JP2016049103A - Transmucosal absorption evaluation device, and evaluation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transmucosal absorption evaluation device by which the measurement of the gingival mucosa epithelium penetration amount of a substance and the efficacy evaluation of the penetrating substance can be carried out simultaneously and conveniently; and to provide an evaluation method thereof.SOLUTION: We found out that the measurement of the mucosal accumulation penetration amount of a substance and the evaluation of efficacy obtained by the effect of the penetrating substance on fibroblasts can be carried out simultaneously and conveniently by using a co-cultivation device combined with the gingival mucosa epithelium penetrating substance and fibroblasts, resulting in the completion of the device by which the efficacy of oral cosmetics, quasi drugs, and pharmaceuticals on gingival tissue can be evaluated.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a transmucosal absorption evaluation apparatus and an evaluation method thereof, which are capable of simultaneously measuring the amount of gingival mucosa permeated by a substance and evaluating the effectiveness of the permeated substance.

  Conventionally, transmucosal absorption of a substance has been to measure the amount of substance permeated using an excised tissue from an animal. On the other hand, evaluation of the effectiveness of a substance has been performed by directly exposing the substance to cells and evaluating its physiological activity. Therefore, there is no way to evaluate how much the substance absorbed in the gingival mucosa is actually delivered to the target cell and whether the delivered substance is really effective, but consider it indirectly It was only.

  By the way, as a technique and an evaluation method using a co-culture, a technique (Patent Document 1) relating to a co-culture that can be applied to screening a substance to be activated as an animal cell culture construct has been reported. In addition, as a device for easily testing the effectiveness and safety of substances in the skin, the sample of reconstituted tissue and the effects of physical, biological and / or chemical stimuli on the sample, or Using a device that correlates the influence of a sample on physical, biological and / or chemical stimulation with a detection system (Patent Document 2), reconstructed skin, and cells to be evaluated, the amount of permeation through and permeation of the substance Although a percutaneous absorption evaluation apparatus (Patent Document 3) characterized in that the effect of the substance obtained can be measured at the same time has been reported, these objects are skin and not gingival mucosa.

  Transmucosal absorption evaluation characterized by the ability to simultaneously measure the amount of gingival mucosa permeated through the gingival mucosa and the effectiveness of the permeated substance as a device for easily testing the effectiveness of the substance using the gingival mucosal epithelial equivalent There has never been a report on the device and its evaluation method.

JP 2008-1119010 A Special table 2008-520199 gazette JP 2011-200284 A

  As described above, a transmucosal absorption evaluation apparatus and an evaluation method thereof that can measure a gingival mucosa permeation amount of a substance and evaluate the effectiveness of the permeated substance at the same time have not been known. Accordingly, it is an object of the present invention to provide a transmucosal absorption evaluation apparatus and its evaluation method capable of simultaneously and simply performing measurement of the amount of gingival mucosa permeation of a novel substance and evaluation of the effectiveness of the permeated substance.

  As a result of intensive research on a transmucosal absorption evaluation apparatus and an evaluation method thereof capable of simultaneously measuring the amount of gingival mucosa permeation of a substance and evaluating the effectiveness of the permeated substance, By using a device using the equivalent and the cell to be evaluated, and more preferably using a device in which the cultured mucosa and the cell to be evaluated are co-cultured as a gingival mucosal epithelial equivalent, The inventors have found that the evaluation of effectiveness can be simultaneously measured, and have completed the present invention.

  Conventionally, it has been difficult to simultaneously measure the amount of gingival mucosa permeating the substance and evaluate the effectiveness of the permeated substance, but by using the transmucosal absorption evaluation apparatus described in the present invention, the substance can be easily and quickly Measurement of the amount of gingival mucosa permeated and evaluation of the effectiveness of the permeated substance can be performed simultaneously. Therefore, since the effect on the gingival mucosa possessed by the substance and / or the external composition containing the substance can be directly measured, the substance and / or the external composition containing the substance that acts effectively when actually applied to the gingival mucosa. Development can be done efficiently.

1 is a transmucosal absorption evaluation apparatus according to the present invention. It is a figure of the transmucosal absorption evaluation apparatus (setting) which concerns on Example 1 of this invention.

Hereinafter, the configuration of the present invention will be described in more detail.
The transmucosal absorption evaluation apparatus according to the present invention uses a gingival mucosal epithelial equivalent and evaluation target cells. The transmucosal absorption evaluation apparatus according to the present invention can simultaneously measure the amount of mucosal permeation of a substance and evaluate the effectiveness of the transmitted substance.

  As the gingival mucosal epithelial equivalent used in the present invention, it is desirable to use a cultured reconstituted gingival mucosa HGE regenerated human mucosa model. In order to co-cultivate different types of monolayer cultured cells, it requires very complicated operations such as medium optimization and pH selection. A co-culture can be created stably and efficiently.

  Examples of cells to be evaluated include fibroblasts, adipocytes, vascular endothelial cells and the like. These should be appropriately selected according to the purpose of evaluating the effectiveness, and fibroblasts are preferred in the present invention. When fibroblasts are used as the cells to be evaluated, the substance absorbed through the mucosa is collagen producing action, hyaluronic acid producing action, elastin producing action, cell growth promoting and / or inhibiting action, various cytokine production promoting and / or Alternatively, evaluation of inhibitory action, fibrillin producing action, collagenase activity and / or production inhibition can be performed. Examples of various cytokines include, but are not limited to, interleukin, interferon, TNF, TGF, FGF, KGF, HGF, IGF, eicosanoid, SCF, MCP, and CSF. By performing these evaluations, the effectiveness of anti-inflammatory action, anti-aging action, etc. can be evaluated.

  Substances to be evaluated mainly target components that can be used for oral cosmetics, quasi drugs and / or pharmaceuticals, and can be applied directly to the gingival mucosal epithelial equivalent, as well as water, oily components, various solvents, etc. It can also be applied as an emulsified preparation as a solution or dispersion. Oral cosmetics, quasi drugs, pharmaceutical preparations and the like can be applied.

  Among the components that can be used in oral cosmetics and / or pharmaceuticals, bioactive ingredients include ascorbic acid, magnesium ascorbate phosphate ester, ascorbyl palmitate, ascorbyl stearate, ascorbyl tetraisopalmitate, ascorbyl glucoside, arbutin, tranexam Acids and derivatives thereof, ellagic acid, lucinol, glycyrrhizic acid, glycyrrhetinic acid, amino acids, saccharides, mucopolysaccharides, ceramides, sterols and derivatives thereof, phospholipids and derivatives thereof, retinol and derivatives thereof, vitamin A acids and derivatives thereof , Tocotrienol, ubiquinone, aloe, ougon, various vitamins and their derivatives, tocopherol, tocopherol acetate, SOD, β-carotene, catechin, polyphenol, caffeine , Cacao, ivy, slimming agents such as visnadine, Kamomira, perilla, carnitine, phospholipids and their derivatives, such as functional polysaccharides and the like.

  In addition, since it can be used as a method for examining the dosage form of oral cosmetics or pharmaceuticals in which physiologically active ingredients are more efficiently absorbed through the mucosa and are more effective, When used for examination, they can be developed very efficiently.

  In order to simultaneously measure the amount of gingival mucosa epithelial permeation of a substance and evaluate the effectiveness of the permeated substance, the medium shown in FIG. 1 may be sampled. The amount of the substance permeated through the mucosa can be quantified using, for example, high performance liquid chromatography or gas chromatography. The effectiveness of the permeated substance can be evaluated by, for example, using a fibroblast as an evaluation target cell to promote collagen production. Can be evaluated by ELISA, and when collagenase activity is evaluated, it can be evaluated by an activity measurement method using a pseudo-substrate.

FIG. 1 is a transmucosal membrane characterized in that measurement of the amount of mucosal permeation of a substance and evaluation of the effectiveness of the permeated substance can be performed simultaneously, using a gingival mucosal epithelial equivalent and a cell to be evaluated. Absorption evaluation device.
The transmucosal absorption evaluation apparatus includes a well 1 that accommodates a basket 2 that supports a gingival mucosal epithelial equivalent 3. The basket 2 in which the well 1 is disposed may be capable of detachably fixing the well 1. The basket 2 includes a bottom wall 7 that supports a gingival mucosal epithelial equivalent 3. The bottom wall 7 has a through hole 8. Some baskets 2 have an insert 9 for fixing the bottom membrane 7. The term basket is not used in a limiting sense and includes any support disposed within the well 1.

The well 1 can fix a part of a plate having a plurality of (for example, 12) wells. The well 2 is filled with a solution 5 (for example, a medium).
The gingival mucosal epithelial equivalent 3 is, for example, the aforementioned cultured mucosa.
The evaluation object cell 4 is the aforementioned fibroblast.
Substances 6 to be evaluated include, for example, components that can be used for oral cosmetics, quasi drugs and / or pharmaceuticals, solutions, solutions or dispersions containing the same, emulsified preparations, cosmetics, oral care products, pharmaceutical preparations, and the like. is there.
The gingival mucosal epithelial equivalent has an area in the range of, for example, 0.2-5 cm ² .

EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

Type I collagen production effect of fibroblasts taking into account transmucosal absorbability by a solution containing L-ascorbyl magnesium phosphate Summary of Test Cumulative permeation amount and tissue content of VCPMg to gingival mucosal epithelial equivalent after application of L-ascorbyl magnesium phosphate (hereinafter referred to as VCPMg) solution, type I collagen production by permeation VCPMg in fibroblast co-culture, Each was measured by a high performance liquid chromatography (HPLC) measurement method and an ELISA method.

2. Experimental Method Fibroblasts were seeded on the day before the transmucosal absorption test, and SkinEthic ^™ HGE (manufactured by SkinEthic, gingival mucosal epithelial equivalent) was preincubated. On the day of the test, SkinEthic ^™ HGE and fibroblasts were co-cultured in cell maintenance medium and samples were applied. FIG. 2 shows the settings of the transmucosal absorption test (gingival mucosa equivalent 3 is SkinEthic ^™ HGE, evaluation target cell 4 is fibroblast, and evaluation target substance 6 is VCPMg solution). After application of the VCPMg solution, incubation was continued up to 48 hours, and the VCPMg cumulative permeation amount was measured by HPLC over time. Further, after 48 hours, SkinEthic ^™ HGE was homogenized, and the amount of VCPMg in the gingival mucosa epithelium was measured by HPLC. In addition, the co-cultured medium was collected, and the amount of type I collagen produced in the culture was measured by ELISA. As a control, phosphate buffered saline (PBS) was used.

3. Results The cumulative amount of SkinEthic ^™ HGE permeation of VCPMg is shown in Table 1, the amount in tissue after 48 hours is shown in Table 2, and the amount of type I collagen produced by co-culture is shown in Table 3. VCPMg cumulative permeation increased in an applied concentration and time dependent manner. After 48 hours, the amount of VCPMg tissue and the amount of collagen produced in the co-culture medium increased depending on the concentration of VCPMg.
From this result, the transmucosal absorption evaluation apparatus can simultaneously and simply evaluate the effect of increasing the amount of type I collagen produced by the amount of the substance permeated through the mucosa and the permeated substance acting on the fibroblasts as the evaluation target cells. Became clear.

Cytokine production inhibitory action taking into account transmucosal absorbability by a solution containing dipotassium glycyrrhizinate. Summary of test Cumulative permeation amount of GK2 to gingival mucosal epithelial equivalent after application of dipotassium glycyrrhizinate (hereinafter referred to as GK2) solution using gingival mucosal epithelial equivalent treated with sodium dodecyl sulfate (hereinafter referred to as SDS) The amount of interleukin-1 alpha (IL-1α) produced by permeabilized GK2 in tissue medium and fibroblast co-culture was measured by high performance liquid chromatography (HPLC) measurement and ELISA, respectively.

2. Experimental Method Fibroblasts were seeded on the day before the transmucosal absorption test, and SkinEthic ^™ HGE (manufactured by SkinEthic, gingival mucosal epithelial equivalent) was preincubated. Inflammation was induced by applying SDS to SkinEthic ^™ HGE for 1 hour on the test day. SkinEthic ^™ HGE and fibroblasts were then co-cultured in cell maintenance medium and the sample applied. FIG. 2 shows the settings of the transmucosal absorption test (gingival mucosa equivalent 3 is SkinEthic ^™ HGE, evaluation target cell 4 is fibroblast, and evaluation target substance 6 is GK2 solution). After application of the GK2 solution, incubation was continued for 24 hours, and the cumulative amount of GK2 permeation was measured with HPLC over time. Also, after 24 hours, SkinEthic ^™ HGE was homogenized, and the amount of GK2 in the gingival mucosal epithelium was measured by HPLC. In addition, the co-cultured medium was collected, and the amount of IL-1α produced in the culture solution was measured by ELISA. As a control, phosphate buffered saline (PBS) was used.

3. Results SkinEthic ^™ HGE cumulative permeation amount of GK2 is shown in Table 4, tissue amount after 24 hours is shown in Table 5, and IL-1α production amount is shown in Table 6. GK2 cumulative permeation increased in an applied concentration and time dependent manner. The amount of GK2 tissue in 24 hours increased depending on the applied concentration. The amount of IL-1α produced in the co-culture medium increased by the induction of inflammation by SDS decreased depending on the concentration of GK2.
From this result, it is clear that the transmucosal absorption evaluation apparatus can simultaneously and easily evaluate the anti-inflammatory action using the amount of IL-1α produced as an index by the amount of the substance permeated through the mucosa and the substance permeated acting on the cells to be evaluated. It became.

1 well 2 basket 3 gingival mucosal epithelial equivalent 4 cell to be evaluated 5 solution 6 substance 7 bottom wall 8 through hole 9 insert

Claims (5)

A transmucosal absorption evaluation apparatus capable of simultaneously measuring the amount of a substance permeated through the mucosa and evaluating the effectiveness of the permeated substance using a gingival mucosal epithelial equivalent and a cell to be evaluated. The transmucosal absorption evaluation apparatus according to claim 1, wherein the gingival mucosal epithelial equivalent is a cultured mucosa. The transmucosal absorption evaluation apparatus according to claim 1, wherein the gingival mucosal epithelial equivalent is a reconstituted mucosa. The transmucosal absorption evaluation apparatus according to any one of claims 1 to 3, wherein the cell to be evaluated is a fibroblast. Using the transmucosal absorption evaluation apparatus according to any one of claims 1 to 4, measurement of transmucosal permeation amount of gingival mucosal epithelial equivalent, and from gingival mucosal epithelial equivalent and / or the cell to be evaluated An evaluation method comprising simultaneously performing one or more types of measurements selected from collagen production amount, hyaluronic acid production amount, various cytokine production amounts, various secretory enzymes and protein amounts and / or activity.

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