Pharmaceutical composition for treating Dowling-Degos disease

Abstract

To provide a hairless Pofut1 hetero junction type knockout mouse, a screening method for a compound for treating Dowling-Degos disease, and a pharmaceutical composition for treating Dowling-Degos disease.SOLUTION: A pharmaceutical composition for treating Dowling-Degos disease includes fucose as an active ingredient.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The disclosure of the present application relates to a hairless Pofut1 heterozygous knockout mouse, a method for screening a compound for treating Dowling-Degos disease, and a pharmaceutical composition for treating Dowling-Degos disease. [Background technology]

[0002] Dowling-Degos disease (hereinafter sometimes referred to as "DDD") is an autosomal dominant inherited hereditary skin disease that causes mesh-like pigmentation in areas where the skin is easily rubbed, such as the neck, armpits, and groin. DDD does not cause serious, life-threatening symptoms, but patients experience mental stress due to cosmetic issues. Following the report of KRT5 in 2006 as the causative gene for DDD, the Pofut1 gene, which modifies the glycosylation of proteins, was reported in 2013.

[0003] As mentioned above, although there are reports on DDD, the pathology of DDD is still unclear, and there is currently no effective treatment method. In research on disease treatment, it is common to prepare model animals such as mice with the disease, use the model animals to examine the pathology of the disease, or administer test compounds to the model animals to screen for therapeutic compounds. Knockout mice are also known for the Pofut1 gene (see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yoshiaki Okamura et al., “Pofut1 is required for the proper localization of the Notch receptor during mouse development”, Mech Dev. 125(8):663-73, 2008. Summary of the Invention [Problem to be solved by the invention]

[0005] As described in Non-Patent Document 1, mice with the Pofut1 gene knocked out are known. However, the Pofut1 knockout mouse described in Non-Patent Document 1 was generated for the purpose of analyzing the function of Pofut1 in mice, particularly the development of the cardiovascular system. Although the Pofut1 gene is knocked out in the Pofut1 knockout mouse described in Non-Patent Document 1, there are problems in that (1) there is no change in coat color as a phenotype, and the mouse does not show pigmented spots on the skin, and (2) the mouse has body hair that is an obstacle when confirming the therapeutic effect of DDD, which is a skin disease.

[0006] The disclosure of this application has been made to solve the above problems, and as a result of intensive research, it has been newly discovered that (1) when a Pofut1 heterozygous knockout mouse is crossed with a hairless mouse, (2) it is possible to produce a knockout mouse that is capable of forming pigmented spots and has no body hair (hairless), and (3) as a result, it can be used in research into the pathology of DDD and in a screening method for compounds for treating DDD.

[0007] That is, the object of the disclosure of the present application is to provide a hairless Pofut1 heterozygous knockout mouse, a method for screening a compound for treating Dowling-Degos disease, and a pharmaceutical composition for treating Dowling-Degos disease. [Means for solving the problem]

[0008] The disclosure of the present application relates to a hairless Pofut1 heterozygous knockout mouse, a method for screening a compound for treating Dowling-Degos disease, and a pharmaceutical composition for treating Dowling-Degos disease, as shown below.

[0009] (1) Hairless Pofut1 heterozygous knockout mice were obtained by crossing Pofut1 heterozygous knockout mice with hairless mice. (2) The hairless Pofut1 heterozygous knockout mouse exhibits a phenotype of forming pigmented spots; A hairless Pofut1 heterozygous knockout mouse described in (1) above. (3) administering a test compound to the hairless Pofut1 heterozygous knockout mouse described in (1) or (2) above; A test compound selection step of selecting a test compound that suppresses pigmentation spots; A method for screening for a compound for treating Dowling-Degos disease, comprising: (4) A pharmaceutical composition for treating Dowling-Degos disease, comprising fucose as an active ingredient. Effect of the Invention

[0010] The hairless Pofut1 heterozygous knockout mouse disclosed in the present application is a new animal model for DDD, and can therefore be used in screening methods for compounds for treating DDD. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a photograph substituting a drawing, in which FIG. 1A is a photograph of the DDD model mouse prepared in Example 1, and FIG. 1B is a photograph of the hairless mouse used to prepare the DDD model mouse. [Diagram 2] FIG. 2 is a photograph substituting a drawing, in which FIG. 2A is a histopathological photograph of a pigmented spot in the DDD model mouse prepared in Example 1, and FIG. 2B is a histopathological photograph of the skin of a hairless mouse (control). [Diagram 3] FIG. 3 is a photograph in place of a drawing. In Example 2, FIG. 3A is a photograph of a DDD model mouse before external application of the DDD pharmaceutical composition, and FIG. 3B is a photograph of the DDD model mouse after 8 weeks of external application of the DDD pharmaceutical composition. [Figure 4]FIG. 4 is a graph showing the results of analyzing the color tone of the rash area using the image analysis software ImageJ for five arbitrarily set locations shown in FIGS. 3A and 3B. [Diagram 5] FIG. 5 is a photograph in place of a drawing. In Example 2, FIG. 5A is a histopathological photograph of a pigmented spot in a DDD model mouse before topical application of the DDD pharmaceutical composition, and FIG. 5B is a histopathological photograph of a pigmented spot in a DDD model mouse after topical application of the DDD pharmaceutical composition. [Figure 6] FIG. 6 is a graph showing that the DDD pharmaceutical composition inhibits the proliferation of human keratinocytes in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The hairless Pofut1 heterozygous knockout mouse (hereinafter sometimes referred to as "DDD model mouse"), the screening method for a compound for treating Dowling-Degos disease (hereinafter sometimes simply referred to as "screening method"), and the pharmaceutical composition for treating Dowling-Degos disease (hereinafter sometimes referred to as "DDD pharmaceutical composition") disclosed in the present application are described in detail below.

[0013] (Embodiment of DDD model mouse) First, the DDD model mouse will be described. The DDD model mouse can be obtained by crossing a Pofut1 heterozygous knockout mouse with a hairless mouse. The Pofut1 heterozygous knockout mouse may be produced by a known method, for example, by the following procedure. (a) Isolate the gene to be knocked out (Pofut1) from the mouse genome, then create a sequence that contains the gene and surrounding parts, but is not identical; it is modified to make it inactive, typically by incorporating a marker gene that provides an observable difference (such as color or fluorescence). (b) Embryonic stem cells are isolated from mouse blastocysts (early mouse embryos in which a sphere of undifferentiated cells is surrounded by extraembryonic cells). Embryonic stem cells can be cultured in vitro, for example, from white mice. (c) The base sequence prepared in (a) above is introduced into the embryonic stem cells obtained in (b) above by means of electroporation or other methods. Next, using the marker gene introduced in (a) above, embryonic stem cells that have actually undergone recombination with the new base sequence (heterozygous type) are isolated. (d) The embryonic stem cells isolated in (c) above that underwent homologous recombination are injected into, for example, a gray mouse blastocyst, which is then injected into the uterus of a female mouse, and a baby mouse is born. This baby mouse will be a chimera, with some of its body derived from the original blastocyst and other parts derived from the genetically engineered cells, and will therefore have a patchy white and gray coat. (e) Only the germ cells (eggs or sperm) of the newly born mice originating from the genetically engineered cells are used. By crossing these mice with white mice, DDD model mice that are completely white and carry at least one functional gene (heterozygous) are obtained.

[0014] The DDD model mouse may be generated according to the procedure described above or the procedure described in Non-Patent Document 1, or may be obtained from an institution such as the National Institute of Genetics.

[0015] A known hairless mouse may be used as the hairless mouse. There is no particular limitation on the hairless mouse as long as a DDD model mouse can be obtained by crossing with a Pofut1 heterozygous knockout mouse. However, the DDD model mouse disclosed in the present application is preferably a healthy mouse except for the phenotypes of (1) having no body hair and (2) having pigmented spots. Therefore, a hairless mouse that has no body hair due to the hairless (Hr) gene being knocked out is more preferable than a hairless mouse (nude mouse) with an inhibited immune system. A hairless mouse may be produced by knocking out the hairless gene according to the above procedure, or one obtained from a research institute or the like may be used.

[0016] A Pofut1 heterozygous knockout mouse and a hairless mouse can be crossed by a general procedure. By crossing, the hairless gene is knocked out and the Pofut1 gene is heterozygous (Pofut1 wt / ko ) is knocked out to obtain a novel genotype DDD model mouse. The obtained DDD model mouse exhibits the phenotypes of (1) having no body hair and (2) forming pigmented spots, but the phenotype does not need to be exhibited immediately after birth. For example, the pigmented spots may be formed after a predetermined period of nurturing. The above explanation has been given as an example of mice that are commonly used as experimental animals, but other animals such as rats and rabbits may also be used.

[0017] (Embodiments of Screening Method) An embodiment of the screening method comprises: administering a test compound to a DDD model mouse; a test compound selection step for selecting a test compound that suppresses pigmentation spots; Includes.

[0018] Test compounds include, for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, antibodies, and peptides, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, and the like.

[0019] The method of administering a test compound to a DDD model mouse is not particularly limited as long as it is taken into the body of the DDD model mouse, such as oral administration, application to the skin, or internal administration by injection, etc. Then, by determining whether or not the administered test compound reduces the number of pigmented spots formed on the skin of the DDD model mouse (lightens the pigment), a compound that suppresses pigmented spots can be selected.

[0020] DDD Pharmaceutical Composition Embodiments The DDD pharmaceutical composition is not particularly limited as long as it contains an ingredient that is effective for treating DDD. The dosage form of the DDD pharmaceutical composition includes, for example, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, gelatin soft and hard capsules, suppositories, sterile injection solutions, and sterile sealed powders.

[0021] The DDD pharmaceutical composition may also include carriers, excipients, and diluents. For example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, phosphate buffered saline (PBS), syrup, methylcellulose, methyl- and propyloxybenzoate, talc, magnesium stearate, and mineral oil. In addition, vitamins, lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners, or flavoring agents may be added. The DDD pharmaceutical composition may also be combined with known drugs.

[0022] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. EXAMPLES

[0023] <Example 1> [Creation of DDD model mice] Pofut1 heterozygous knockout mice were obtained from the National Institute of Genetics. In this example, "Pofut1 null mice" in which the Pofut1 gene was knocked out throughout the body were used. In addition, "Hos:HRM-2" hairless mice were used, which were obtained from the Hoshino Laboratory Animal Breeding Center. The obtained Pofut1 heterozygous knockout mice and hairless mice were mated and bred in the usual manner to produce DDD model mice. Figure 1A is a photograph of the DDD model mouse after about 8 months of breeding. In addition, Figure 1B is a photograph of the hairless mouse used for mating.

[0024] In humans, it is said that when the Pofut1 gene is knocked out in a heterozygous form, DDD develops. However, in the Pofut1 heterozygous knockout mouse, even though the Pofut1 gene is knocked out, no pigmented spots were observed even when the hair was shaved. On the other hand, in the DDD model mouse produced in Example 1, the formation of pigmented spots was confirmed as shown by the arrow in FIG. 1A. From the above results, by crossing the Pofut1 heterozygous knockout mouse with a hairless mouse, it was possible to produce a novel mouse that not only shows a phenotype of a Pofut1 heterozygous knockout mouse simply being made hairless, but also shows an unexpected phenotype of pigmented spots being formed.

[0025] [Identifying pigment cells in the skin] Next, we investigated pigment cells in the skin of the DDD model mouse and the hairless mouse prepared in Example 1. Skin samples were prepared by the following procedure. (a) Skin was taken by punch biopsy from the pigmented spot on the back of a DDD model mouse and from the same location (normal skin) of a control hairless mouse. (b) The collected skin was fixed in 5% paraformaldehyde, embedded in paraffin, and mounted on a slide glass at a thickness of 4 μm to prepare an unstained specimen. (c) Unstained specimens were stained with hematoxylin and eosin and observed.

[0026] FIG. 2A is a histopathological photograph of a pigmented spot of a DDD model mouse prepared in Example 1. The cells with gaps (with transparent nuclei) indicated by the arrow in the photograph are pigment cells. FIG. 2B is a histopathological photograph of the skin of a hairless mouse (control), with the arrow (black) indicating a pigmented cell. As shown in FIG. 2A and FIG. 2B, it was confirmed that the pigmented spot area of ​​the DDD model mouse prepared in Example 1 in which the pigmented spot was formed had a greater number of pigment cells in the basal layer of the epidermis than the hairless mouse used as a comparison, and that there was variation between areas with high and low density of pigment cells. In addition, the epidermis of the DDD model mouse was thickened compared to the epidermis of the hairless mouse.

[0027] <Example 2> [Confirmation of the effect of DDD pharmaceutical composition] (1) Preparation of pharmaceutical compositions A 10% fucose solution was prepared by mixing 0.5 g of fucose (Sigma-Aldrich) with 5 mL of ddw (double distilled water). When applying fucose to DDD model mice, 0.16 g of hydrophilic cream (Nikko Pharmaceutical Co., Ltd., hydrophilic cream "Nikko") was mixed with 40 μL of 10% fucose solution to prepare a 2% fucose cream just before use, and the DDD pharmaceutical composition of Example 2 was prepared. (2) External application of DDD pharmaceutical composition 0.2 g of the freshly prepared DDD pharmaceutical composition was applied externally to an area of ​​approximately 4 square centimeters of pigmented spots on the backs of DDD model mice, once a day, 5 days a week, and the effect was evaluated after 8 weeks.

[0028] (3) Visual confirmation of effect Figure 3A is a photograph of the DDD pharmaceutical composition before application, and Figure 3B is a photograph of the DDD pharmaceutical composition after 8 weeks of application. The five squares in the photograph are arbitrarily set contrast areas. As is clear from the photographs shown in Figures 3A and 3B, after 8 weeks of application of the DDD pharmaceutical composition, a reduction in pigmentation spots was visually confirmed.

[0029] (4) Confirmation of effectiveness through image analysis The color tone of the rash was analyzed using the image analysis software ImageJ for the five arbitrarily set locations shown in Figures 3A and 3B. Specifically, the color photographs taken before and after external application of the DDD pharmaceutical composition were converted to grayscale, and the brightness was quantified using ImageJ, with white being 256 and black being 1. One point was the average value of the area of ​​201x201 points. Figure 4 is a graph showing the analysis results using ImageJ. Since the values ​​after external application of the DDD pharmaceutical composition increased for all five arbitrarily set locations, it was also confirmed from image analysis that the pigmented spots were lighter due to external application of the DDD pharmaceutical composition. In addition, when the values ​​of the five set locations were tested using a paired t-test, p = 0.006 was obtained. Therefore, it was confirmed that the pigmented spots were significantly improved by external application of the DDD pharmaceutical composition.

[0030] (5) Effect confirmation by sectioning Next, skin sections before and after topical application of the DDD pharmaceutical composition were prepared in the same manner as in [Confirmation of pigment cells in the skin] above. Figure 5A is a histopathological photograph of a pigmented spot in a DDD model mouse before topical application of the DDD pharmaceutical composition, and Figure 5B is a histopathological photograph of a pigmented spot in a DDD model mouse after topical application of the DDD pharmaceutical composition. Comparing Figures 5A and 5B, before topical application of the DDD pharmaceutical composition, the number of pigment cells (arrows in the photograph) was large and the distribution of the pigment cells was uneven. On the other hand, after topical application of the DDD pharmaceutical composition, the number of pigment cells decreased and there were no areas with high density distribution. From the above results, it was confirmed from the observation of pigment cells in the histopathological photograph that the pigmented spot was significantly improved by topical application of the DDD pharmaceutical composition.

[0031] From the above results, it was confirmed that fucose is useful for treating DDD. In addition, it was confirmed that the DDD model mouse generated in the examples can be used in a method for screening compounds for treating DDD.

[0032] (6) Confirmation of in vitro proliferation inhibition using human keratinocytes As described above, it was confirmed that the DDD pharmaceutical composition shown in Example 2 significantly improved pigmentation spots in DDD model mice. In order to investigate the effects of the DDD pharmaceutical composition on humans, an in vitro experiment was also conducted using human cells according to the following procedure. (a) HaCaT cells (manufactured by Cell Lines Service) were used as human keratinocytes. In DDD patients, epidermal thickening due to proliferation of the epidermis in the affected area is significant. In other words, inhibition of proliferation of keratinocytes, which are cells that make up the epidermis, is considered to be useful as one of the therapeutic effects. HaCaT cells are immortalized keratinocytes established from the skin of an adult male. Since fucose has been shown to inhibit the proliferation of HaCaT cells, external application to humans may inhibit epidermal thickening in the affected area of ​​the patient, leading to the normalization of the skin. (b) HaCaT cells were cultured in a 96-well plate at 1500 cells / well for 24 hours in a standard manner. Low dose fucose was added to the medium at a concentration of 10 μg / mL, and high dose fucose was added to the medium at a concentration of 100 μg / mL. The proliferation ability was quantified using the cell counting kit-8 (CCK-8) (Dojindo Laboratories) according to the manufacturer's instructions. (c) Figure 6 is a graph showing the results of measuring proliferation ability. As shown in Figure 6, it was confirmed that the DDD pharmaceutical composition shown in Example 2 inhibits the proliferation ability of human epidermal cells. [Industrial Applicability]

[0033] The DDD model mouse disclosed in the present application exhibits a phenotype of hairlessness and pigmented spots, and can therefore be used to elucidate the pathology of DDD and screen for compounds for treating DDD. Therefore, it is useful for the development of compounds for treating DDD in universities, medical institutions, pharmaceutical companies, etc.

Claims

[Claim 1] A pharmaceutical composition for treating Dowling-Degos disease, comprising fucose as an active ingredient.

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