JP6052719B2 - Caspase 14 synthesis promoter - Google Patents

Description

  The present invention relates to a caspase 14 synthesis accelerator and a skin external preparation containing the same.

  It has been clarified that 14 kinds of caspases exist so far. Among these, caspase 14 is specific in that it does not participate in apoptosis. It has been clarified that caspase 14 is mainly present in epidermal keratinocytes of the skin and maintains the stratum corneum barrier function and water retention function (Non-patent Document 1).

However, there are still many unclear points regarding caspase-14. As a few reported examples, there is a study that the synthesis of caspase 14 is induced by vitamin D ₃ (Non-patent document 1) and by green tea polyphenols (Non-patent document 2). Furthermore, there is a case where the activity of caspase 14 is stimulated by a predetermined urea derivative (Patent Document 1).

  By the way, it is known that caspase 3 and caspase 9 which are also caspase associates are activated in apoptosis induced by phytosphingosine (Non-patent Document 3). It is also known that phytosphingosine promotes skin differentiation and regulates apoptosis to inhibit epidermal hyperplasia (Non-patent Document 4). Utilizing this action of sphingosines, it has been proposed to add sphingosines to an external preparation for skin to suppress enhancement of skin turnover, for example, epidermal thickening (epidermal hyperplasia) (Patent Document 2). ).

JP 2007-23038 A JP-A-5-85924

Lippens S. et al .: Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures. Am. J. Pathol. (2004) 165,833-841 Hsu S. et al .: Green tea polyphenol induces caspase 14 in epidermal keratinocytes via MAPK pathways and reduces psoriasiform lesions in the flaky skin mouse model.Exp. Dermatol. (2007) 16,678-684 Nagahara Y. et al .: Phytosphingosine induced mitochondria-involved apoptosis. Cancer Sci. (2005) 96,83-92 Kim S. et al .: Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.Mol. Med. (2006) 12,17-24.

  If the synthesis of caspase 14 can be promoted, it is very useful for maintaining the skin moisturizing function, preventing the deterioration of the function, and improving the function. However, as described above, there are few examples for that purpose. Therefore, an object of the present invention is to provide a synthesis accelerator for caspase-14.

  The present invention is a caspase 14 synthesis promoter containing at least one compound selected from the group consisting of sphingoids and N-acetylsphingosines. The present invention also provides a skin external preparation containing the caspase 14 synthesis accelerator in an amount such that the concentration of at least one compound selected from the group consisting of sphingoids and N-acetylsphingosines is 0.1 to 20 μM. It is.

  As described above, caspase 14 is different from caspase 3 and caspase 9 in that it does not participate in apoptosis. Therefore, it is unlikely that caspase 14 is activated by sphingosine. However, contrary to this expectation, it was found that caspase 14 was promoted by sphingoids and N-acetylsphingosine. The caspase 14 synthesis promoter promotes the synthesis of a skin moisturizing component through promoting the expression of procaspase 14, which is different from the conventional apoptosis control mechanism. Thereby, the moisture retention function of skin can be improved permanently.

It is a figure which shows the cytotoxicity of phytosphingosine measured by MTT method. It is a figure which shows the cytotoxicity of sphingosine measured by MTT method. It is a figure which shows the cytotoxicity of sphinganine measured by MTT method. Was measured by the MTT method, C 2 _- illustrates the cytotoxicity of ceramide. It is a figure which shows the expression effect of the sphingoid procaspase 14 by Western blotting. It is a figure which shows the expression effect of the sphingoid procaspase 14 by Western blotting. It is a figure which shows the mRNA expression effect of caspase 14 of sphingoids by RT-PCR method.

  As described above, caspase 14 is present in epidermal keratinocytes of the skin. Epidermal keratinocytes, in humans, occupy about 95% of cells constituting the skin epidermis, and are composed of four layers from the deepest to the basal layer, spinous layer, granule layer, and stratum corneum. The keratinocytes are born in the basal layer, gradually repeat differentiation, pass through the spinous layer, then the granular layer, finally reach the stratum corneum, and fall into the horny piece and fall off the skin.

 In a steady state, caspase 14 is localized as a precursor (pro) body in the basal layer constituted by keratinocytes. Therefore, if the expression of procaspase 14 in keratinocytes is promoted, caspase 14 can be increased. Activation of caspase 14 by cleavage of procaspase 14 is observed at a later stage of epidermal differentiation. Caspase 14 degrades filaggrin precursor, a structural protein involved in skin moisturization. The filaggrin released by the degradation aggregates keratin fibers in the cytoplasm of the keratinocytes and then degrades into amino acids in the upper stratum corneum, but these have water retention function and ultraviolet absorption ability, Called to maintain the stratum corneum barrier function and moisture retention function.

The caspase 14 synthesis promoter of the present invention contains at least one compound selected from the group consisting of sphingoids and N-acetylsphingosines as an active ingredient. The sphingoids are long-chain base parts of sphingolipids, that is, long-chain amino alcohols having 16 to 20 carbon atoms, and are synonymous with sphingoid bases. Usually, sphingoids have a trans double bond at the C4 position. Examples of the sphingoid include the following, and may be a mixture of these two types.
(1) Sphingosine or sphingenin having a C18 long chain fatty chain with a double bond at the C4 position:
(2) Sphinganin or dihydrosphingosine, a molecule in which the sphingosine double bond is saturated:
(3) Phytosphingosine in which the sphingosine double bond is monohydroxylated:
(4) 4,8-sphingadienin in which sphingosine is dehydrogenated at position C8:

(5) 4-hydroxy-8-sphingenin in which phytosphingosine is dehydrogenated at the C8 position:

  Of these, phytosphingosine, sphingosine, and sphinganine are preferable, and phytosphingosine is particularly preferable.

 The sphingoid can be obtained by a method of performing alkaline hydrolysis of sphingolipids from animals or plants, or a method of performing purification with silica gel using a chloroform-methanol mixture after extraction with an alkaline alcohol solution. Alternatively, it can also be obtained by chemical synthesis described in, for example, JP-A-7-258178. Moreover, you may use what is marketed.

In the present invention, N-acetylsphingosines are compounds in which the acyl group is an acetyl group among N-acylsphingosines in which the amino group of the sphingoid is acylated. N-acyl sphingosine is also generally called ceramide. Examples of the N-acetylsphingosine include C ₂ -ceramide represented by the following formula (6):

  N-acetyl sphingosines can be obtained by a method of performing alkaline hydrolysis of sphingolipids from animals or plants, or a method of performing purification with silica gel using a chloroform-methanol mixture after extraction with an alkaline alcohol solution. Alternatively, it can be obtained by chemical synthesis. Moreover, you may use what is marketed.

  The caspase 14 synthesis promoter of the present invention may contain a pharmaceutically acceptable carrier. Examples of the carrier include excipients, binders, solvents, and solubilizing agents. Furthermore, you may use suitably additives, such as a normal preservative and an antioxidant, as needed. Examples of the excipient include sugar starch and corn starch, and examples of the binder include celluloses and dextrin. Examples of the solvent include alcohols, fats and oils, vegetable oils and the like, and among them, vegetable oils such as macadamia nut oil, olive oil, corn oil and the like are preferable. Moreover, when using these vegetable oils, you may mix | blend phytosterol etc. as a solubilizing agent.

  Regarding the formulation of the caspase 14 synthesis accelerator, application to an external preparation for skin will be described below as an example. That is, the present invention also relates to a skin external preparation containing the caspase 14 synthesis accelerator as a medicinal ingredient. The external preparation for skin contains the caspase 14 synthesis accelerator at a concentration of 0.1 to 20 μM. When the amount of the caspase 14 synthesis accelerator is less than the lower limit, a satisfactory effect cannot be obtained. When the amount exceeds the upper limit, the synthesis promotion effect is weakened as shown in the examples described later. Preferably, the concentration of the caspase 14 synthesis promoter in the external preparation for skin is 1 μM or more from the viewpoint that the action time is short, and is 15 μM or less from the viewpoint of high synthesis promotion efficiency. More preferably, the concentration is 5-15 μM.

  As a base for the external preparation for skin, any of liquid, semi-solid or paste may be used as long as it is used for normal external preparation for skin. As the base material, olive oil, soybean oil, sesame oil, camellia oil and other vegetable oils, waxes and other hydrocarbons, fats and oils, waxes, hardened oils, esters, fatty acids, higher alcohols, silicone oils are used. can do.

  Moreover, it is good also as an emulsion form containing water, In that case, an anionic, cationic, amphoteric, and nonionic surfactant used for emulsification, solubilization, etc. can be used.

  In addition, as long as the effects of the present invention are not impaired, components that are usually used in formulations such as cosmetics, quasi-drugs, and external medicines, that is, gelling agents, powders, alcohols, water-soluble polymers, Film-forming agents, salts, pH adjusters, chelating agents, plant extracts, antibacterial agents, blood circulation promoters, vitamins, preservatives, fragrances and the like can be added.

  As the product form of the external preparation for skin, it may be in the form of liquid, emulsion, paste, or poultice, etc., and cosmetics such as lotions, emulsions and complaints, and external medicines such as ointments. It may be.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.

[Example]
I. In the examples, the following cells and cell culture methods were used.
(1) Cells Human keratinocyte cell line HaCaT cells were purchased from the German Cancer Research Center (Eppelheim, Germany) (Boukamp et al: “Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.” J. Cell Biol. 106: 761-771 (1988)).
(2) Cell culture As cell culture medium, 3.5 μL / L 2-mercaptoethanol (Wako Pure Chemical Industries), 10 mL / L streptomycin-penicillin (× 100) solution (Wako Pure Chemical Industries), 10% fetal calf serum (Valley The cells were seeded in a petri dish using a DMEM (Dulbecco Modified Eagle Medium) (Nippon Pharmaceutical Co., Ltd.) medium containing Biomedical, and cultured in a 37 ° C., 5% CO ₂ incubator (ESPEC).

II. The cytotoxicity of sphingoids was examined by the MTT method shown below.
<MTT method>
1.0 × 10 ⁴ cells of HaCaT cells were seeded at 80 μL each in a 96-well microplate and incubated overnight so that the HaCaT cells adhere. After incubation, an organic solvent in which phytosphingosine (PHS), sphingosine (SPH), sphinganine (SPG), and C ₂ -ceramide (Cer) are dissolved so that the final concentrations are 0, 5, 15, and 25 μM, respectively. 20 μL of each was added. The action time was 24, 48, and 72 hours. After acting in a CO ₂ incubator at 37 ° C., 10 μL of MTT solution was added to each well 1 hour before the measurement, and further incubated for 1 hour. After the action, discard the supernatant, add 100 μL each of DMSO, measure the mitochondrial NADH content by measuring the absorbance at 570 nm using a microplate reader, and measure the percentage of NADH content in the test group relative to the NADH content of the unadded control. Was defined as cell viability.

  The result of the said test is shown to FIGS. From these results, the concentration of sphingoids or N-acetylsphingosines (hereinafter sometimes collectively referred to as “sphingoids”) is 25 μM regardless of the action time, and the survival rate may drop sharply. I understood. Although the reason for this is unclear, it is suggested that apoptosis may be induced by sphingoids. In addition to the above test, when the same test was carried out for PHS, SPH, SPG, and Cer at a concentration of 20 μM, the survival rate was about 30 to 80% of the initial value, which was about twice that of 25 μM. It was. From this, it was found that the concentration of the caspase synthesis accelerator is preferably 20 μM or less.

The expression of procaspase 14 by sphingoids was examined by Western blotting according to the following procedure.
(I) Cell preparation
Prepare HaCaT cells at 1.0 × 10 ⁵ cells / mL, add 0, 5, 15 μL of organic solvent in which PHS, SPH, SPG, and Cer are dissolved so that the final concentration is 0, ⁵ , 15 μM. It was allowed to act for 24, 48, 72 hours in a CO ₂ incubator. Thereafter, the cells were collected by centrifugation at 250 × g for 5 minutes, and the precipitate was washed with PBS, transferred to an Eppendorf tube, and centrifuged at 500 × g for 3 minutes. Thereafter, the supernatant was removed, Lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2, 1 mM EGTA, × 100 protease inhibitor cocktail (manufactured by Sigma)) and 80 μL was added to suspend, ice-cooled for 20 minutes, and centrifuged at 14000 × g for 15 minutes to collect the supernatant as a cell lysate.
(Ii) Protein quantification
Dilute the BSA standard solution with Lysis Buffer to 0, 0.25, 0.5 mg / mL, and the cell lysate obtained by cell preparation using Pierce BCA Protein Assay Reagent with a microplate reader at 570 nm absorbance. Was measured. A calibration curve was prepared from the absorbance of the BSA standard solution, and the protein concentration was adjusted to 1 mg / mL.
(Iii) SDS-polyacrylamide gel electrophoresis 20 μL of protein solution prepared by protein quantification and Sample Application Buffer (10% sodium dodecyl sulfate (SDS), 125 mM Tris-HCl, pH 6.8, 20% glycerol, bromophenol blue , 10% 2-mercaptoethanol) was mixed, and incubated at 100 ° C. for 3 minutes using a block incubator. For protein separation, a 12% SDS polyacrylamide gel was used, the gel was fixed in an electrophoresis tank, and Electrode Buffer (3 g / L Tris, 14 g / L glycine, 1 g / L SDS) was poured. Samples and molecular weight markers were placed in the wells and run at 50 V for 30 minutes, and then run at 100 V for 90 minutes.
(Iv) Transfer After completion of the electrophoresis, the gel was taken out and transferred using a PVDF membrane for 30 minutes.
(V) Reaction of primary antibody and secondary antibody After completion of the transfer, the PVDF membrane was immersed in skim milk (30% skim milk powder, 0.1% NaN ₃ ) and shaken at room temperature for 60 minutes using a shaker to perform blocking. Thereafter, a primary antibody solution (x1000-fold Caspase-14 antibody (manufactured by Santa Cruz) or x1000-fold β-actin antibody (manufactured by Cell Signaling)) was added, and the mixture was allowed to react at room temperature overnight. After washing, a secondary antibody solution (x2000-fold Anti-rabbit IgG antibody (manufactured by Cell Signaling)) was added and allowed to react at room temperature for 1 hour with shaking. After the reaction, it was washed.
(Vi) ECL reaction
Using ECL Western Blotting Detection Reagent, it set to the image analyzer (Image Quant LAS4000) (made by Fuji Film), and detected.

  The results obtained with the image analyzer are shown in FIGS. As can be seen from FIG. 5, the expression of procaspase-14 was not observed at 24 hours of PHS action, but the expression of procaspase 14 was confirmed when PHS 5 μM was allowed to act for 48 hours and 72 hours. Moreover, as shown in FIG. 6, the expression of procaspase 14 was also confirmed when SPH, SPG, and Cer were allowed to act. At a drug concentration of 3 μM, an excellent procaspase 14 expression effect of PHS was observed.

The expression of caspase 14 mRNA by sphingoids was examined by the following RT-PCR method.
(I) Addition of sphingoids (PHS, SPH, SPG, Cer)
Three 10 mL petri dishes were prepared so that HaCaT cells were 1.0 × 10 ⁵ cells / mL, and 0 μL (control), 1 μL, and 3 μL of sphingoids were added to each, and incubated for 48 hours.

(Ii) RNA extraction RNA was extracted by the following procedure.
1) Trizol (manufactured by Invitrogen) was added to the petri dish and suspended uniformly.
2) 0.2 mL of chloroform was added and stirred with a vortex mixer for 20 seconds.
3) The sample was left for 2 to 3 minutes and centrifuged at 12000 × g and 4 ° C. for 10 minutes.
4) The upper layer was transferred to a new Eppendorf tube with a 400 μL micropipette, and 500 μL of Trizol was added to suspend uniformly.
5) 0.2 mL of chloroform was added and stirred with a vortex mixer for 20 seconds.
6) The sample was left for 2 to 3 minutes and centrifuged at 12000 × g and 4 ° C. for 10 minutes.
7) 400 μL of the upper layer was collected with a micropipette, transferred to a new Eppendorf tube, 500 μL of 2-propanol was added, and the mixture was stirred with a vortex mixer and allowed to stand for 5 minutes.
8) Centrifugation was performed at 12000 × g and 4 ° C. for 10 minutes, and the supernatant was discarded.
9) 1 mL of 70% ethanol was added to the precipitate and centrifuged at 12000 × g at 4 ° C. for 10 minutes.
10) The supernatant was discarded and 20 μL of milliQ water was added to suspend well.

(Iii) RT-PCR method RT-PCR was performed according to the following procedure. The protocol of TaKaRa RNA PCR ^™ Kit (AMV) Ver.3.0 from Takara Bio Inc. was mainly followed.

1) Reverse transcription reaction solution [25 mM MgCl ₂ (2 μL), reverse transcription buffer (1 μL), milliQ water (3.75 μL), 1 mM dNTP mixture (1 μL), 1 U / μL RNase inhibitor (0.25 μL), 0.25 U / μL AMV reverse transcriptase XL (0.5 μL), 2.5 μM Oligo dT-adapter primer (0.5 μL)] were prepared.
2) 1 μL of the RNA sample obtained in (ii) above and 9 μL of the reverse transcription reaction solution were added to the PCR tube. Each PCR tube was set in a thermal cycler and incubated at 42 ° C., 50 minutes → 99 ° C., 5 minutes → 5 ° C., 5 minutes.
3) During reverse transcription, a reaction solution (5 × PCR buffer 10 μL, Milli Q water 27.75 μL, Ex Taq 0.25 μL, 40 μM primer solution 1.0 μL) was prepared and added to each PCR tube. The primers used at this time were the following for GAPDH as internal standard and caspase 14:
[Caspase 14 (NM_012114.2) (172bp)]
Sense: 5'-AGGAGGAGCTTTCCTTCCAG-3 '
Antisense: 5'-GCTAAGTTTTGGCTGGCTTG-3 '
[GAPDH (NM_001256799.1) (365bp)]
Sense: 5'-ATCATCAGCAATGCCTCCTG -3 '
Antisense: 5'-CTGCTTCACCACCTTCTTGA-3 '
4) Flashed for 10 seconds.
5) The tube was set in a thermal cycler and subjected to 30 cycles of 94 ° C., 30 seconds → 60 ° C., 30 seconds → 72 ° C., 2 minutes.
6) Agarose gel electrophoresis was performed on a 5% agarose gel, and the band was confirmed. The results are shown in FIG.

  As can be seen from FIG. 7, GAPDH mRNA is expressed at a constant level regardless of the concentration of each sphingoid, whereas caspase 14 mRNA shows a higher expression level as the sphingoid concentration is higher. It was. From this, it was confirmed that the expression of caspase 14 is promoted by sphingoids.

  A topical skin preparation containing Cer was prepared. Cer was dissolved in macadamia nut oil to a concentration of 50 μM. The obtained oil solution was added to a base containing beeswax: olive oil in a weight ratio of 20:80 to prepare a moisturizing ointment containing 5 μM of Cer. When 5 subjects applied an appropriate amount of the ointment to the right hand once a day and applied nothing to the left hand and evaluated both hands after 30 days, 4 out of 5 However, it was evaluated that the skin of the right hand was improved in terms of freshness compared to the skin of the left hand.

  The caspase 14 synthesis promoter of the present invention is useful for applications that utilize the promotion of caspase 14 expression, and is particularly useful as a skin external preparation for moisturizing.

Claims (5)

Phytosphingosine, sphingosine, sphinganine, and C ₂ - Caspase 14 in keratinocytes by at least one viewing containing an amount of a compound 3μM below, contact or coating with the skin of the keratinocytes selected from the group consisting of ceramides A caspase 14 synthesis promoter for promoting synthesis.   The caspase 14 synthesis promoter according to claim 1, wherein the compound is sphingosine.   The caspase 14 synthesis promoter according to claim 1, wherein the compound is phytosphingosine. The concentration of at least one compound selected from the group consisting of phytosphingosine, sphingosine, sphinganine, and C ₂ -ceramide as the caspase 14 synthesis accelerator according to any one of claims 1 to 3 is 0.1 to 3 µM. A skin external preparation for promoting caspase 14 synthesis, comprising:   The external preparation for skin according to claim 4, wherein the external preparation for skin is in the form of liquid, emulsion, paste, or poultice.