candidate transdermal drug. A method for screening for a substance that enhances or inhibits skin permeability of a transdermal drug or a candidate transdermal drug, comprising measuring the degree of skin permeability of the skin and comparing and evaluating the degree of skin permeability.  [13] The solution on the subcutaneous tissue side is maintained at body temperature and the solution on the epidermis side is maintained at room temperature, and after a predetermined time, the degree of skin permeability of the transdermal drug or the candidate transdermal drug through the skin transporter is reduced. 13. The screening method for a substance that promotes or inhibits skin permeability of a transdermal drug or a transdermal candidate drug according to claim 12, wherein the measurement is performed.  14. The method according to claim 12, wherein the measurement of the degree of skin permeability is an evaluation of one or more measurements of saturation, inhibition effect, directionality and energy dependence of skin penetration. Or a method for screening a substance that enhances or suppresses skin permeability of a transdermal drug or a transdermal candidate drug according to item 13. 15. The transdermal drug according to any one of claims 12 to 14, wherein a transdermal drug or a transdermal drug candidate labeled with a radioisotope or a fluorescent substance is used as the transdermal drug or the transdermal drug candidate. A method for screening a substance that enhances or suppresses skin permeability of a drug or a transdermal candidate drug. [16] As a solution in which the transdermal drug or the transdermal candidate drug is dissolved, a solution containing an energy source in which the subcutaneous drug or the transdermal candidate drug is dissolved in the subcutaneous tissue side, and a transdermal drug or a transdermal candidate drug in the epidermis side are used. 16. The method for screening a substance that promotes or inhibits skin permeability of a transdermal drug or a candidate transdermal drug according to any one of claims 12 to 15, wherein a solution containing a dissolved polyhydric alcohol is used.  [17] The method for assaying skin permeability of a transdermal drug or a candidate transdermal drug according to claim 16, wherein the polyhydric alcohol is propylene glycol.  [18] The solution in which the transdermal drug or the transdermal candidate drug is dissolved is a solution containing an energy source in which the transdermal drug or the transdermal candidate drug is dissolved on both the subcutaneous tissue side and the epidermis side. 12. A method for screening a substance that promotes or suppresses skin permeability of a transdermal drug or a transdermal candidate drug according to any of 12 to 15.  [19] The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a transdermal candidate drug according to any one of claims 16 to 18, which is a solution-based Hanks solution containing an energy source.  [20] Unlabeled transdermal drug or transdermal candidate drug is used for both the subcutaneous tissue side and epidermis side, and the radioactive isotope or fluorescent substance is used to label the transdermal drug or the saturated transdermal drug of the transdermal candidate drug. 20. The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a transdermal candidate drug according to any one of claims 12 to 19, wherein the penetration is measured and evaluated.  [21] The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or a candidate transdermal drug according to any one of claims 12 to 19, wherein the pH of the solution on the epidermis side is changed. [22] Transdermal drug or transdermal candidate drug using NaN and NaF for both subcutaneous tissue and epidermis  Three  20. A substance that promotes or inhibits skin permeability of a transdermal drug or a transdermal candidate drug according to any one of claims 12 to 19, characterized in that whether the penetration of the drug is energy-dependent is measured and evaluated. Screening method.