JP4714807B2 - Method for testing skin permeability of transdermal drugs via skin transporter - Google Patents

Description

  The present invention relates to a method for assaying the skin permeability of a transdermal drug or a transdermal candidate drug, and more specifically, using a chamber divided into a subcutaneous tissue side and an epidermal side in a skin section, The present invention relates to a method for assaying skin permeability of a transdermal drug or transdermal candidate drug that measures and evaluates the degree of skin permeability through a skin transporter (transporter provided in skin tissue).

  Transdermal drug delivery is often used as a route of administration for effective low molecular weight therapeutics, avoiding first-pass metabolism, reducing pain, and slow drug release compared to conventional dosage forms such as tablets and injections. It has excellent effects including the possibility of. For example, administration of non-steroidal anti-inflammatory drugs (NSAIDs) by the transdermal route has been introduced to avoid the disadvantages of the oral route such as gastrointestinal inflammation and ulceration (eg, J. Pharm. Sci. 86: 503-508, 1997). However, transdermal delivery has limited application due to low skin penetration.

  The skin is a physical and biochemical barrier on both sides. The stratum corneum (SC), the outermost layer of the skin, is said to be the greatest barrier when various substances penetrate due to the physical structure. Furthermore, foreign body metabolic enzymes in the skin are the second biochemical barrier. For example, the expression and activity of glutathione S transferase (GST) P1-1 has been detected in human epidermal cells (see, for example, J. Dermatol. Sci. 30: 205-214, 2002). Xenobiotic metabolizing enzyme expression is also 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).

  Recently, the expression of several transporters in the skin has been reported. The neuronal glutamate transporter EAAC1 and the glial glutamate transporter (GLT-1) are detected in keratinocytes (see, for example, J. Invest. Dermatol. 112: 337-342, 1994), a sodium-dependent multivitamin transporter (SMVT) Has been found to be expressed in the skin (see, for example, 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 xenobiotics transporters has also been reported recently. They transport numerous organic ionic drugs and are present in human normal keratinocytes, multidrug resistance-related proteins (MRPs) (see, for example, J. Invest. Dermatol. 116: 541-548, 2001) and organic Includes anion transport polypeptide (OATP) family members (see, eg, J. Invest. Dermatol. 120: 285-291, 2003). The expression of such transporters in the skin may be an active barrier when the drug penetrates transdermally. However, at present, the involvement of transporters in the transdermal penetration of drugs has not been clarified.

  On the other hand, indomethacin, an NSAID, is administered transdermally and is used as a hydrophobic model compound for investigating new enhancers in transdermal delivery (see, for example, J. Pharm. Sci. 84: 482-488, 1995, J. Control Release 90: 335-343, 2003, J. Control Release 88: 243-252, 2003). In fact, 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). Mannitol has also been used as a paracellular marker.

  The object of the present invention is to clarify that a carrier-mediated transport is involved in the percutaneous penetration of a therapeutic agent, and to permeate the skin 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 skin permeability of a transdermal drug or transdermal candidate drug that measures and evaluates the degree of sex.

In order to investigate the characteristics of the transdermal transport mechanism, the present inventors used indomethacin, an NSAID transported transdermally, as a hydrophobic model drug. When the transdermal permeability of [ ¹⁴ C] indomethacin and [ ³ H] mannitol was measured from both the absorption and secretion directions, the transdermal transport of [ ¹⁴ C] indomethacin was from the paracellular marker [ ³ H] mannitol. It was found that the transdermal transport of [ ¹⁴ C] indomethacin is not a paracellular transport but a transcellular transport. In addition, the effect of unlabeled indomethacin on the permeability of [ ¹⁴ C] indomethacin and [ ³ H] mannitol was compared to determine whether the transdermal penetration of [ ¹⁴ C] indomethacin was saturable. When unlabeled indomethacin (500 μM) was added, it was found that the permeability of [ ¹⁴ C] indomethacin in the direction of secretion increased, but it did not affect the permeability of [ ³ H] mannitol. It has been found that it is specific for [ ¹⁴ C] indomethacin, and that the percutaneous permeability of [ ¹⁴ C] indomethacin involves a saturation process. Furthermore, the effect of NaN ₃ and NaF was observed to investigate whether the saturation penetration of [ ¹⁴ C] indomethacin is energy dependent. As a result, it was found that the penetration of [ ¹⁴ C] indomethacin was also increased by NaN ₃ and NaF, and the penetration step in the absorption direction was ATP-dependent. These results for unidirectional, ATP-dependent and saturable penetration of [ ¹⁴ C] indomethacin indicate that transporter (s) may be involved in indomethacin percutaneous penetration. . 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.

  That is, the present invention is (1) injecting a solution in which a transdermal drug or transdermal candidate drug is dissolved into one of chambers divided into a subcutaneous tissue side and an epidermis side by a skin section, A solution is injected, and the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug is measured and evaluated after a predetermined time under the living conditions of the skin section. (2) Maintain the subcutaneous tissue side solution at body temperature and the epidermis side solution at room temperature, and after a predetermined time, The present invention relates to a method for assaying skin permeability of a transdermal drug or transdermal candidate drug as described in (1) above, wherein the degree of skin permeability through a skin transporter is measured and evaluated.

Further, the present invention is characterized in that (3) the measurement / evaluation of the degree of skin permeability is one or more of the measurement / evaluation of skin permeability saturation, inhibitory effect, directionality and energy dependence. (1) The method for assaying the skin permeability of the transdermal drug or transdermal candidate drug described in (1) or (2) above, or (4) a radioisotope or fluorescent substance labeled as the transdermal drug or transdermal drug candidate A method for testing the skin permeability of a transdermal drug or transdermal candidate drug according to any one of the above (1) to (3), wherein a transdermal drug or transdermal candidate drug is used, or (5) transdermal As a solution in which the drug or transdermal candidate drug is dissolved, a solution containing an energy source in which the subcutaneous tissue side dissolves the transdermal drug or transdermal candidate drug, and a polyhydric alcohol in which the epidermal side dissolves the transdermal drug or transdermal candidate drug Each of the above solutions is used (1) to (4), wherein the transdermal drug or transdermal candidate drug has a skin permeability assay method, or (6) the polyhydric alcohol is propylene glycol (5), (7) A solution in which a transdermal drug or transdermal candidate drug is dissolved is a transdermal drug or transdermal candidate on both the subcutaneous tissue side and the epidermis side. (8) A method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of (1) to (4) above, which is a solution containing an energy source in which the drug is dissolved; The solution containing an energy source is Hanks' liquid, the method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of (5) to (7) above, (9) Unlabeled transdermal drug or transdermal candidate drug on the subcutaneous tissue side and epidermal side The transdermal drug according to any one of the above (1) to (8), which is used together to examine the saturated transdermal penetration of a transdermal drug or transdermal candidate drug labeled with a radioisotope or fluorescent substance Alternatively, a method for testing skin permeability of a transdermal candidate drug, or (10) the transdermal drug or transdermal system according to any one of (1) to (8) above, wherein the pH of the solution on the epidermis side is changed Whether the permeation of the transdermal drug or transdermal candidate drug is energy-dependent, using (11) NaN ₃ and NaF both on the subcutaneous tissue side and the epidermis side The method for assaying the skin permeability of a transdermal drug or transdermal candidate drug according to any one of the above (1) to (8), wherein

  Furthermore, the present invention relates to (12) a solution in which a transdermal drug or transdermal candidate drug is dissolved, and a solution in which the transdermal drug or transdermal candidate drug and a test substance are dissolved, and the skin tissue section and the epidermis side. A skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time under the living conditions of the skin section, by injecting into the subcutaneous tissue side in the chamber partitioned into the side, and injecting a predetermined solution into the other A method for screening a substance that promotes or suppresses skin permeability of a transdermal drug or a transdermal candidate drug, characterized by measuring the degree of skin permeability through each skin, and comparing and evaluating the degree of skin permeability, 13) Maintain the subcutaneous tissue side solution at body temperature and the epidermis side solution at room temperature, and measure the degree of skin permeability of the transdermal drug or transdermal candidate drug through the skin transporter after a predetermined time. It is characterized by Serial (12) a method of screening for skin permeation promoting agent or an inhibitory substance for transdermal drug or transdermal drug candidates according.

The present invention is also characterized in that (14) the measurement / evaluation of the degree of skin permeability is one or more of the measurement / evaluation of skin permeability saturation, inhibitory effect, directionality and energy dependence. (12) A method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to (12) or (13), or (15) a radioisotope or fluorescence as a transdermal drug or transdermal candidate drug. Screening of a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to any one of (12) to (14), wherein the transdermal drug or transdermal candidate drug labeled with a substance is used. (16) As a solution in which the transdermal drug or transdermal drug candidate is dissolved, a solution containing an energy source in which the subcutaneous tissue side dissolves the transdermal drug or transdermal drug candidate, and the epidermal side is transdermal drug or transdermal Candidate drug The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to any one of (12) to (15), wherein the polyhydric alcohol-containing solution obtained is used, (17) The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to (16) above, wherein the polyhydric alcohol is propylene glycol, or (18) the transdermal drug or transdermal candidate Any of the above (12) to (15), wherein the solution in which the drug is dissolved is a solution containing an energy source in which the transdermal drug or transdermal candidate drug is dissolved on both the subcutaneous tissue side and the epidermis side Or (19) a solution containing an energy source is a Hank's solution, To (18) a method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to any one of (18), or (20) a non-labeled transdermal drug or transdermal drug candidate on the subcutaneous tissue side and the epidermis Any one of the above (12) to (19), wherein the saturated percutaneous penetration of a transdermal drug or transdermal candidate drug labeled with a radioisotope or fluorescent substance is measured and evaluated. (21) A method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug, or (21) changing the pH of a solution on the epidermis side of (12) to (19) above and a screening method of skin permeation enhancers or inhibitors of transdermal drug or transdermal candidate agent according to any one, (22) a NaN ₃ and NaF used together and subcutaneous tissue side and a skin side, transdermal drug, or through The penetration of skin candidate drugs The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to any one of the above (12) to (19), characterized in that it is measured / evaluated as to whether or not it is dependent on rugi. .

FIG. 1 is a diagram showing an outline of a percutaneous permeation experiment of indomethacin by the Ussing-type Chamber method using hairless mouse skin. FIG. 2 shows the results of percutaneous penetration of [ ¹⁴ C] indomethacin (A) and [ ³ H] mannitol (B) in hairless mouse skin. FIG. 3 shows the effect of unlabeled indomethacin on the transdermal penetration of [ ¹⁴ C] indomethacin (A) and [ ³ H] mannitol (B). FIG. 4 is a diagram showing the influence of NaN ₃ and NaF on the percutaneous penetration of [ ¹⁴ C] indomethacin (A) and [ ³ H] mannitol (B) in the secretory direction. FIG. 5 is a graph showing the influence of pH value on [ ¹⁴ C] indomethacin (white) and [ ³ H] mannitol (black). FIG. 6 is a graph showing the non-linear transdermal permeability of [ ¹⁴ C] indomethacin in the absorption direction. FIG. 7 shows the results of percutaneous penetration of Fluo-3 in the hairless mouse skin. FIG. 8 shows the results of mRNA expression of transporters in hairless mouse skin and normal human skin.

  As a method for assaying the skin permeability of the transdermal drug or transdermal candidate drug of the present invention, a solution in which the transdermal drug or transdermal candidate drug is dissolved is divided into a skin section and a subcutaneous tissue side and an epidermal side. Injecting into one of them, injecting a predetermined solution into the other, and skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time under the living conditions of the skin section The method is not particularly limited as long as it is a method for measuring and evaluating the degree of the skin, and the temperature (liquid temperature) of the solution on the subcutaneous tissue side or the epidermis side is not particularly limited. For example, the solution on the subcutaneous tissue side Maintain the body temperature and the epidermal solution at room temperature, or maintain both the subcutaneous tissue side and epidermal side solution at body temperature. Degree of skin permeability through Can be measured, the body temperature of a solution of the subcutaneous tissue side, it is preferred to maintain the solution of the epidermis side to room temperature. Furthermore, as the measurement / evaluation of the above-mentioned degree of skin permeability, one or more measurement / evaluation of saturation of skin permeation, inhibitory effect, direction directivity and energy dependence can be preferably mentioned. A method of measuring and evaluating a transdermal drug or transdermal drug candidate at a low concentration using another highly sensitive detector is also included in the present invention.

    Examples of the transdermal drug or transdermal drug candidate include not only existing transdermal drugs but also those used as oral drugs and candidate substances that will be developed as transdermal drugs in the future. The skin candidate drug may be a chemical substance or a composition such as an animal or plant extract or a microorganism extract. For example, centrally acting drugs (sedatives / antipruritics / brain metabolism improving drugs, anti-anxiety drugs, etc.), analgesics, antipruritics, astringents, anti-inflammatory drugs (NSAIDs, steroids, etc.), otachoids, urinary incontinence drugs, bronchi Dilators, antibiotics and corticosteroids, antifungal agents, antiviral agents, cardiovascular agents, parasitic skin disease agents, antineoplastic agents, local anesthetics, eye drops / nasal drops, peripheral vasodilators (E.g. prickly), skin disinfectant, wound dermatology, hormonal, antihistamine, purulent skin disease, topical enzyme, skin ulcer, cosmetics, hair restorer, hair restorer, animal and plant Examples include extracts, crude drugs, nucleic acids, polypeptides and the like. Examples of these transdermal drugs or transdermal candidate drugs are shown below.

  Analgesic, antipruritic, astringent, anti-inflammatory drugs (NSAIDs, steroids, etc.) include: amsinonide, prednisolone valerate acetate, diflucortron valerate, dexamethasone valerate, betamethasone valerate, diflorazone acetate, hydrocortisone acetate, difluprednate, di Betamethasone propionate, dexamethasone, triamcinolone acetonide, harsinonide, flumethasone pivalate, mometasone furancarboxylate, fluocinonide, fluocinolone acetonide, fludroxycortide, prednisolone, alclomethasone propionate, clobetasol propionate, dexamethas propionate, propionate dexamethasone propionate Deprodon, beclomethasone propionate, clobetasone butyrate, hydrocortisone butyrate, hydrocortisone butyrate propionate, propiobutyrate Betamethasone acid, Prednisolone acetate, Zinc white starch, Zinc white, Ibuprofen piconol, Indomethacin, Ufenamate, Ketoprofen, Glycyrrhetinic acid, Zinc oxide, Diclofenac sodium, Suprofen, Piroxicam, Felbinac, Bufexamac, Flurbiprofen, Bendazac, Heparin Substance gel, hydrocortisone, crotamiton and the like can be mentioned.

  Antibiotic and corticosteroid mixed preparations include oxytetracycline hydrochloride / hydrocortisone hydrochloride, tetracycline hydrochloride / hydrocortisone acetate, betamethasone valerate / gentamicin, betamethasone valerate / valanomycin valerate, triamcinolone / fradiomycin, fradiomycin / fluocinolone acetate Nido, fradiomycin sulfate / prednisolone sulfate, erythromycin, pimaricin, acyclovir, bleomycin sulfate, hydrocortisone / fradiomycin solution, oxytetracycline hydrochloride / polymyxin sulfate B, chloramphenicol / fradiomycin solution, and the like.

  Parasitic skin disease / antifungal / antiviral agents include salicylic acid, croconazole hydrochloride, neticonazole hydrochloride, clotrimazole, ketoconazole, isoconazole nitrate, econazole nitrate, oxyconazole nitrate, sulconazole nitrate, miconazole nitrate, bifonazole , Lanoconazole, siccanin, ofloxacin, minosacrine hydrochloride, terbinafine hydrochloride, butenafine hydrochloride, tolnaphthalate, nadifloxacin, acyclovir, vidarabine and the like.

  In addition, lidocaine, ethyl aminobenzoate, atropine sulfate, and naphazoline sulfate are used as local anesthetics and ophthalmic agents, isosorbide nitrate and nitroglycerin are used as vasodilators, tulobuterol is used as a bronchodilator, and purulent. Examples of the dermatological agent include sulfadiazine, kanamycin sulfate, erythromycin, tetracycline hydrochloride, chloramphenicol, gentamicin sulfate, fradiosulfate, colistin / fradiomycin, bacitracin / fradiomycin sulfate.

  In addition, diphenhydramine, diphenhydramine lauryl sulfate and crotamiton are used as antihistamines, estradiol is used as hormones, popidone iodine and iodine are used as disinfectants for skin, and lysozyme chloride and bromelain are used as external enzymes. Fluorouracil as an antineoplastic agent, sucrose / povidone iodine combination agent, alprostadil alphadex as skin ulcer drug, chlorhexidine hydrochloride / diphenhydramine combination agent, Diflucortron / Lidocaine herbate, sicon extract, tribenoside lidocaine, urea, tacrolimus hydrate, gelatin, hydrocortisone / hinokitiol acetate, etretinate, calcipotriol, tacalcito Mention may be made of Le respectively.

  Examples of the dosage form of the transdermal drug or transdermal drug candidate include haps, tapes, plasters, ointments, creams, solutions, lotions, sprays, mousse types, aerosol types, and the like.

  The skin section to be used is preferably a hairless skin section, and the origin thereof is not particularly limited. However, a skin section of a mammal such as a mouse, rat, dog, cow, or human, particularly a hairless mouse skin section that is easy to prepare. preferable. The skin section can be prepared by a conventional method such as lightly separating the subcutaneous fat of the excised skin. Moreover, as a skin section, a skin section in which expression of a specific skin transporter is suppressed or a skin section in which expression of a specific skin transporter is amplified can be used.

As the chamber partitioned by the skin section on the subcutaneous tissue side and the epidermis side, a Ussing-type Chamber in which the skin section can be mounted in the vertical direction can be preferably exemplified. A solution in which a transdermal drug or a transdermal drug is dissolved is stored in either the subcutaneous tissue side or the epidermis side of these compartments, and a predetermined solution is stored in the other. Is maintained at body temperature (about 36-37 ° C.), and the epidermal solution is maintained at room temperature. In addition, the degree of skin permeability is measured and evaluated under the survival condition of the skin section, such as supplying 95% O ₂ /5% CO ₂ gas to the solution on both sides of the chamber.

In order to facilitate measurement of skin permeability, a transdermal drug or transdermal candidate drug labeled with a radioisotope or a fluorescent substance can be advantageously used. For example, as a transdermal drug or transdermal candidate drug, ^A transdermal drug chemical or a transdermal drug candidate labeled with a radioisotope such as ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ I can be advantageously used. In addition, in order to perform skin permeability measurement by confocal microscopy, a fluorescent dye such as FM4-64 can also coexist as a cell membrane marker.

  The solution on the subcutaneous tissue side in which the transdermal drug or transdermal candidate drug is dissolved is preferably a solution containing an energy source, that is, a solution containing glucose or other nutrients that serve as an energy source. Hank's solution, Ringer's solution, Krebs-Henseleit solution can be specifically exemplified, and the epidermal side solution in which the transdermal drug or transdermal candidate drug is dissolved may be a transdermal drug or transdermal drug. The solution is not particularly limited as long as it is a solution capable of dissolving the skin candidate drug. In addition to the solution containing the above energy source, a liquid containing a polyhydric alcohol such as propylene glycol, glycerol, ethylene glycol, sorbitol, or an ethanol-containing solution is specifically used. Among them, a propylene glycol-containing liquid or Hanks liquid can be advantageously used.Therefore, as a preferred embodiment, as a solution in which the transdermal drug or transdermal drug candidate is dissolved, a solution containing an energy source in which the subcutaneous tissue side dissolves the transdermal drug or transdermal drug candidate, and the epidermal side is transdermal drug or transdermal The percutaneous drug or transdermal candidate drug skin permeability assay method using the polyhydric alcohol-containing solution in which the candidate drug is dissolved, or the transdermal drug or transdermal candidate drug dissolved solution are used for the subcutaneous tissue side and the epidermis. Examples of the method for assaying the skin permeability of the transdermal drug or transdermal candidate drug, which are solutions containing an energy source in which the transdermal drug or transdermal drug candidate is dissolved on both sides, can be mentioned.

When testing the skin permeability of a transdermal drug or a transdermal candidate drug, as described in the Examples below, the unlabeled transdermal drug or transdermal candidate drug is applied to both the subcutaneous tissue side and the epidermis side. Use it to examine the saturation transdermal penetration of transdermal drugs or transdermal candidate drugs labeled with radioisotopes or fluorescent substances, or to change the pH of the solution on the epidermis side. It is preferable to examine the influence or to examine whether penetration of the transdermal drug or transdermal candidate drug is energy-dependent using NaN ₃ and NaF on both the subcutaneous tissue side and the epidermis side.

  Next, as a method for screening a substance for promoting or suppressing skin permeability of the transdermal drug or transdermal candidate drug of the present invention, a solution in which the transdermal drug or transdermal candidate drug is dissolved, and the transdermal drug or transdermal candidate A solution in which a drug and a test substance are dissolved is injected into a subcutaneous tissue side in a chamber divided into a subcutaneous tissue side and an epidermis side by a skin section, and a predetermined solution is injected into the other side of the skin section. The method is particularly limited if it is a method for measuring the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time under the living conditions, and comparing and evaluating the degree of skin permeability. The test substance may be a chemical substance or a composition such as an extract of animals or plants or microorganisms. For example, it transports an organic acid (anion) present on the basal side of epithelial cells. Pro is a carrier inhibitor Neshido (probenecid), FCCP to uncouple mitochondrial oxidative phosphorylation (carbonyl cyanide p- trifluoromethoxyphenyl hydrazone), blocker TEA (tetraethylammonium) of adrenaline and cholinergic nerves and the like. Further, the temperature (liquid temperature) of the solution on the subcutaneous tissue side or 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 are maintained. The temperature of the side solution can be maintained at body temperature, and the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug can be measured after a predetermined time. Is preferably maintained at body temperature and the epidermal solution at room temperature. Furthermore, as the measurement / evaluation of the above-mentioned degree of skin permeability, one or more measurement / evaluation of saturation of skin permeation, inhibitory effect, direction directivity and energy dependence can be preferably mentioned. A method of measuring and evaluating a transdermal drug or transdermal drug candidate at a low concentration using another high-sensitivity detector is also included in the present invention, and the evaluation should be performed by comparing with a control in the absence of a test substance. Is preferred.

In the method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or transdermal candidate drug of the present invention, the transdermal drug or transdermal drug labeled with a radioisotope or fluorescent substance is used as the transdermal drug or transdermal candidate drug. It is preferable to use a skin candidate drug. Further, as a solution in which the transdermal drug or transdermal candidate drug is dissolved, the subcutaneous tissue side contains an energy source such as Hanks' solution in which the transdermal drug or transdermal candidate drug is dissolved, as in the above-described assay method of the present invention. A solution can be advantageously used, and a solution containing an energy source such as a polyhydric alcohol-containing liquid such as propylene glycol or Hank's liquid in which the epidermal side is dissolved with a transdermal drug or transdermal candidate drug can be advantageously used. Further, at the time of screening, as in the above-described assay method of the present invention, unlabeled transdermal drug or transdermal candidate drug was used for both the subcutaneous tissue side and the epidermis side and labeled with a radioisotope or fluorescent substance. Measure and evaluate the saturation transdermal penetration of transdermal drugs or drug candidates, change the pH of the solution on the epidermis side, measure and evaluate the effect of skin permeability due to pH differences, NaN It is preferable to measure and evaluate whether penetration of the transdermal drug or transdermal candidate drug is energy-dependent using ₃ and NaF on both the subcutaneous tissue side and the epidermis side.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations. In the examples, all data are shown as mean ± standard error, and statistical analysis was performed by Student's t test. Differences between the mean values were considered significant when the p-value was less than 0.05.

[Materials and animals]
[ ¹⁴ C] Indomethacin (740 MBq / mol) and [ ³ H] mannitol (740 GBq / mol) were purchased from PerkinElmer Life Sciences, Inc. and American Radiolabeled Chemicals Inc., respectively. Fluo-3-AM and FM4-64 were purchased from Dojindo Laboratories Co., Ltd. and Molecular Probe, respectively. SUPERSCRIPT ^™ II RNase H ^- was purchased from Invitrogen Corp. Normal human adult skin cDNA was 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 Co., Ltd. and 5 to 7-week-old male hairless mice (FVB) were purchased from Clea Japan Co., Ltd. . FVB / Mrp1 (− / −) mice were prepared according to the method described in the literature (Nature Med. 3: 1275-1279, 1997). Animal experiments were conducted according to the guidelines for animal experiments at Kanazawa University Takaramachi Campus.
[Reverse transcription-polymerase chain reaction (RT-PCR)]
Hairless mouse skin was collected, RLT buffer added with β-mercaptoethanol was added, and total RNA was extracted using RNeasy Mini Kit (manufactured by QIAGEN). One microgram of total RNA was reverse transcribed using oligo (dT) _12-18 (Life technologies) and SUPERSCRIPT ^™ II RNase H ^- Reverse Transcriptase 200 U to produce cDNA. The mixture after the reaction was subjected to 30 cycles of PCR using an appropriate set of mouse primers (Table 1) and human primers (Table 2). PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. The amount of each PCR product was measured with an AE-6955 Light Capture instrument (manufactured by ATTO).

[Penetration experiment by Ussing-type Chamber Method]
The permeation experiment was conducted by the method described in the literature (J. Pharm. Pharmacol. 49: 108-112, 1997, J. Pharm. Sci. 92: 1502-1508, 2003). Briefly, a hairless mouse was euthanized, and a skin section obtained by directly excising the skin and lightly separating the subcutaneous fat was longitudinally placed in a Ussing-type Chamber with an exposed area of 0.766 cm ^2. Attached to. As shown in FIG. 1, Hanks's solution (HBSS, 136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl ₂ , 0.812 mM MgSO is contained in a chamber on the subcutaneous tissue side partitioned by a skin section. _{4, KH} 2 _PO 4 of 0.441mM, _Na 2 _PO 4 of 0.385 mm, 25 mM of D- glucose and 10mM of HEPES, pH 7.4) and 1.2 mL, into the chamber of the epidermis side, propylene glycol: water 1.2 mL of the (1: 1) mixed solution was added, and the temperature of the solution was maintained at 37 ° C. on the subcutaneous tissue side and at room temperature on the epidermis side. To maintain skin tissue viability, 95% O ₂ /5% CO ₂ gas was supplied to the solution on both sides of the chamber during the transport experiment. O ₂ / CO ₂ bubbles not only supplied oxygen to the tissue, but also mixed the solution around the tissue.

In order to measure the percutaneous permeability of [ ¹⁴ C] indomethacin and [ ³ H] mannitol, a test compound was used on the subcutaneous tissue side or epidermal side, and 0.4 mL of solution was added from the opposite side at a predetermined time. It was collected 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 percutaneous 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 propylene glycol: 20 mM phosphorus. Consists of acid buffer (1: 1). The values of permeability coefficient (μL / cm ² / hr, P _sec and P _abs ) were determined from the slope of the linear part of the permeability plot with respect to time (hr) (μL / cm ² ).
[Thin layer chromatography (TLC) method]
Using TLC, it was investigated whether or not indometha single clonide was generated during the experiment according to the method described in the literature (Pharm. Res. 17: 432-438, 2000). Briefly, after a [ ¹⁴ C] indomethacin percutaneous penetration experiment, the medium opposite the test compound was extracted with diethyl 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 chloroform: acetic acid (95: 5 v / v) and quantified using a radioisotope scanner. The strength obtained was compared with that of standard [ ¹⁴ C] indomethacin.
[Confocal microscopy]
Skins of hairless mice and FVB and FVB / Mrp1 (− / −) mice that had been depilated with a hair removal cream and recovered for 24 hours were each cut on skin with a microslicer (DTK-2000, Dosaka EM Co., LTD.). (Thickness: 100 μm) was produced. The skin section was infiltrated with 4 ° C Hanks solution (pH 7.4) supplemented with Fluo-3-AM (10 μM) and FM4-64 (1 μM) for 2 hours, and then the skin section was LabTek chambered micro cover glass (24 × 60 mm). ) And mounted on a confocal microscope (Zeiss Axiovert 100M LSM510), and fluorescence was observed with a water immersion lens (630 ×) at room temperature (20-22 ° C.) using 488 nm and 543 nm lasers.
[Directed percutaneous penetration of indomethacin]
Percutaneous permeability of [ ¹⁴ C] indomethacin (47 μM) and [ ³ H] mannitol (0.25 μM) using the Ussing-type chamber method, from subcutaneous to epidermal side (square), and subcutaneous from the epidermis The hairless mouse skin was examined for 4 hours toward the tissue side (circle). The results are shown in FIG. 2 (each point represents an average value ± standard error in 3 to 4 experiments). The percutaneous penetration of [ ¹⁴ C] indomethacin is linear and the permeability in the direction of secretion (from the subcutaneous tissue side to the epidermis side; P _sec ) is compared to the absorption direction (from the epidermis to the subcutaneous tissue side; P _abs ). It was 4 times higher (FIG. 2-A). In contrast, unidirectional transport was not observed in the transdermal penetration of [ ³ H] mannitol (FIG. 2-B). The permeability of [ ¹⁴ C] indomethacin was much higher (more than 5 times) compared to [ ³ H] mannitol.
[The radioactivity appearing on the opposite side is mainly due to the parent compound. ]
Indomethacin clonide is a substrate of cMOAT / MRP2 (Pharm. Res. 17: 432-438, 2000), and indomethacin is OAT1 (J. Pharmacol. Exp. Ther. 303: 534-539, 2002) and 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 not by cMOAT / MRP2 (Pharm. Res. 17: 432-438, 2000). In order to examine whether indomethacin was metabolized during the percutaneous penetration experiment, after [ ¹⁴ C] indomethacin was added to the subcutaneous tissue side, the radioactivity appearing on the epidermis side was analyzed by 6-hour thin layer chromatography (TLC). ). The recovery rate of [ ¹⁴ C] indomethacin was 80% or more. More than 95% of the labeled compounds were [ ¹⁴ C] indomethacin. Therefore, indomethacin metabolism in this experiment was considered to be negligible.
[Saturable transdermal penetration of indomethacin]
Since one of the important features of active transport is saturation, it was investigated whether the transdermal penetration of [ ¹⁴ C] indomethacin was saturable. [ ¹⁴ C] Indomethacin (47 μM) and [ ³ H] mannitol (0.25 μM) percutaneous infiltration in the presence of unlabeled indomethacin (500 μM) added to both sides of the Ussing type chamber (white column), in the absence FIG. 3 shows the results obtained by examining (black pillars) for 4 hours (each pillar represents an average value ± standard error in 3 to 4 experiments). As a result, unlabeled indomethacin (500 μM) increased P _sec of [ ¹⁴ C] indomethacin, but did not affect P _abs (FIG. 3-A). In contrast, the effect of unlabeled indomethacin on [ ³ H] mannitol permeability was not observed (FIG. 3-B). Therefore, the effect of unlabeled indomethacin is specific for penetration ^{[14 C]} indomethacin, ^{[14 C]} transdermal penetration of indomethacin is saturable secretion direction.
[ATP-dependent percutaneous penetration of indomethacin]
In order to investigate whether the penetration of [ ¹⁴ C] indomethacin is energy dependent, the effect of ATP depletor on the penetration of [ ¹⁴ C] indomethacin was examined. 30 minutes after loading NaN ₃ and NaF (10 mM) on both sides of the Ussing-type chamber, the transdermal permeability of [ ¹⁴ C] indomethacin (47 μM) and [ ³ H] mannitol (0.25 μM) The results observed after 4 hours in the presence (white circle) and absence (black circle) of added NaN ₃ and NaF (10 mM) are shown in FIG. 4 (each point is an average value in 4 to 5 experiments). ± standard error.) As a result, it was found that P _sec of [ ¹⁴ C] indomethacin increases in the presence of NaN ₃ and NaF (FIG. 4-A), but for the permeability of [ ³ H] mannitol, No effect was observed (FIG. 4-B). These results indicated that the penetration of [ ¹⁴ C] indomethacin in the absorption direction was energy dependent.
[Effect of pH on percutaneous penetration of indomethacin]
Since the skin environment is weakly acidic, the effect of pH on the percutaneous 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 the pH of human skin ( pH 4-6) (21). On the epidermal side of the chamber, [ ¹⁴ C] indomethacin (47 μM) and [ ³ H] mannitol (0.25 μM) transdermal permeability coefficients at different pH values (pH 5.0, 6.0, 7.4). The results observed in the absorption direction (A) and the secretion direction (B) are shown in FIG. 5 (each column represents an average value ± standard error in 4 to 5 experiments). Since the pKa value of indomethacin is 4.5, changes in the pH (5.0, 6.0 and 7.4) of the epidermal medium changed the degree of ionization of indomethacin. It was found that the P _abs of [ ¹⁴ C] indomethacin increases when the pH value on the epidermis side decreases. In contrast, no changes were observed in the P _abs of [ ³ H] mannitol (FIG. 5-A). Conversely, on the epidermal side, no effect of pH was observed on P _sec of [ ¹⁴ C] indomethacin or [ ³ H] mannitol (FIG. 5-B). As a result, the unidirectional penetration of [ ¹⁴ C] indomethacin disappeared as the pH value decreased on the epidermis side.
[Saturable penetration of indomethacin at pH 5.0 on the epidermis side]
On the epidermal side, [ ¹⁴ C] indomethacin P _abs at pH 5.0 was examined in the absence or presence of unlabeled indomethacin. In the presence of unlabeled indomethacin (500 μM), the transdermal permeability of [ ¹⁴ C] indomethacin (47 μM) and [ ³ H] mannitol (0.25 μM) (B) added to both sides of the Ussing-type chamber (white) And the result investigated after 4 hours in absence (black) is shown in FIG. 6 (each point represents the average value ± standard error in 3-5 experiments). Note that the pH value on the epidermal side of the chamber was maintained at 5.0 in the presence and absence of unlabeled indomethacin. As a result, on the epidermal side, at pH 5.0, [ ¹⁴ C] indomethacin P _abs was increased by unlabeled indomethacin (500 μM) (FIG. 6A). However, the effect of unlabeled indomethacin (500 μM) on [ ³ H] mannitol P _abs was not observed (FIG. 6-B). The penetration of [ ¹⁴ C] indomethacin in the absorption direction was considered saturable.
[Directed percutaneous penetration of Fluo-3]
Fluo-3 directional percutaneous penetration of Fluo-3 is permeabilized by converting the carboxyl group of Fluo-3 into a fat-soluble acetoxymethyl ester, and is permeabilized by intracellular esterase to become Fluo-3. AM (1- [2-Amino-5- (2,7-dichloro-6-hydroxy-3-
oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid, pentaacetoxymethyl ester; 10 μM) and fluorescent dye FM4-64 (1 μM) The added Hanks solution was permeated and placed in a Ussing-type chamber, and Fluo-3 excreted from both sides of the chamber was examined. Similarly, directional transdermal penetration of Fluo-3 was also examined for specimens obtained by adding probenecid 5 mM, FCCP 5 μM, and TEA 5 mM to Fluo-3-AM. The results are shown in FIG. As a result, the percutaneous penetration of Fluo-3 produced in the skin tissue is linear, and the absorption direction (from the skin tissue to the subcutaneous tissue side; control on the left in FIG. 7) is the permeability in the direction of secretion (skin From inside the tissue to the epidermis side; the control on the right in FIG. In probenecid, FCCP, and TEA, when FCCP was coexisted, the absorption was about twice that of Fluo-3 alone.
[Expression of transporter in skin]
RT-PCR and PCR were performed on hairless mouse skin total RNA (panel A) and normal human skin cDNA (panel B), respectively. Human cDNA is a mixture of 3 ages of age 80 / female (panel BI), age 44 / male (panel B-II) and age 44 / male, age 58 / female, age 65 / female. (Panel B-III) was used. PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The results are shown in FIG.

  The lanes 1-32 in Panel A are: 1, Mdr1a; 2, Mdr1b; 3, Mrp1; 4, Mrp2; 5, Mrp3; 6, Mrp4; 7, Mrp5; 8, Mrp6; 9, Smvt; 10, PepT1; , PepT2; 12, Mct1; 13, Mct2; 14, Mct3; 15, Mct4; 16, Npt1; 17, Oatt1; 18, Oapt2; 19, Oapt3; 20, OAtp4; 21, Oatt5; 22, Oatt11; 24, mPGT; 25, Oct1; 26, Oct2; 27, Oct3; 28, Octn1; 29, Octn2; 30, Octn3; 31, Oat1; 32, Oat2.

  In addition, lanes 1 to 38 of panel B are 1, β-actin; 2, Urat1; 3, Mrp1; 4, Mrp2; 5, Mrp3; 6, Mrp4; 7, Mrp5; 8, Mrp6; 9, Mrp7; 10 11, Oat2; 12, Oat3; 13, Oat4; 14, Oatp-A; 15, Oatp-B; 16, Oatp-C; 17, Oatp-D; 18, Oatp-E; , Mdr1; 21, Oct1; 22, Oct2; 23, Oct3; 24, Octn1; 25, Octn2; 26, Npt1; 27, Npt; 28, Pept1; 29, Pept2; 30, Ae2; 31, Nhe3; 32, Glut5 33, Ent1; 34, Mct1; 35, Mct2; 26, Mct3; 37, Mct4; 38, Mct5, respectively It is.

  In hairless mouse skin, MRP1, MRP3, MRP4, MRP5, peptide transporter 1 (PEPT1), PEPT2, monocarboxylic acid transporter 1 (MCT1), MCT2, MCT4, OATP3, OATP11, prostaglandin transporter (PGT) The expression of organic cation transporter 3 (OCT3), organic cation / carnitine transporter 1 (OCTN1), OCTN2 and OCTN3 was observed (FIG. 8-A).

In addition, the expression of the transporter was observed in cDNA of normal human skin (manufactured by ResGen Invitrogen and Biochain Institute). Expression of MRP1, MRP3, MRP4, MRP5, MRP6, GLUT5, AE2, MCT1, MCT4, MCT5, OATP-B, OATP-D, OATP-E, OCTN1, OCTN2, and ENT1 was observed in normal human skin (FIG. 8). -B). The expression of MRP2, MDR1, PEPT1, PEPT2, MCT2, MCT3, OCT1, OCT3 and URAT1 was detected depending on the donor, suggesting that there is an individual difference in expression.
[Discussion]
Saturation with unlabeled indomethacin (500 μM) was clearly observed in the secretory direction, whereas saturated penetration of [ ¹⁴ C] indomethacin was also found in the epidermal compartment at low pH and in the absorption direction. These results can be explained if multiple transport systems are involved in indomethacin transport. From the viewpoint of the efficiency of percutaneous absorption, such non-linear dynamics in the absorption direction are important. In order to saturate the percutaneous penetration of indomethacin in the absorption direction, the concentration on the skin surface when indomethacin is administered transdermally is usually very high compared to the concentration of unlabeled indomethacin (500 μM) used in the above examples. Thus, the results of the above examples suggest that the permeability of drugs administered transdermally depends on the concentration administered.

It was found that a decrease in pH value in the epidermal compartment increases [ ¹⁴ C] indomethacin P _abs but does not affect P _sec . As a result, unidirectional penetration of [ ¹⁴ C] indomethacin disappears at pH 5.0 in the epidermal compartment. One possible increase in P _abs is the increased distribution of non-ionized [ ¹⁴ C] indomethacin in the stratum corneum (SC). Thereby causing a high concentration gradient of the subcutaneous tissue side from the stratum corneum ^(SC), leads to a non-linear _{P abs} of ^[14 C] Indomethacin (Figure 6-A). However, there is another hypothesis that a proton-dependent transport system is involved in [ ¹⁴ C] indomethacin P _abs . In previous studies we have demonstrated that monocarboxylic acid transporter 1 (MCT1) is 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: 1113-1121, 1999). On the other hand, the expression of OATP family transporters that transport organic anionic compounds was also confirmed.

In order to identify transporter (s) that may be involved in percutaneous penetration of [ ¹⁴ C] indomethacin, transporter expression in hairless mouse skin was examined by RT-PCR. In addition to confirming the expression of MRP1, MRP3, MRP4 and MRP5, expression of PEPT1, PEPT2, MCT1, MCT2, MCT4, OATP3, OATP11, mPGT, OCT3, OCTN1, OCTN2 and OCTN3 was also observed in the hairless mouse skin (FIG. 8). . Indomethacin has been identified as OAT1 (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) has been previously reported. However, in the results of this example, the expression of OAT1, OAT3 and NPT1 was not detected, suggesting that other transporters may be involved in indomethacin penetration.

  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 is similar to that in normal human skin, suggesting that hairless mouse skin can be a good model for examining the function of these transporters. Similar to recent studies, expression of MRP family members other than MRP2 was detected in all individuals (J. Invest. Dermatol. 116: 541-548, 2001). In hairless mouse skin, the expression of Mrp1, Mrp3, Mrp4 and Mrp5 was observed, but Mrp2 was not observed, indicating that the characteristics of expression 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 accepts drugs such as steroid sulfate conjugates, pravastatin, and fexofenadine as substrates, but does not accept glucuronide conjugates and mediates gastrointestinal absorption of anionic compounds (J Pharmacol. Exp. Ther. 306: 703-708, 2003). OATP-C was considered to transport both types of steroid conjugates (Pharm. Res. 18: 1262-1269, 2001), while OATP-D transfects prostaglandins into special tissues and cells. It plays an important role in locating (Am. J. Physiol. Renal Physiol 285: F1188-1197, 2003). Staining by immunohistochemistry revealed that OATP-B was expressed in all layers of the epidermis but not subcutaneously. Furthermore, uptake of estrone sulfate by normal human epidermal keratinocytes was reduced by 33% by taurocholic acid, a substrate of the OATP family (J. Invest. Dermatol. 120: 285-291, 2003). These findings suggest that organic anion transport systems may be related to their substrate uptake by keratinocytes, and it is clear whether such transporters play an important role in the transdermal transport of drugs Suggests the need for further analysis. While expression of MCT1, MCT2 and MCT5 was detected in human skin, expression of MCT2 and MCT3 could only be detected in some 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 have shown that MCT1 plays 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 MCT in the skin may indicate a role in skin pH regulation and weak organic acid transport. The expression of OCTN family members was observed in all individuals (Figure 8). The OCTN family is involved in the transport of carnitine, a cofactor essential for the oxidation of long chain fatty acids. An in vitro carnitine transport system in human cultured dermal fibroblasts has been characterized. Km for uptake of carnitine was 5 μM 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 is a multi-selective, pH-dependent organic cation transporter that can function in the apical membrane of the kidney and other tissues as a proton / organic cation antiporter and / or organic cation / 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 compounds. Thus, although further research on the function in the skin is necessary, the expression of these transporters in the skin, as an active barrier system of the skin, has the potential to be involved in substrate transport.

    The present invention revealed that the transporter (s) is involved in the percutaneous penetration of transdermal drugs such as indomethacin or transdermal candidate drugs. Moreover, since the mRNA expression of several transporters of MRP, OATP, MCT and OCTN family was observed in both hairless mouse skin and normal human skin, the physiological meaning of transporter expression in these skins is It is necessary to elucidate further, but the existence of various types of transporters in this way indicates a possible role in controlling the transdermal penetration of xenobiotics as an active barrier. The present invention not only advances further elucidation of the physiological role of transporters in the skin, but also enables the development of superior transdermal delivery systems for transdermal drugs and cosmetics.

Claims (22)

A solution in which the transdermal drug or transdermal candidate drug is dissolved is injected into one of the chambers divided into the subcutaneous tissue side and the epidermis side by a skin section, and a predetermined solution is injected into the other, and the skin A transdermal drug or transdermal candidate drug characterized by measuring and evaluating the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time under the survival conditions of the section Test method for skin permeability. Maintain the subcutaneous tissue side solution at body temperature and the epidermis side solution at room temperature, and measure and evaluate the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to claim 1, wherein The measurement / evaluation of the degree of skin permeability is one or more of the measurement / evaluation of saturation of skin permeation, inhibitory effect, direction directivity and energy dependence. Method for assaying skin permeability of transdermal drug or transdermal candidate drug. The transdermal drug or transdermal drug according to any one of claims 1 to 3, wherein a transdermal drug or transdermal candidate drug labeled with a radioisotope or a fluorescent substance is used as the transdermal drug or transdermal drug candidate. Test method for skin permeability of candidate drugs. As a solution in which the transdermal drug or transdermal drug candidate is dissolved, a solution containing an energy source in which the subcutaneous tissue side dissolves the transdermal drug or transdermal drug candidate, or in which the epidermal side dissolves the transdermal drug or transdermal drug candidate A method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 1 to 4, wherein a solution containing a monohydric alcohol is used. 6. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to claim 5, wherein the polyhydric alcohol is propylene glycol. The solution in which the transdermal drug or transdermal candidate drug is dissolved is a solution containing an energy source in which the transdermal drug or transdermal candidate drug is dissolved on both the subcutaneous tissue side and the epidermis side. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of the above. 8. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 5 to 7, wherein the solution containing an energy source is Hanks' liquid. To investigate the saturation percutaneous penetration of a transdermal drug or transdermal candidate drug labeled with a radioisotope or fluorescent substance, using an unlabeled transdermal drug or transdermal candidate drug on both the subcutaneous tissue side and the epidermis side. A method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 1 to 8. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 1 to 8, wherein the pH of the epidermal solution is changed. The NaN ₃ and NaF used together and subcutaneous tissue side and a skin side, osmotic transdermal drug or transdermal candidate agent, any of the preceding claims, characterized in that to determine whether an energy dependent A method for assaying the skin permeability of the transdermal drug or transdermal candidate drug. In a chamber where a transdermal drug or a transdermal drug candidate and a transdermal drug or transdermal drug candidate and a test substance are dissolved are divided into a skin section and a subcutaneous tissue side and an epidermis side. Injecting into the subcutaneous tissue side of the skin, and injecting a predetermined solution into the other, the skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time under the condition of survival of the skin section. A screening method for a substance that promotes or suppresses skin permeability of a transdermal drug or transdermal candidate drug, characterized by measuring the degree of each and comparing and evaluating the degree of skin permeability. Maintaining the solution on the subcutaneous tissue side at body temperature and the solution on the epidermis side at room temperature, and measuring the degree of skin permeability through the skin transporter of the transdermal drug or transdermal candidate drug after a predetermined time, respectively 13. A screening method for a substance that promotes or suppresses skin permeability of a transdermal drug or transdermal candidate drug according to claim 12. 14. The measurement / evaluation of the degree of skin permeability is one or more of the measurement / evaluation of skin permeability saturation, inhibitory effect, direction directivity and energy dependence. A screening method for a substance that promotes or suppresses skin permeability of a transdermal drug or transdermal candidate drug. The transdermal drug or transdermal drug according to any one of claims 12 to 14, wherein a transdermal drug or transdermal drug candidate labeled with a radioisotope or a fluorescent substance is used as the transdermal drug or transdermal drug candidate. A screening method for a substance that promotes or suppresses skin permeability of a candidate drug. As a solution in which the transdermal drug or transdermal drug candidate is dissolved, a solution containing an energy source in which the subcutaneous tissue side dissolves the transdermal drug or transdermal drug candidate, or in which the epidermal side dissolves the transdermal drug or transdermal drug candidate A method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or transdermal drug candidate according to any one of claims 12 to 15, wherein a solution containing a monohydric alcohol is used. 17. The method for assaying skin permeability of a transdermal drug or transdermal candidate drug according to claim 16, wherein the polyhydric alcohol is propylene glycol. 16. The solution in which the transdermal drug or transdermal candidate drug is dissolved is a solution containing an energy source in which the transdermal drug or transdermal candidate drug is dissolved on both the subcutaneous tissue side and the epidermis side. A screening method for a substance that promotes or suppresses skin permeability of a transdermal drug or transdermal candidate drug according to any one of the above. The method for screening a substance for promoting or suppressing skin permeability of a transdermal drug or transdermal drug candidate according to any one of claims 16 to 18, wherein the solution containing an energy source is Hanks' liquid. Measurement of saturation transdermal penetration of transdermal drug or transdermal candidate drug labeled with radioisotope or fluorescent substance using unlabeled transdermal drug or transdermal candidate drug on both subcutaneous tissue and epidermal side 20. The screening method for a substance that promotes or suppresses skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 12 to 19, wherein evaluation is performed. 20. The method for screening a substance for promoting or inhibiting skin permeability of a transdermal drug or transdermal candidate drug according to any one of claims 12 to 19, wherein the pH of the solution on the epidermis side is changed. 13. Using NaN ₃ and NaF on both the subcutaneous tissue side and the epidermis side, it is measured and evaluated whether the penetration of the transdermal drug or transdermal candidate drug is energy-dependent or not. 20. A screening method for a substance that promotes or inhibits skin permeability of a transdermal drug or transdermal candidate drug according to any one of 19 items.